CN110286118A - A method of antibiotic mechanism of action is determined using bioluminescence reporting system - Google Patents

Abstract

The invention discloses a kind of methods that antibiotic mechanism of action is determined using bioluminescence reporting system.It is using the preparation staphylococcus aureus lux report strain of bioluminescence reporting system, it arranges on the agarose double-layer plate containing the bacterial strain filter paper of the solution to be checked containing antibiotic, it cultivates and obtains luminous picture, luminosity curve is converted by luminous picture, it extracts luminosity curve feature and compares progress hierarchical clustering with known antibiotic profile luminosity curve, if detected antibiotic luminosity curve determines that antibiotic mechanism of action to be checked is identical as the antibiotic of known action mechanism together with the luminosity curve of the antibiotic of known action mechanism cluster.The method of the present invention can realize the judgement to antibiotic mechanism of action, reduce the cost of antibiotic mechanism of action identification, improve the speed of antibiotic mechanism of action class identification, improve the ability of antibiotic-screening, have a extensive future.

Description

A method of antibiotic mechanism of action is determined using bioluminescence reporting system

Technical field

The present invention relates to a kind of methods that antibiotic mechanism of action is determined using bioluminescence reporting system.Belong to biological skill Art field.

Background technique

Antibiotic is primary armament of the mankind to bacterial-infection resisting.Being widely used for antibiotic causes serious antibiotic Drug resistance problems, so that existing antibiotic fails rapidly, bacterium infection becomes the significant threat of human health again.According to statistics, Present Global is because of about 700,000 people of antibiotics resistance death.In the year two thousand fifty, it is contemplated that because the antibiotics resistance whole world every year will Dead 10,000,000 people, becomes the No.1 cause of the death of the mankind.Because of the presence of this threat, so that the importance day of antibiotics discovery Benefit improves, and also becomes scientific circles to the discovery of antibiotics and the world of medicine studies and the emphasis of work.

Antibiotic can realize the killing to bacterium by number of mechanisms, such as prevent Cell wall synthesis, protein is prevented to close At, prevent DNA replication dna etc..Different bactericidal mechanisms results in different bactericidal effects.Therefore antibiotic bactericidal mechanism is sentenced Disconnected is one of the important indicator screened to antibiotic.However, the identification of antibiotic bactericidal mechanism needs complicated, time-consuming point Sub- Mechanism Study still can not often determine that it reduce the efficiency of antibiotic-screening, also reduce completely after antibiotic listing To the effect of antibiotic usage.

The luciferase of lux coded by said gene can be used to realize bioluminescence in bacterium.In the lux gene of bacterium, The effect of luxA and luxB is the subunit for encoding catalytic luminescence, and the related egg of luxC, luxD and luxE then encoding substrate synthesis It is white.The luciferase of bacterium is widely used in various reporting systems, wherein also including can detect in the system of antibiotic.

So far, the method for existing identification antibiotic mechanism of action depends critically upon complicated biochemistry, molecule Biology, genetics research, take time and effort, and dependent on high-level scientific research, do not have one kind that can quickly determine antibiosis yet The method of plain mechanism of action.This status has seriously affected grinding for the quick screening of antibiotic, discovery, identification and mechanism of action Study carefully.Through retrieving, there has been no the promoter fragments and and molecular chaperones that use by molecular chaperone clpX, groE, dnaJ, dnaK The built-up luminous reporting system of the connected luxABCDE genetic fragment of gene promoter carries out the judgement of antibiotic mechanism of action Report occur.

Summary of the invention

The object of the present invention is to provide a kind of methods that antibiotic mechanism of action is determined using bioluminescence reporting system.

The method of the present invention that antibiotic mechanism of action is determined using bioluminescence reporting system, step is:

(1) strain is reported using bioluminescence reporting system preparation staphylococcus aureus lux；Wherein, the luminous report System is by pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, pAmilux-dnaK- LuxABCDE plasmid, pAmilux-groE-luxABCDE plasmid composition；The core of the pAmilux-clpX-luxABCDE plasmid Nucleotide sequence is as shown in SEQ ID NO:1, the nucleotide sequence of the pAmilux-dnaJ-luxABCDE plasmid such as SEQ ID Shown in NO:2, the nucleotide sequence of the pAmilux-dnaK-luxABCDE plasmid is described as shown in SEQ ID NO:3 The nucleotide sequence of pAmilux-groE-luxABCDE plasmid is as shown in SEQ ID NO:4；The staphylococcus aureus lux Report strain be by four kinds of plasmid pAmilux-clpX-luxABCDE plasmids, pAmilux-dnaJ-luxABCDE plasmid, PAmilux-dnaK-luxABCDE plasmid, pAmilux-groE-luxABCDE plasmid are transformed into staphylococcus aureus respectively In, manufactured four plants of staphylococcus aureuses report strain, are respectively designated as: reporting bacterial strain S1, reporting bacterial strain S2, reporting bacterial strain S3, reporting bacterial strain S4；

(2) the agarose double-layer plate containing four plants of staphylococcus aureus report strains is prepared, the filter paper of sterilizing is existed Agarose double-layer plate center or media surface ordered arrangement are simultaneously closely affixed with culture medium；

(3) solution to be checked containing antibiotic is added drop-wise on filter paper, continues to cultivate and shoot bacterial luminescence picture；

(4) luminosity curve is converted by luminous picture, extracts luminosity curve feature with known antibiotic profile luminosity curve It compares and carries out hierarchical clustering, if the luminosity curve of detected antibiotic luminosity curve and the antibiotic of known action mechanism cluster exists Together, then determine that antibiotic mechanism of action to be checked is identical as the antibiotic of known action mechanism.

In the above-mentioned method for determining antibiotic mechanism of action using bioluminescence reporting system: step (2) described filter paper No. 1 qualitative filter paper of Xinhua is preferably broken into punch the small scraps of paper of circle of diameter 6mm.

In the above-mentioned method for determining antibiotic mechanism of action using bioluminescence reporting system: step (3) is described will be to be checked After solution containing antibiotic is added drop-wise on filter paper, preferably plate is inverted in incubator, 37 DEG C of 20~25h of culture.

In the above-mentioned method for determining antibiotic mechanism of action using bioluminescence reporting system, step (4) will be described to shine Picture is converted into luminosity curve or the method for drafting of known antibiotic profile luminosity curve is:

1) using plate center as origin (x₀,y₀), plate radius is divided into a specific number (such as 300 or 1200 parts), Average distance is denoted as its midpoint to the maximum distance of origin and the average value d of minimum range in obtained each annulus；

2) A is set as the average canbdle power of all the points in some annulus, then average canbdle power is the function of d: A=f (d)；

3) function of application settings is depicted as luminosity curve.

In the above-mentioned method for determining antibiotic mechanism of action using bioluminescence reporting system: the known antibiotic is to reach Tobramycin (DAP), polymyxin B (PMB), Imipenem (IPM), benzyl penicillin (PEN), vancomycin (VAN), erythromycin (ERY), kanamycins (KAN), spectinomycin (SPE), streptomysin (STR), tetracycline (TET), mitomycin C (MMC), cyclopropyl Sha Xing (CIP), gatifloxacin (GAT), trimethoprim (TMP), rifampin (RIF), phleomycin (PHL).

In the above-mentioned method for determining antibiotic mechanism of action using bioluminescence reporting system: step (4) is described according to special The method that light curve of levying carries out hierarchical clustering analysis is:

1) the characteristic luminescence curve by each from inhibition zone edge to plate edge part is denoted as by 300 luminous intensity institutes A vector (luminous intensity characterization vector) for characterization, each luminous intensity represent in the picture that shines from inhibition zone edge to flat Average canbdle power in each annulus of plate edge portion, that is, inhibition zone edge to plate edge part 1/300 annulus；

2) by the feature of four plants of staphylococcus aureus report strains in bio-luminescence system corresponding to each antibiotic Luminosity curve is merged into one and characterizes vector by the merging luminous intensity that 1200 luminous intensities are characterized；

3) use Euclidean distance calculation method, calculate antibiotic mergings luminous intensity characterize vector between away from From, and construct the distance matrix of antibiotic；

4) vector is characterized to the merging luminous intensity of antibiotic with UPGMA algorithm using the hclust function in R language Between carry out clustering, whether with the antibiotic cluster of known action mechanism judge that it is anti-together according to antibiotic to be checked The type of raw element mechanism of action.

Method disclosed by the invention can quick easy judgement antibiotic mechanism of action type, clinic with higher Application value.It is identified for antibiotic discovery, antibiotic mechanism, and is further used for instructing the side such as bacterial infection treatment scheme Mask is significant.

The present invention is that bioluminescence reporting system is transformed into gold using bioluminescence reporting system and data analysing method After staphylococcus aureus, the antibiotic scraps of paper are being loaded with according to the staphylococcus aureus containing bioluminescence reporting system The phenomenon to shine on double-layer plate shoots the picture that shines, and converts luminosity curve for luminous picture, and it is bent to calculate luminous feature Line, and clustering is carried out to indicatrix.By the luminescence feature curve to different antibiotic analysis shows: this method can The luminescence feature curve of the antibiotic of mechanism of action having the same to be clustered by the clustering of luminescence feature curve, from Which and clustered by the antibiotic luminescence feature curve of the luminescence feature curve and known action mechanism that judge antibiotic one It rises and determines antibiotic mechanism of action, the sensitivity and specificity of detection have reached 0.89 or more, to demonstrate of the invention Effect.

Compared with prior art, the beneficial effects of the present invention are:

Complicated base mechanisms research method is avoided, quickly antibiotic mechanism of action can be determined, to mention The discovery speed of high antibiotics improves the identification speed of antibiotics mechanism of action, the antibiosis found using the method for the present invention Element can be used clinically for treatment bacterium infection, including multi-drug resistant bacteria infection, to bring huge social and economical benefits.

Detailed description of the invention

Fig. 1: antibiotic (Ciprofloxacin) makes bioluminescence reporting system (pAmilux-clpX-luxABCDE) to shine.

Fig. 2: the characteristic luminescence curve of different antibiotic.

Wherein: clpX, dnaJ, dnaK, groE refer to four kinds of plasmids in luminous reporting system of the present invention.Three Line (from left to right) indicates respectively scraps of paper radius, inhibition zone radius, plate radius.RLU, relative light units.CIP, cyclopropyl are husky Star；DAP, Daptomycin；ERY, erythromycin；GAT, gatifloxacin；IPM, Imipenem；KAN, kanamycins；MMC, mitogen are mould Plain C；PEN, penicillin；PHL, phleomycin；PMB, polymyxin B；RIF, rifampin；SPE, spectinomycin；STR, streptomysin； TET, tetracycline；TMP, trimethoprim；VAN, vancomycin.

Fig. 3: the clustering determined for antibiotic mechanism of action.

Specific embodiment

Below will by specific embodiment, the present invention is further illustrated, but be discussed further below example only and be the present invention Better embodiment, be not intended to limit the present invention in any form, it is right according to the technical essence of the invention Any simple modification that embodiment is made, equivalent variations and modification, belong in the range of technical solution of the present invention.

It is routinely experimental method if method content is without specified otherwise described in following embodiments.The reagent that is related to, If carrier, bacterial strain without specified otherwise, are that well known Sales Channel obtains.

Embodiment 1

Experimental strain and plasmid:

Escherichia coli DH5 α: preparing for competence, and China General Microbiological culture presevation administrative center obtains Take (CGMCC1.12873).

Escherichia coli DH10B: preparing for competence, and ThermoFisherScientific company obtains (article No. 18297010)

Staphylococcus aureus RN4220: prepare for competence and shine detection, and BEI Resources is obtained It takes.

PMD19-T: it is purchased from Dalian treasured biotech firm (TaKaRa)

PAmilux: from University of British Columbia (document: Mesak LR, Yim G, Davies J, Improved lux reporters for use in Staphylococcus aureus,Plasmid,2009,61(3): 182-187.)

Culture medium needed for testing and its preparation:

LB culture medium: every liter of culture medium is configured, tryptone (10g), sodium chloride is added in Ying 950ml deionized water (10g), yeast extract (5g), and extremely dissolved with magnetic stirrer.5mol/L sodium hydroxide is added dropwise and is adjusted to pH7.0, uses After deionized water is settled to 1L, dispenses and high pressure steam sterilization 20min is spare under the conditions of 121 DEG C.If needing solid medium, Agar powder need to be added in 1.5% ratio.

NYE culture medium: 1 liter of culture medium is prepared, casein hydrolysate (10g), yeast is added in Ying 950ml deionized water Extract (5g), sodium chloride (5g), with magnetic stirrer to dissolution.5mol/L sodium hydroxide is added dropwise and is adjusted to 7.2 left side pH The right side after being settled to 1L with deionized water, dispenses and high pressure steam sterilization 20min is spare under the conditions of 121 DEG C.If needing solid culture Base then needs that agar powder is added in 1.5% ratio.

Antibiotic needed for testing and its preparation:

Ampicillin (AMP) solution: weighing 100mg ampicillin, is dissolved in the sterile ultrapure water of 1ml and the vibration that is vortexed Mixing is swung, its final concentration of 100mg/ml is made, is deposited in spare in -20 DEG C of refrigerators.In use, training is added in the ratio of 1:1000 It supports in base.

Chloramphenicol (CHL) solution: weighing 10mg chloramphenicol, is dissolved in 1ml dehydrated alcohol and vortex oscillation mixes, make it Final concentration of 10mg/ml is deposited in spare in -20 DEG C of refrigerators.In use, being added in culture medium in the ratio of 1:1000.

Solution needed for testing and its preparation:

Competence buffer: with electronic balance weigh 0.333g CaCl2,0.105g 3-N-morpholinopropanesulfonic acid sodium salt (MOPS), 0.148g MnCl24H2O is dissolved in 40ml deionized water, after measuring the above-mentioned solution of 7.5ml glycerol addition with liquid-transfering gun, It is settled to 50ml with deionized water, and is adjusted to pH to 7 or so with NaOH.After 0.22 μm of membrane filtration degerming of buffer, turn It moves on in 50ml sterile centrifugation tube and is pre-chilled on ice, it is spare.The buffer matching while using.

80% (v/v) glycerol: measuring 80ml glycerol and be dissolved in 20ml ultrapure water, high pressure steam sterilization under the conditions of 121 DEG C 20min is spare.

50 × TAE electrophoretic buffer: weighing Tris 242g, and 700ml ultrapure water is added in beaker in EDTA 18.612g With the glacial acetic acid of 57.1ml, dissolution is sufficiently stirred.Then, NaOH is added and adjusts pH to 8.3, be finally settled to 1L, stored up under room temperature Deposit spare, 50 times of used time dilution.

Reagent needed for testing and instrument:

Reagent needed for testing: Ezup pillar bacterial genomes DNA extraction agent box (Sangong biotech, Shanghai, China), EZ-10 pillar small amount plasmid extraction agent box (Sangong biotech, Shanghai, China), EZ-10 pillar DNA plastic recovery kit (Sangong biotech, Shanghai, China), pMD^TM19-T Vector Cloning Kit (Takara, Dalian, China), the quick restriction endonuclease of FlyCut BamHI (TransGene Biotech, Beijing, China), T4DNA Ligase (Takara, Dalian, China), the nontoxic nucleic acid dye of 4S Green Plus (Sangong biotech,Shanghai,China)、NormalRunTM prestained 250bp-I DNA ladder (Generay biotech, Shanghai, China), 1 × T3Super PCR Mix (Tsingke, Beijing, China), nothing Bacterium filter membrane (0.22 μm).

Instrument needed for testing:

DNP-9082 type constant incubator (the upper macro experimental facilities Co., Ltd of Nereid), (Jiangsu is too for constant-temperature shaking incubator Cang Shiyanyiqichang), HH-S thermostat water bath (Medical Instruments factory, Community of Jin Tan County city), micro-wave oven (Glanz), superclean bench (Jiangsu purifies experimental facilities factory), assay balance (Sai Duolisi scientific instrument Co., Ltd), turbula shaker (its woods of Jiangsu Province Bell's instrument manufacturing Co., Ltd), centrifuge (Co., Ltd in Ai Bende), PCR instrument (Bole's life medical product (Shanghai) Co., Ltd), DYY-6C type electrophoresis apparatus (Liuyi Instruments Plant, Beijing).

Experimental procedure:

The activation of step 1. bacterial strain and purifying

The Escherichia coli (15% glycerol) of the glycerol stocks frozen are linked into LB culture medium in the ratio of 1:300,37 Shaken cultivation is stayed overnight under the conditions of DEG C.

Escherichia coli use the culture of LB culture medium, and staphylococcus aureus uses the culture of NYE culture medium.It draws activated 10 μ l of bacterium solution is added drop-wise on culture dish, then burns red oese, is dipped bacterium solution after its cooling and is crossed.When paying attention to crossing not Wanted dredge or it is overstocked, be otherwise difficult to isolated single colonie.Culture 1h is first just put under the conditions of 37 DEG C, after bacterium solution fully absorbs It is inverted overnight incubation.

Step 2. bacterial genomes DNA is extracted

This experiment uses Ezup pillar bacterial genomes DNA extraction agent box, and steps are as follows:

Microorganism collection:

Staphylococcus aureus: the bacterial solution for taking 1ml to be incubated overnight into 1.5ml centrifuge tube, 8000-12000rpm from Heart 1min as far as possible removes supernatant clean.(Enzymatic is added using preceding in lysozyme by the lysozyme soln that 180 μ l are added In lysis buffer, it is configured to the lysozyme soln of 30mg/ml), it is blown and beaten repeatedly with liquid-transfering gun and thallus is resuspended, in 37 DEG C of conditions Lower water-bath at least 3h.Then, 20 μ l Proteinase K are added, overturns or oscillation mixes, be incubated at least 1h in 56 DEG C of water-baths Liquid clarification such as egg white shape into EP pipe.This step is very crucial, because of aureus cell wall thickness, if cracking is endless Entirely, it is difficult to extract its genomic DNA.

200 μ l Buffer BD are added, mix.

200 μ l dehydrated alcohols are added, mix.

Adsorption column in kit is put in collecting pipe, the liquid after BufferBD and dehydrated alcohol are added with liquid-transfering gun It is fully transferred in adsorption column, stands 2-3min, subsequent 12000rpm is centrifuged 3min, discards the waste liquid in collecting pipe.

Adsorption column is put back into collecting pipe, 500 μ l PW Solution, 12000rpm centrifugation 1min are added dropwise, discard collecting pipe In waste liquid.

Adsorption column is put back into collecting pipe, 500 μ l Wssh solution, 12000rpm is added to be centrifuged 1min.

Adsorption column is put back in collecting pipe, 12000rpm is centrifuged 5min, to remove remaining rinsing liquid.

Adsorption column is taken out, is placed in new 1.5ml EP pipe, the sterile ultrapure water of 50-100 μ l is added and stands 2min, 12000rpm centrifugation 2min obtains DNA solution.

The building of step 3. promoter-carrier T

The amplification of gene groES-EL, dnaK, dnaJ, clpX promoter fragment:

Using staphylococcus aureus gene group DNA as template, accordingly drawn using 5.0 software design of Primer Premier (clpX, dnaK, dnaJ, groE promoter sequence are directed to) after object, add I enzyme of BamH respectively at 5 ' ends of upstream, downstream primer Enzyme site: GGATCC.Further, since many restriction endonucleases are difficult to cut the identification sequence close to DNA molecular end, In order to solve this problem, additional protection base (such as CGC) should be designed at 5 ' ends of Oligonucleolide primers, primer sequence is such as Shown in table 1.

Primer needed for 1 construction of recombinant plasmid of table and verifying

PCR reaction system is to refer to (table 2) with Super PCR Mix specification:

2 PCR reaction system of table

Annealing temperature in PCR program can be adjusted according to amplification situation, annealing temperature be improved if miscellaneous band is more, if mesh Band be difficult to expand, reduce annealing temperature.Extension of time is related with the clip size of target gene, normal amplification rate For 1Kb/min.

3 PCR response procedures of table

Staphylococcus aureus RN4220 agarose gel electrophoresis detects PCR product:

(1) Ago-Gel for preparing 0.8%, weighs 0.8g agarose, mixes with 100ml 1 × TAE electrophoretic buffer Afterwards in microwave stove heating until agarose melts completely, until obtaining clear and transparent solution.

(2) it is cooled to 50 DEG C or so at room temperature, nucleic acid dye is added in the ratio of 1:1000 and mixes, pours into mold simultaneously Suitable comb is plugged, its condensation is then waited.It the position of comb teeth bottom should be apart from pallet face 0.5-1mm, if comb teeth connects very much Nearly pallet face, the loading wells of gel is easily destroyed when extracting comb, sample is caused to reveal.

(3) wait be gelled it is solid after, be placed into the electrophoresis tank equipped with 1 × TAE running buffer, with liquid-transfering gun by PCR product and DNA marker is put in glue hole respectively.Electrophoresis slot cover is shut, electrode plug is connected, carries out electrophoresis under 120V voltage.

It is as far as possible same batch with the electrophoresis liquid placed in electrophoresis liquid used in glue and electrophoresis tank, because even being very little Ionic strength change is likely to cause gelling performance abnormal, influences the migration rate of DNA.

(4) Ago-Gel after the completion of electrophoresis is put into imager, observes and take pictures under the ultraviolet lamp of 300nm.

PCR product purifying:

(1) Ago-Gel is observed using gel imager, the blob of viscose containing purpose band is cut with clean blade, Edge is cut, keeps blob of viscose volume as small as possible.Then, blob of viscose weight is weighed, and blob of viscose is transferred in 1.5ml EP pipe.

(2) 300 μ l Binding buffer II are added in the blob of viscose of every 100mg, are incubated in 50-60 DEG C of water-bath During which 10min constantly gently shakes EP pipe, until blob of viscose melts.

(3) collecting pipe is placed into the EP pipe of 2ml, mixed liquor obtained in the previous step is transferred in collecting pipe, in room Temperature is lower to stand 2min, and 12000rpm is centrifuged 1min, discards the waste liquid in EP pipe.

(4) 500 μ l Wash Solution are added into collecting pipe, 12000rpm is centrifuged 1min, discards useless in EP pipe Liquid.

(5) step (4) are repeated.

(6) collecting pipe is put back in EP pipe, 12000rpm is centrifuged 1min to remove remaining Wash Solution.

(7) collecting pipe is placed in new 1.5ml EP pipe, the addition sterile ultrapure water of 30-40 μ l on filter membrane, 50 DEG C 10min is incubated in water-bath, target DNA solution can be obtained in 12000rpm centrifugation 1min.

The connection of promoter and carrier T:

Constructing intermediate vector is to keep promoter sequence digestion complete.Using pMD^TM19-T Vector Cloning Kit, linked system are following (10 μ l):

4. linked system of table

The above linked system is flicked into mixing, is placed in 4 DEG C of refrigerator overnights.

The production of competent escherichia coli cell:

(1) into fresh 3ml LB test tube, 37 DEG C are incubated overnight 10 μ l glycerol tube thallus of inoculation.

(2) thallus after activation is inoculated in fresh 100ml LB culture medium in the ratio of 1%-5%, 37 DEG C, 150r revolving speed shake culture to OD value is 0.3-0.6, is subsequently placed at least 30min on ice, terminates the fast-growth of bacterium, this step Suddenly it is the key that competence is successful, can not omits.

(3) bacterium solution is transferred in 50ml sterile EP tube, 10min is centrifuged using 4 DEG C of refrigerated centrifuge 5000rpm, in abandoning Clearly.

(4) the competence buffer of 5ml pre-cooling is added, thallus, ice bath 10min is gently resuspended with pipette tips.

(5) it is centrifuged 10min using 4 DEG C of refrigerated centrifuge 5000rpm, abandons supernatant.The competence buffering of 1ml pre-cooling is added Thallus is gently resuspended with pipette tips in liquid, is then sub-packed in sterile EP tube, is frozen in -80 DEG C of refrigerators by the volume of every 100 μ l of pipe In it is spare.

(6) it detects: taking 10 μ l competence bacterium solutions, be inoculated into 3ml and contain in the fresh LB of AMP, 37 DEG C of trainings overnight It supports.If culture medium had no thalli growth in second day, illustrates the non-microbiological contamination of competence, can be used normally.If culture medium becomes cloudy, Then this batch of competence should give it up, and remake.

It converts (thermal shock method):

(1) conical flask containing 100ml LB agar medium is placed in micro-wave oven and is heated to melting completely, in room It is cooled to 60 DEG C or so under temperature, 100 μ l ampicillins (100mg/ml) are added into culture medium.Conical flask is gently shaken, is infused Meaning not have bubble, be then poured in sterilizes culture dish and amicillin resistance plate is made.

(2) ice will be filled in ice chest, be placed in beside -80 DEG C of refrigerators.The competent cell of preservation is taken out from refrigerator, and Make its quick-thawing using the temperature of palm.When competence is just melted, it is immediately inserted into ice.

(3) reaction system connecting 10 μ l promoters with carrier T after the reaction was completed is added to 100 μ l competent cells In, flick the mixing of EP pipe, ice bath 30min.

(4) the thermal shock 90s in 42 DEG C of water-baths of the mixed liquor after ice bath, the temperature and time of this step is all very crucial, Pay attention to that EP pipe can not be shaken when thermal shock.

(5) after thermal shock, EP pipe is transferred on ice rapidly, ice bath 5min.

(6) the fresh not antibiotic LB culture medium of 800 μ l, 150r renewal cultivation under the conditions of 37 DEG C are added into EP pipe 1h.Then, 3000r is centrifuged, and mixes after discarding part supernatant, 150 μ l is taken to be coated on the LB plate containing corresponding antibiotic. Culture 1h is first just set under the conditions of 37 DEG C, after bacterium solution is cultured base absorption completely, then is inverted overnight incubation.

The identification of positive colony:

The 10 sterile ultrapure waters of μ l are added in EP pipe, the monoclonal on picking plate mixes in water.1 μ l is taken to be used as PCR Template carries out bacterium colony PCR, and 9 μ l of residue are for being inoculated with (table 5).

5 PCR response procedures of table:

The PCR product that amplification obtains is obtained into purpose band after electrophoresis, then can be sequenced, sequencing result utilizes After Bioedit software carries out sequence alignment, correct positive colony can be filtered out, to be successfully prepared bioluminescence report system System, by pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, pAmilux-dnaK- LuxABCDE plasmid, pAmilux-groE-luxABCDE plasmid composition；The core of the pAmilux-clpX-luxABCDE plasmid Nucleotide sequence is as shown in SEQ ID NO:1, the nucleotide sequence of the pAmilux-dnaJ-luxABCDE plasmid such as SEQ ID Shown in NO:2, the nucleotide sequence of the pAmilux-dnaK-luxABCDE plasmid is described as shown in SEQ ID NO:3 The nucleotide sequence of pAmilux-groE-luxABCDE plasmid is as shown in SEQ ID NO:4.

The T plasmid that table 6 constructs

Step 4. shines the building of reporting system

Digestion: plasmid pAmilux, PM1, PM2, PM3, PM4 are subjected to digestion with BamH I.Reaction system is following (table 7):

7 digestion system of table

Above-mentioned mixed liquor is incubated for 1h at 37 DEG C, and subsequent 80 DEG C of incubations 20min inactivates restriction endonuclease, and deactivation step can not save Slightly, in order to avoid star activity occurs in restriction endonuclease.It is recommended to use PCR instrument and carries out temperature control.

Carrier dephosphorylation: for plasmid pAmilux after single endonuclease digestion, the phosphate group at 5 ' end of removal is able to suppress Plasmid DNA Occur to connect and be cyclized certainly.In vitro in connection reaction, only when a nucleotide contains 5 '-phosphate groups and another has When 3 '-C-terminal, it could be catalyzed to form phosphodiester bond by DNA ligase.Therefore, alkaline phosphatase treatment plasmid is utilized DNA makes it slough the phosphate group at 5 '-ends, the generation of recirculation can be effectively prevented.Reaction system is following (table 8):

8 carrier dephosphorylation system of table

1h is reacted under the conditions of 37 DEG C, subsequent 75 DEG C of heating 5min inactivates alkaline phosphatase.Promoter fragment is not necessarily to dephosphorization Acidification, the carrier dephosphorylation time is also unsuitable too long, in order to avoid reduce joint efficiency.

DNA purifying: kits process is referred to.

Connection: the promoter fragment by digestion, after purification is connected with plasmid pAmilux with T4 ligase, the following (table of system 9):

9 linked system of table

After linked system is mixed, it is placed in 4 DEG C of refrigerator overnight connections.

Conversion and the screening of positive colony: since aureus cell wall is very thick, the conversion operation of plasmid is more Difficulty, so to be first transformed into connection product in E.coli DH5 α with thermal shock method, convenient for the screening of positive colony.Then from I site two sides design primer (LuxF, LuxR) of BamH, to carry out the verifying of segment direction of insertion.

The preparation of staphylococcus aureus electrotransformation competence:

Staphylococcus aureus competence whole process will be prepared at normal temperature.

(1) the S.aureus RN4220 for taking overnight growth is inoculated into fresh 100ml NYE liquid in 2% ratio and trains Support base in, 37 DEG C, 150r shaken cultivation to OD be 0.2-0.3.

(2) by microorganism collection into 50ml sterile EP tube, 5000rpm is centrifuged 10min.

(3) liquid is discarded supernatant, the sterile ultrapure water of 25ml is added, gently being blown and beaten with pipette tips is resuspended thallus, 5000rpm centrifugation Ten minutes.

(4) liquid is discarded supernatant, the sterile ultrapure water of 10ml is added, thallus is resuspended, 5000rpm is centrifuged 10min.

(5) liquid is discarded supernatant, 10% glycerol of 5ml is added, thallus is resuspended, 5000rpm is centrifuged 10min.

(6) step (5) are repeated.

(7) glycerol of 1ml 10% is added into thallus, is resuspended, is dispensed into sterile EP tube by the volume of every 70 μ l of pipe ,- 80 DEG C save backup.

During making electrotransformation competence, it is necessary to the abundant washing thalline of deionized water, to remove institute in culture medium The salt ion contained, quick-fried cup when preventing from shocking by electricity.

Staphylococcus aureus electrotransformation:

(1) conical flask containing 100ml NYE agar medium is placed in micro-wave oven and is heated to melting completely, in room It is cooled to 60 DEG C or so under temperature, 100 μ l chloramphenicol (10mg/ml) are added into culture medium.Conical flask is gently shaken, is careful not to There is bubble, is then poured in sterilizes culture dish and chlorampenicol resistant plate is made.

(2) an appropriate number of freezing competent cell is taken out from -80 DEG C of refrigerators is placed in room temperature, when it just thaws, 10 μ l pAmilux-clpX-luxABCDE plasmids or pAmilux-groE-luxABCDE plasmid are added thereto, or PAmilux-dnaJ-luxABCDE plasmid or pAmilux-dnaK-luxABCDE plasmid flick the mixing of EP tube wall, at normal temperature It is incubated for 30min.There is document report incubation time to be preferred with 30min, be more than or transformation efficiency will be reduced less than 30min.

(3) mixed liquor is transferred in 2mm electricity revolving cup, is preferably inserted into suction pipette head along electric revolving cup wall, it will be thin Born of the same parents are added to one end of slot, and the tapping electricity revolving cup on smooth desktop, to shake the bubble contained in liquid out.Then by electric revolving cup It closes the lid and is fitted into electroporation, select pre-set programs: StA presses Pulse key, rapidly takes electric revolving cup after hearing buzzer Out, the fresh NYE culture medium of 800 μ l is added into electric revolving cup in 30s.

The ion contained in the mixed liquor of competent cell and DNA will increase the conductivity of solution, lead to the generation of electric arc, At this moment electric revolving cup can burst, and greatly reduce the efficiency of electrotransformation.If there is such case, to consider that competent cell makes When whether wash not exclusively, or dissolution Plasmid DNA solution in whether contain certain ions, should check and solve one by one.

(4) liquid is transferred in sterile EP tube, 100r renewal cultivation 1.5h under the conditions of 37 DEG C.Microorganism is non-after electric shock It is often fragile, it can not acutely shake.

(5) the bacterium solution 3000rpm after recovery is centrifuged 5min, discards part supernatant, remaining 450 μ l supernatant and thallus It is used to coated plate after resuspension, every plate is coated with 150 μ l.37 DEG C are just set culture 1h, and culture 24-48h is inverted after bacterium solution absorption, can be gone out Existing macroscopic bacterium colony.It is not recommended that the bacterial suspension after centrifugal concentrating is all coated on a plate, because of electrotransformation meeting A large amount of dead cells are generated, these cells are attached to the transformant growth that media surface may inhibit originally rare.

Above-mentioned four kinds of plasmid pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, PAmilux-dnaK-luxABCDE plasmid, pAmilux-groE-luxABCDE plasmid are transformed into staphylococcus aureus respectively Obtain four plants of staphylococcus aureus lux report strain be respectively designated as: reporting bacterial strain S1, reporting bacterial strain S2, reporting bacterial strain S3, Reporting bacterial strain S4.

The drafting of 2 characteristic luminescence curve of embodiment

Experimental strain: four plants of staphylococcus aureus lux report strain that experiment bacterial strain uses therefor is: S1, S2, S3, S4.

Culture medium needed for testing and its preparation

1.NYE agar medium

2. 0.7% agar: it weighs 0.7g agar in the balance and is added in conical flask, add 100ml deionized water, 121 High pressure steam sterilization 20min is spare under the conditions of DEG C.

Antibiotic needed for testing and its preparation

Daptomycin 1. (DAP) solution: taking 30mg Daptomycin to be dissolved in 1ml ultrapure water, make its final concentration of 30mg/ Ml, 0.22 μm of membrane filtration degerming；

Polymyxin B 2. (PMB) solution: taking 100mg polymyxin B to be dissolved in 1ml ultrapure water, keep its final concentration of 100mg/ml, 0.22 μm of membrane filtration degerming；

Imipenem 3. (IPM) solution: taking 10mg Imipenem to be dissolved in 1ml ultrapure water, make its final concentration of 10mg/ Ml, 0.22 μm of membrane filtration degerming；

Benzyl penicillin 4. (PEN) solution: taking 10mg benzyl penicillin to be dissolved in 1ml ultrapure water, make its final concentration of 10mg/ Ml, 0.22 μm of membrane filtration degerming；

Vancomycin 5. (VAN) solution: taking 30mg vancomycin to be dissolved in 1ml ultrapure water, make its final concentration of 30mg/ Ml, 0.22 μm of membrane filtration degerming；

Erythromycin 6. (ERY) solution: taking 15mg erythromycin to be dissolved in 1ml dehydrated alcohol, make its final concentration of 30mg/ Ml, 0.22 μm of membrane filtration degerming；

Kanamycins 7. (KAN) solution: taking 30mg kanamycins to be dissolved in 1ml ultrapure water, make its final concentration of 30mg/ Ml, 0.22 μm of membrane filtration degerming；

Spectinomycin 8. (SPE) solution: taking 100mg spectinomycin to be dissolved in 1ml ultrapure water, keep its final concentration of 100mg/ml, 0.22 μm of membrane filtration degerming；

Streptomysin 9. (STR) solution: it takes 10mg streptomysin to be dissolved in 1ml ultrapure water, makes its final concentration of 10mg/ml, 0.22 μm of membrane filtration degerming；

Tetracycline 10. (TET) solution: it takes 30mg tetracycline to be dissolved in 1ml ultrapure water, makes its final concentration of 30mg/ml, 0.22 μm of membrane filtration degerming；

Mitomycin C 11. (MMC) solution: taking 5mg mitomycin C to be dissolved in 1ml ultrapure water, keep its final concentration of 5mg/ml, 0.22 μm of membrane filtration degerming；

Ciprofloxacin 12. (CIP) solution: taking 5mg Ciprofloxacin to be dissolved in 1ml ultrapure water, make its final concentration of 5mg/ Ml, 0.22 μm of membrane filtration degerming；

Gatifloxacin 13. (GAT) solution: taking 5mg gatifloxacin to be dissolved in 1ml ultrapure water, make its final concentration of 5mg/ Ml, 0.22 μm of membrane filtration degerming；

Trimethoprim 14. (TMP) solution: it takes 5mg trimethoprim in 500ml ultrapure water, dilute hydrochloric acid is slowly added dropwise, until All dissolutions, are settled to 1ml with ultrapure water, make its final concentration of 5mg/ml, 0.22 μm of membrane filtration degerming；

Rifampin 15. (RIF) solution: it takes 10mg rifampin to be dissolved in 1ml DMSO, makes its final concentration of 10mg/ml, 0.22 μm of membrane filtration degerming；

Phleomycin 16. (PHL) solution: taking 5mg phleomycin to be dissolved in 1ml ultrapure water, make its final concentration of 5mg/ Ml, 0.22 μm of membrane filtration degerming.

Instrument needed for testing: superclean bench (Jiangsu purifies experimental facilities factory), electro-heating standing-temperature cultivator (the upper macro reality of Nereid Test equipment Co., Ltd), Lumazone PyLoN 2048B plant living body imaging system (Bo Yi great achievement Instrument Ltd.).

Experimental procedure

Step 1, plate preparation

1. by reporting bacterial strain S1, reporting bacterial strain S2, reporting bacterial strain S3, reporting bacterial strain S4 in the flat lining out of NYE, 37 DEG C of mistakes Night culture, is then wrapped with sealed membrane, it is spare to be put into 4 DEG C of refrigerators.

2. taking No. 1 qualitative filter paper of Xinhua, the small scraps of paper of circle of diameter 6mm are broken into punch, are placed on glass culture dish In, it is wrapped with brown paper, 121 DEG C of sterilizing 20min are dried for standby in an oven.

3. NYE agar medium is heated to after agar melts completely with micro-wave oven, it is placed in room temperature and is cooled to 60 DEG C of left sides The right side, pour plate, the thickness of every block of plate uniformity as far as possible.

4. picking reporting bacterial strain S1 or reporting bacterial strain S2 or reporting bacterial strain S3 or reporting bacterial strain S4 single colonie are dissolved in In the 200 sterile ultrapure waters of μ l, then bacteria suspension is added in 0.7% agar in the ratio of 1:1000, NYE is poured over after mixing Pair containing bacterial strain S0, reporting bacterial strain S1, reporting bacterial strain S2, reporting bacterial strain S3, reporting bacterial strain S4 is accordingly made in the upper layer of plate Plate each 16, layer.

5. by tweezers, calcination once, clamps filter paper after cooling and is gently attached to training on flame after double-layer plate solidification Support primary surface.In order to be affixed the scraps of paper closely with culture medium, the scraps of paper can be pressed lightly on tweezers.In order to accurately observe knot Fruit, the scraps of paper should be orderly distributed in the position of media surface.

6. culture medium, during cooling, surface is likely to occur condensing drip.To prevent condensed water from drug is broken up shadow The lid of culture medium can be opened first before scraps of paper dosing, be evaporated using the filtrated air in super-clean bench by the shape for ringing inhibition zone Excessive moisture.

7. taking 1 μ l antibiotic solution to drip with liquid-transfering gun in the prepared double-layer plate (totally 16) containing bacterial strain S1 It is added on filter paper preset in each plate, plate is inverted in incubator, 37 DEG C of culture 20h.Wherein, the antibiotic It is CIP, DAP, ERY, GAT, IPM, KAN, MMC, PEN, PHL, PMB, RIF, SPE, STR, TET, TMP or VAN, by each 1 μ l of plate is added dropwise respectively.

8. taking 1 μ l antibiotic solution to drip with liquid-transfering gun in the prepared double-layer plate (totally 16) containing bacterial strain S2 It is added on filter paper preset in each plate, plate is inverted in incubator, 37 DEG C of culture 20h.Wherein, the antibiotic It is CIP, DAP, ERY, GAT, IPM, KAN, MMC, PEN, PHL, PMB, RIF, SPE, STR, TET, TMP or VAN, by each 1 μ l of plate is added dropwise respectively.

9. taking 1 μ l antibiotic solution to drip with liquid-transfering gun in the prepared double-layer plate (totally 16) containing bacterial strain S3 It is added on filter paper preset in each plate, plate is inverted in incubator, 37 DEG C of culture 20h.Wherein, the antibiotic It is CIP, DAP, ERY, GAT, IPM, KAN, MMC, PEN, PHL, PMB, RIF, SPE, STR, TET, TMP or VAN, by each 1 μ l of plate is added dropwise respectively.

10. taking 1 μ l antibiotic solution to drip with liquid-transfering gun in the prepared double-layer plate (totally 16) containing bacterial strain S4 It is added on filter paper preset in each plate, plate is inverted in incubator, 37 DEG C of culture 20h.Wherein, the antibiotic It is CIP, DAP, ERY, GAT, IPM, KAN, MMC, PEN, PHL, PMB, RIF, SPE, STR, TET, TMP or VAN, by each 1 μ l of plate is added dropwise respectively.

Step 2, flat panel imaging

Lumazone imaging system consists of three parts, and is CCD camera, camera bellows and light source respectively.Concrete operation step is such as Under:

1. opening CCD camera power switch, camera bellows switch, power switch, computer and software are opened.

2. opening liquid nitrogen switch, start to fill liquid nitrogen into CCD camera at this time, temperature can be down to -110 after about 2h ℃。

3. the culture dish being imaged will be needed to be placed in camera bellows, " Acquire " option of software is opened, is selected " light field ", Time for exposure is 100ms, sample frequency 1MHz, then clicks " Acquire " and obtains bright field image, saves as TIF format.

4. obtain bioluminescence image, " Acquire " option of software being opened, being selected " Luc ", the time for exposure is Then 10min, sample frequency 50KHz click " Acquire " and obtain image, save as TIF format.

5. after use, being first shut off analysis software, it is then shut off camera bellows power supply and light source power.Note: CCD camera Power supply can not close at once, to wait for 24 hours after CCD camera temperature be raised to room temperature after turn off.

The reporting system that shines is as shown in Figure 1 to the citing of the luminescence response of antibiotic.

Step 3 draws characteristic luminescence curve

1. using plate center as origin (x₀,y₀), plate radius is divided into a specific number (such as 300 or 1200 parts), Average distance is denoted as its midpoint to the maximum distance of origin and the average value d of minimum range in obtained each annulus.

2. setting A as the average canbdle power of all the points in some annulus, then average canbdle power is the function of d: A=f (d)。

3. the function of application settings is depicted as being characterized luminosity curve.

It is as shown in Figure 2 to draw resulting characteristic luminescence curve.

The clustering of 3 characteristic luminescence curve of embodiment

Experimental procedure

Step 1, clustering

1. being denoted as each characteristic luminescence curve (part from inhibition zone edge to plate edge) by 300 luminous intensities The vector (luminous intensity characterization vector) characterized, each luminous intensity represent embodiment 2 and shine in picture from antibacterial Edge is enclosed to the average hair in each annulus in plate edge part (span is inhibition zone edge to the 1/300 of plate edge part) Luminous intensity.

2. the characteristic luminescence curve of four reporting bacterial strains corresponding to each antibiotic is merged into one by 1200 The merging luminous intensity that luminous intensity is characterized characterizes vector.

3. use Euclidean distance calculation method, calculate antibiotic mergings luminous intensity characterize vector between away from From, and construct the distance matrix of antibiotic.

4. using the hclust function in R language, using UPGMA algorithm, to the merging luminous intensity of antibiotic characterize to Clustering is carried out between amount.

5. obtaining the merging luminous intensity characterization vector (each 3 parallel) of 16 kinds of antibiotic: CIP, Ciprofloxacin；DAP reaches Tobramycin；ERY, erythromycin；GAT, gatifloxacin；IPM, Imipenem；KAN, kanamycins；MMC, mitomycin C；PEN, it is green Mycin；PHL, phleomycin；PMB, polymyxin B；RIF, rifampin；SPE, spectinomycin；STR, streptomysin；TET, Fourth Ring Element；TMP, trimethoprim；VAN, vancomycin.

6. the merging luminous intensity characterization vector of pair 16 kinds of antibiotic carries out clustering, find for different report bacterium Strain, the antibiotic cluster of the same mechanism of action is together.Cluster result is as shown in Figure 3.

7. the cluster analysis result of the antibiotic of pair identical mechanism of action is analyzed (table 10), illustrate to pass through clustering The sensitivity and specificity for carrying out the judgement of antibiotic mechanism of action are both greater than equal to 0.89.

The sensitivity and specificity of 10 antibiotic mechanism of action of table judgement

The judgement of step 2, antibiotic mechanism of action

Same antibiotic solution is detected using four kinds of reporting bacterial strains S1, S2, S3, S4.

Reporting strain containing four plants of staphylococcus aureuses using the preparation of the method as described in embodiment 2 and embodiment 3 Agarose double-layer plate, by the filter paper of sterilizing agarose double-layer plate center or media surface ordered arrangement and with culture Base is closely affixed；Solution to be checked containing antibiotic is added drop-wise on filter paper, continues to cultivate and shoot bacterial luminescence picture；It will Luminous picture is converted into luminosity curve, and it is poly- with known antibiotic profile luminosity curve comparison progress level to extract luminosity curve feature Class, if together with the luminosity curve cluster of detected antibiotic luminosity curve and the antibiotic of known action mechanism, determine to The antibiotic mechanism of action of inspection is identical as the antibiotic of known action mechanism.

Wherein, above-mentioned filter paper is the small scraps of paper of circle that No. 1 qualitative filter paper of Xinhua is broken into diameter 6mm with punch.On It states after the solution to be checked containing antibiotic is added drop-wise on filter paper, plate is inverted in incubator, 37 DEG C of culture 25h.

The above-mentioned method for drafting for converting luminosity curve for luminous picture is:

1) using plate center as origin (x₀,y₀), plate radius is divided into one such as 300 or 1200 parts of specific number, Average distance is denoted as its midpoint to the maximum distance of origin and the average value d of minimum range in obtained each annulus；

2) A is set as the average canbdle power of all the points in some annulus, then average canbdle power is the function of d: A=f (d)；

3) function of application settings is depicted as luminosity curve.

The above-mentioned method for carrying out hierarchical clustering analysis according to characteristic luminescence curve is:

1) the characteristic luminescence curve by each from inhibition zone edge to plate edge part is denoted as by 300 luminous intensity institutes One vector of characterization, each luminous intensity represent in the picture that shines from inhibition zone edge to each annulus in plate edge part That is inhibition zone edge is to the average canbdle power in the annulus of plate edge part 1/300；

2) by the feature of four plants of staphylococcus aureus report strains in bio-luminescence system corresponding to each antibiotic Luminosity curve is merged into one and characterizes vector by the merging luminous intensity that 1200 luminous intensities are characterized；

3) use Euclidean distance calculation method, calculate antibiotic mergings luminous intensity characterize vector between away from From, and construct the distance matrix of antibiotic；

4) vector is characterized to the merging luminous intensity of antibiotic with UPGMA algorithm using the hclust function in R language Between carry out clustering, whether with the antibiotic cluster of known action mechanism judge that it is anti-together according to antibiotic to be checked The type of raw element mechanism of action.

Sequence table

<110>Shandong University

<120>a kind of method that antibiotic mechanism of action is determined using bioluminescence reporting system

<141> 2019-6-11

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12432

<212> DNA

<213>artificial sequence

<221>pAmilux-clpX-luxABCDE plasmid sequence

<400> 1

aattcgacgc tgcaggatat caactatcaa acgcttcggt taagcttaaa gcacaccctt 60

tctgcgtcct cgtattgacg cgacgtaaaa tttcaacgag cacgccggga tacttaccat 120

attctctgct aattatcccg acatcatcgg taacaataaa tgctggataa ctggttgctg 180

acgcatccat ataactcatc aaccccggcg ttccatcagg tacaggtttc aacgtttcag 240

gatcaagcgc tcgcgcatat acccacggcg gaacatgttt acgctgcatt tcatcctcaa 300

agaaacaagt gttgagttca acttgattaa atatatctcg gatctgacta atatcactga 360

gattgaaagt atcaaataaa agatgattga aatcatcacg tttcagagat tctttttcgt 420

aacttttcca gccgcctccg gttatgatat aaaggctttt atctccagaa aatgagattt 480

ttttatcttt catataatgg cagagtaaat aaataaagta tggcgaacca ataagacaaa 540

gatctttccc ttgatttttt attcgttcaa gactattcaa tgttttaaca aaatctattc 600

gttcttctgt tacggtaaat gtcgtaggat ataacaattc caccaaactc ataacatatt 660

taaaccaaat attatgagca ttaaatctat ctggtcccaa attgactaat tctatttgat 720

gatcaaacca actaccaaca tatttcatgc cataactcac agagcctaag agtctctcaa 780

tacttaatct gtcacgcgcc acctgacttt ttaaaccatt cgtgccgcta ctggtaaacc 840

aactttcaat ctcgttttcc tgagaagtta ataagcgagt aaacttaaaa accgatgttg 900

ggaatacagg tatgtcatca atttccgtaa tattgtcatc tactttgtgt gcctgacagt 960

agtgacgata ttctcgacaa tgtttataat gattacgaaa tgcatcaagc acaagtttct 1020

ttctgatttt ttcctgctcg tcgtaagacc acactaatgg atcgctcgaa aaaatcaaat 1080

catcaatttc tgagcttgct gtaatttctt gtttatcaac atatgaagtc atacctgttt 1140

tcctcctcaa gatcctttaa gacagagaaa ttgcttgatt ttcaatctca attctcattc 1200

ggcgttcatt gactgtcgca atagttaaat gttcaaatga cggttcagta atatcaacat 1260

caatatccag atgatcatta tccatcgcga tagcggcttt cgtaaccgat tgataaaaat 1320

tgcgcaggac cactaaattt tcactcaagt catgcgaact tcctaacaaa gaatatatct 1380

tgcatcgatt actacgaata tttgataaca atgtgataac ttcatcttgc ttgacccaat 1440

tatcgttatt tgcagtaaaa gcaataaacg gtatatcaag atacatcatg ttattaattg 1500

tagaagctaa atcttcccaa ccaaaatcaa gacaatctct cgcaaagact tcagcaccca 1560

atttatggcc ttcaaaatct agattatccg gcaattcatt aatgggtaga ctgagataat 1620

caaaccctaa agctctttca agagaatatc ttaagttaac aacaccgact gcggtgatta 1680

aaaacgaagc attgatttca gataggcttg cataagctat ccgcgcagat aagcttgaag 1740

ccaacatacc gaagttattt atttttcgtg tagttaacca atcaaccact gctaacaagc 1800

tctgctttcc tatagacatt gtaaattcat caattgtccc tgaactcaat ccaacgtggt 1860

gaagcgaatc atagcggatc acatgaaatc cattccgcga taaatattcc gccagaccag 1920

caaaatgatc catcctgcgg gcaaaaccag acgcaataat aatggcattc tttctctttg 1980

ggctgttttc ttctggcagc gtttcccaaa catgaatttt tttatttcct tcaacacaaa 2040

taacgtggtc gatggtttta tattttgatt cattttccat acttttacct attatgggac 2100

aaatacaagg aacttatctt cttccaggaa tcgagtctgt tctatttcaa ccgcaacatc 2160

cttagccgta tagttagatg gcctttcatg agaaatatat gtcactaatc gttgcaacgg 2220

tctcattccg tcatgagatc caccaactcg aaatatgtta ttcattcctg cttctacaat 2280

cctttccgca ccttttaatg ctaacgcatc tcgatattta aatgatgact cccaaggaaa 2340

aatagatatg gtttgcgtct tatttttttg aacataaggc aatatttgct caatattatc 2400

gacgtgatga aggtacacac atctgccaag tggttgatta aattccacac ctgcatttga 2460

ctcaataatc atccaacgtt gatgaatatc cacctctact tttaatccag caaacaagct 2520

ttctttttga actaaagaat aggccgcctt ttcatcaaaa tcttttttgg cattcggtaa 2580

tatatgcgca tatagattaa gtttttctat caacgctaac ttaaattcct cataatgatt 2640

tcccatgtaa tatatgtttt gggcagaaaa acaagctcgc tgatcgtaaa aacaaacatc 2700

atgagccgca cctgtcgctg cggacgtcaa atcaacagga ttatcgataa tgcaaagact 2760

ctttttagaa ccaaatttaa tcacatcagc ataagatggc gcatgctcta ccgcccaatt 2820

aatcgcatct ggccctcccc aagcgacaat aacatccgca tgtcgcataa tttcttttgc 2880

gagtgatgta tcaccttggt ggggccaata tataacagat aaagagcgcg ttatcggatg 2940

attagggtct acatcaataa aacttaacgc taatgcatta gcggtaaaag gatcggttga 3000

cgatgttttt ataatacact gattcttagt taaaattgcg cgtaatatag acatgatccc 3060

agataatgga acattacctg ccaacagatg tacagattta cctttcggaa aagcccgaac 3120

ataactttca tcctgaggta gccattcatc catgatatgg cgagaaccaa gttcattttc 3180

tacaacatca taaaggccgc ctttagaaca taaaatcata gatatccaat tggcctctag 3240

cttagccatt tcttctgaat atcccatata tttttttaag tcacgaatgt atgtcctgcg 3300

tcttgagtat tcttcatttt tccatctttg ccctaccgta tagagaaaat tgacaatgtt 3360

atgcaaccgt aattcgttat ttccattaca atcaataatg ttttttacat gagagtcatt 3420

caatattggc aggtaaacac tattatcacc aaaattaatg gattgcacta aatcatcact 3480

ttcgggaaag atttcaacct ggccgttaat aatgaatgaa atttttttag tcatatttgc 3540

cttcctcctg gtaccccatt atatcacatt atccattaaa aagcaaacaa attttcggta 3600

cctattaggt atattccatg tggtacttct taatattatc atcaacaata ttgattacat 3660

ttttttggct catcaaatca ttcattggtt caaaggacag caatacactt ttcgcaccac 3720

acttttcaat tgccaactta gccgcagtta tacactccgt ataatttccg acagcgtttt 3780

ctgcaattat ttcttcaagt ttattttcga aattttcatt agggtgcatt tcaagaacat 3840

aatcactaat aaatgcacgc gtctcttgtt tagctttatt actatcttcg ttatagttaa 3900

ctaatatcat taactgatgg tctatctctg ataggtcaac gtcatattta tccgcaacgg 3960

ctttatatct ttcagcatat tcatatctaa catcattaga atcatcccac ttaaagatga 4020

gaggaatacc ttttttggcc gcccactcaa caatatgatg actggttgct gttacatatt 4080

tccgaggtcc gcctggcgta taagcatggg gatttacaga tattttaggg aagctataaa 4140

aatcgttatc tggattacaa tagcctgttg ttaaagcatc gttaatgatt tcataacact 4200

cttcaaatag ttgctgttga tattcaaccg ggcgattaaa aaaatgcatt tcatcttttt 4260

tttcgcaatc actaaaccct aaaataaatc tcccttcact taactgatcc aataagcaag 4320

cttcctccgc tatggcgaca ggatgatgag ttgtaatgat gtgatttaat gaaccaattt 4380

taattttctc tgttaaaccg agcagaaaac cagaaacagt cagaggagcg ccgacaacac 4440

cattatctga aaaatgattt tcatacacta aaatctgttc aaaattcaac ttatcaacat 4500

actccgttat ttcctgcatg cgaactatac tttgttcttg aacagttgtt gaattgatga 4560

agttaaggaa gaacaatcca aatttcattt ctttctcctt agctaatata atagcgaacg 4620

ttgtttttct ttaagaaatg gcatgacatc agactggaag agcttcatgg aagcaataat 4680

ttcgtctact gttccattag cttcaaatcc acaacaaata tttgatattc ctgtagcatc 4740

aatgtctttt tgaattatgt caatacattc ctgcggcgtt cccacgggat tgatttcgta 4800

actgtaatca atacggcgat tagtatcttt atgtcctttt aatacaaagt cacgccactg 4860

ccctttattg aaatcataac ctcttgtttg gtctgaatca tcaaaaatag tcgtagcatt 4920

cacataagaa tcataccaat gccccagaaa tttccggcaa atctctttcg ctttaattga 4980

gtcatgatct acagatgtta tatatgataa gcaatggtcg atattatgaa tatcgtgccc 5040

atattcttga gccacttcat tataaagctc aagttgtgct ttcttttcgt tagtatttat 5100

aatccaactt aatatcatcg gtaggccaaa ttgagcagcc cactcagtcg tcgaagctga 5160

ttcagccacc acataaaccg gtgcgccacc tctgctatac gccgcggggt ttacttttac 5220

cttatggaac ttgatatgtt cattatcagc ttccatatat ccctctgtca tgccattctt 5280

tatcagcccg taccagcatt ccgctaaggc gcgactgtta ttcatatctg tgccgaatac 5340

gcgaaagtcc ttgttgtaaa gccctcggca aataccaaac cgaaatcgtc cttttgacat 5400

ttgatccaat aaattcacat cttcaagttg gcgtactgga tgggctgtgg gaagaacaat 5460

agcggcagtt cctacattca attttttagt cgcgccaagt aaatatgcag cagcgacata 5520

agggttacca agcaaaccaa actccgtgaa atgatgctcc agtaaccata cggtatcaaa 5580

accacactcc tcagagatgc gacctaattt aaccaaacgt ttcattacct ctgtttgaga 5640

aaattgggga ggttggtatg taagcaaaaa gtttccaaat ttcatagaga gtcctcctgt 5700

cgacggatcc acaacaaggt ttagatttac aaacgtactt ccaaatctca ggtcaagatg 5760

aaactcaatt aagagagcaa atgaaagacg atgcagaaca acgtgttaaa actaacttaa 5820

ctttaactgc gatcgctgaa gctgaaaaaa tcgaagctac tgatgaagat atcgataaag 5880

aattagaaaa aatgagtaaa caatttaata tctcagttga agatatcaaa aatactttag 5940

gtaatactga tatcattaaa aatgatgttc gtatccaaaa agttatcgat ttattaagag 6000

ataacgcaaa gttcgttgaa ggaactaaag aagattaatc ttcattaaat attaaattac 6060

aaaaatgagt agcagatgca tagcttatgt atctgctact attctttaag caaaaagttt 6120

gtatgttaat atgttgcatt gtaacatcca atctagtata gtctttaacg aataggggtg 6180

taaaaagaat gtttaaattc aatgaagatg aagaaaattt gaaatgctct ttctgcggaa 6240

aagaccaaga tcaagtaaaa aaacttgtag caggaagtgg tgtatatatt tgtaatgagt 6300

gtattgaatt atgctcagaa atcgtcgaag aagaattagc tcaaaacact tctgaagcga 6360

tgacagaatt acctactcct aaagaaatta tggatcattt aaacgaatat gttattggtc 6420

aagaaaaagc taaaaaatct ttagctgtag ctgtttataa ccactataag cgtattcaac 6480

aattaggacc aaaagaagat gatgttgaat tacaaaaaag taacattgca ttaattgggc 6540

caacaggtag tggtaaaaca tggatccccg ggaattcctg gcagtttatg gcgggcgtcc 6600

tgcccgccac cctccgggcc gttgcttcgc aacgttcaaa tccgctcccg gcggatttgt 6660

cctactcagg agagcgttca ccgacaaaca acagataaaa cgaaaggccc agtctttcga 6720

ctgagccttt cgttttattt gatgcctggg gaattcttga agacgaaagg gcctcgtgat 6780

acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt cttcaactaa 6840

agcacccatt agttcaacaa acgaaaattg gataaagtgg gatattttta aaatatatat 6900

ttatgttaca gtaatattga cttttaaaaa aggattgatt ctaatgaaga aagcagacaa 6960

gtaagcctcc taaattcact ttagataaaa atttaggagg catatcaaat gaactttaat 7020

aaaattgatt tagacaattg gaagagaaaa gagatattta atcattattt gaaccaacaa 7080

acgactttta gtataaccac agaaattgat attagtgttt tataccgaaa cataaaacaa 7140

gaaggatata aattttaccc tgcatttatt ttcttagtga caagggtgat aaactcaaat 7200

acagctttta gaactggtta caatagcgac ggagagttag gttattggga taagttagag 7260

ccactttata caatttttga tggtgtatct aaaacattct ctggtatttg gactcctgta 7320

aagaatgact tcaaagagtt ttatgattta tacctttctg atgtagagaa atataatggt 7380

tcggggaaat tgtttcccaa aacacctata cctgaaaatg ctttttctct ttctattatt 7440

ccatggactt catttactgg gtttaactta aatatcaata ataatagtaa ttaccttcta 7500

cccattatta cagcaggaaa attcattaat aaaggtaatt caatatattt accgctatct 7560

ttacaggtac atcattctgt ttgtgatggt tatcatgcag gattgtttat gaactctatt 7620

caggaattgt cagataggcc taatgactgg cttttataat atgagataat gccgactgta 7680

ctttttacag tcggttttct aatgtcacta acctgccccg ttagttgaag aaggttttta 7740

tattacagct ccagatccat atccttcaga tccctagata attcttctga taatttagtt 7800

tttgttagag tttaacataa ctgatgatta tcagaagttt ctttaatata aataaaagca 7860

ctataagcga cttaacttat agtgcttatt taatatattt tttcaaacaa tcattagcca 7920

ccaaacaaac gactccagaa gcctttttta gtttgggttt cttctatagc aatatcttta 7980

tcattttgat taaatgaatc atcttgttgt tgtgattttt catcttgttg actctcagat 8040

atatctttag gttgaacctc tggtttcttt tcttctttct gatattgatc agtatttaca 8100

gaatccgaag tatatgatgc ttcttgtaca tcaaagtttt ctcgatcatt agtagattta 8160

tcaaaggaat aacttaattg tctctcttcc tctaattgat gttccaattt ttgaattttt 8220

ttattgcttt ctaaagctaa aatttgttga ttctctaata atttatttaa tgtattaaca 8280

ttgctatttt gttggtctag ttgtttagtt aaattctcaa tatacttttc atcattttta 8340

gctctagttt caaagatttc tacctgcttt tctaattcgt ttacttttgt ttttaatgtc 8400

tcaaagccat ctgaattatt atatctattc ttttcagtgt tttcataagt tatttctgat 8460

tttttatttt ctttattttg agttgttctt tctttcttag tgattttttc tactattttc 8520

aaataatcat tatcatcaat ataattcact ccattttctt tttcaaaaga tatattcaaa 8580

tttttagcat tattaacaac agtttgttta gttacattca attcatcagc aaccattttt 8640

atagttttca tatcatccac ccttaggcac ctaatttact acctaatttt accacctaac 8700

aatttttctt tcatcttttt taccgcctaa atatatcatt tttactacct aagtacctaa 8760

tttactacct aattagccac taaaataata aattttgttg ttttaaggtc ataacatgat 8820

tctgatctgt acttaaaacg ctttatatac acctttaaaa atgttaatat tatcttatga 8880

attttaaaaa gccaatgcta ttggcgtagc atcggctctg gtaattaaaa cgatttgcgt 8940

tcgtttattt atatattttt ttgatacttg tattatatat atctactcgt ctaagtgcaa 9000

gcacaaaaca tataacttac gtaaaaattg ttttattacc tcaactctaa gtaaaaggaa 9060

atgaggtttt ttattatgtc taaatttaaa aaaatatctg caagtgaatt cgaaacatta 9120

cgtttttatc aattacccaa atttttattt gaagatgaat acttttctaa aatgcccaca 9180

gatgcaaaag ttatgtatgc tttattaaaa gatcgctttg aactatcaag attaaataac 9240

tgggtagatt cagaaaataa tatttatctt ttatatacca ataaacagtt atgctcaatt 9300

ttaaattatg cagaaccaaa aatcattaaa ttaaaaaaag aattagaaaa atacaatttg 9360

attataaacg aaagacaagg tttaaataaa cctaacaaaa tttatttact cgaacccaca 9420

tatgacaagg aactaataaa ttctaagttc cagaacaaag aatttattag ttccagaact 9480

aataaatcat cagttcaaga actaataaat tctaagtcaa gtgatactga ttttaataat 9540

actgaatata tagagactaa gaataatgat acgaattata cgaatgatac atctaacatg 9600

atttctaaga attctcattc gaatcataca aatcatcaac aaaccgaatt taataatgat 9660

gccttaaaat tccaggcgct tgaagaatta ccttcgcaaa tcaaatctta tgtaagtaat 9720

tttgaaatta aagacatccg tattattaaa agtatcttac tcaaggggaa aaagtcattt 9780

aataatacac atgatacata ttaccgttta gaagacgtcg aatttgaact tgtaagtgtt 9840

ttaaaacgtt ttaaagccat gttgctacaa aaaaatgaaa ccgttgaaac tatgcaaggc 9900

tatttaatgc aatcaattaa agctgaactt gaagaaatac atgcattaaa tatgcgtcgt 9960

caaaacatac ctcaatacaa tatctttaat caataactca aataatctta caacaatcaa 10020

aacaacatca aaatttggaa ttaagtcaac agaaaaggat ctccccaggt ggcacttttc 10080

ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 10140

cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 10200

gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 10260

ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 10320

tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 10380

aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg 10440

ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 10500

agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 10560

gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 10620

gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 10680

gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 10740

cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 10800

ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 10860

cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 10920

gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 10980

cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 11040

tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 11100

aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 11160

aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 11220

gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 11280

cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 11340

ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 11400

accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 11460

tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 11520

cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 11580

gaacgaccta caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc 11640

ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 11700

cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 11760

tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 11820

ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 11880

ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 11940

ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 12000

gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca 12060

ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct 12120

acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg 12180

ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat 12240

gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg caggggttaa aattccttca 12300

ttacactctt ggcggtttca cttatcaact tatcatttgg cttatcactt ttattgtctt 12360

tattcgtaaa aatgactaaa acaataggtt cagattggcc cttaggataa acaaaagcaa 12420

catcatttct ag 12432

<210> 2

<211> 12421

<212> DNA

<213>artificial sequence

<221>pAmilux-dnaJ-luxABCDE plasmid sequence

<400> 2

aattcgacgc tgcaggatat caactatcaa acgcttcggt taagcttaaa gcacaccctt 60

tctgcgtcct cgtattgacg cgacgtaaaa tttcaacgag cacgccggga tacttaccat 120

attctctgct aattatcccg acatcatcgg taacaataaa tgctggataa ctggttgctg 180

acgcatccat ataactcatc aaccccggcg ttccatcagg tacaggtttc aacgtttcag 240

gatcaagcgc tcgcgcatat acccacggcg gaacatgttt acgctgcatt tcatcctcaa 300

agaaacaagt gttgagttca acttgattaa atatatctcg gatctgacta atatcactga 360

gattgaaagt atcaaataaa agatgattga aatcatcacg tttcagagat tctttttcgt 420

aacttttcca gccgcctccg gttatgatat aaaggctttt atctccagaa aatgagattt 480

ttttatcttt catataatgg cagagtaaat aaataaagta tggcgaacca ataagacaaa 540

gatctttccc ttgatttttt attcgttcaa gactattcaa tgttttaaca aaatctattc 600

gttcttctgt tacggtaaat gtcgtaggat ataacaattc caccaaactc ataacatatt 660

taaaccaaat attatgagca ttaaatctat ctggtcccaa attgactaat tctatttgat 720

gatcaaacca actaccaaca tatttcatgc cataactcac agagcctaag agtctctcaa 780

tacttaatct gtcacgcgcc acctgacttt ttaaaccatt cgtgccgcta ctggtaaacc 840

aactttcaat ctcgttttcc tgagaagtta ataagcgagt aaacttaaaa accgatgttg 900

ggaatacagg tatgtcatca atttccgtaa tattgtcatc tactttgtgt gcctgacagt 960

agtgacgata ttctcgacaa tgtttataat gattacgaaa tgcatcaagc acaagtttct 1020

ttctgatttt ttcctgctcg tcgtaagacc acactaatgg atcgctcgaa aaaatcaaat 1080

catcaatttc tgagcttgct gtaatttctt gtttatcaac atatgaagtc atacctgttt 1140

tcctcctcaa gatcctttaa gacagagaaa ttgcttgatt ttcaatctca attctcattc 1200

ggcgttcatt gactgtcgca atagttaaat gttcaaatga cggttcagta atatcaacat 1260

caatatccag atgatcatta tccatcgcga tagcggcttt cgtaaccgat tgataaaaat 1320

tgcgcaggac cactaaattt tcactcaagt catgcgaact tcctaacaaa gaatatatct 1380

tgcatcgatt actacgaata tttgataaca atgtgataac ttcatcttgc ttgacccaat 1440

tatcgttatt tgcagtaaaa gcaataaacg gtatatcaag atacatcatg ttattaattg 1500

tagaagctaa atcttcccaa ccaaaatcaa gacaatctct cgcaaagact tcagcaccca 1560

atttatggcc ttcaaaatct agattatccg gcaattcatt aatgggtaga ctgagataat 1620

caaaccctaa agctctttca agagaatatc ttaagttaac aacaccgact gcggtgatta 1680

aaaacgaagc attgatttca gataggcttg cataagctat ccgcgcagat aagcttgaag 1740

ccaacatacc gaagttattt atttttcgtg tagttaacca atcaaccact gctaacaagc 1800

tctgctttcc tatagacatt gtaaattcat caattgtccc tgaactcaat ccaacgtggt 1860

gaagcgaatc atagcggatc acatgaaatc cattccgcga taaatattcc gccagaccag 1920

caaaatgatc catcctgcgg gcaaaaccag acgcaataat aatggcattc tttctctttg 1980

ggctgttttc ttctggcagc gtttcccaaa catgaatttt tttatttcct tcaacacaaa 2040

taacgtggtc gatggtttta tattttgatt cattttccat acttttacct attatgggac 2100

aaatacaagg aacttatctt cttccaggaa tcgagtctgt tctatttcaa ccgcaacatc 2160

cttagccgta tagttagatg gcctttcatg agaaatatat gtcactaatc gttgcaacgg 2220

tctcattccg tcatgagatc caccaactcg aaatatgtta ttcattcctg cttctacaat 2280

cctttccgca ccttttaatg ctaacgcatc tcgatattta aatgatgact cccaaggaaa 2340

aatagatatg gtttgcgtct tatttttttg aacataaggc aatatttgct caatattatc 2400

gacgtgatga aggtacacac atctgccaag tggttgatta aattccacac ctgcatttga 2460

ctcaataatc atccaacgtt gatgaatatc cacctctact tttaatccag caaacaagct 2520

ttctttttga actaaagaat aggccgcctt ttcatcaaaa tcttttttgg cattcggtaa 2580

tatatgcgca tatagattaa gtttttctat caacgctaac ttaaattcct cataatgatt 2640

tcccatgtaa tatatgtttt gggcagaaaa acaagctcgc tgatcgtaaa aacaaacatc 2700

atgagccgca cctgtcgctg cggacgtcaa atcaacagga ttatcgataa tgcaaagact 2760

ctttttagaa ccaaatttaa tcacatcagc ataagatggc gcatgctcta ccgcccaatt 2820

aatcgcatct ggccctcccc aagcgacaat aacatccgca tgtcgcataa tttcttttgc 2880

gagtgatgta tcaccttggt ggggccaata tataacagat aaagagcgcg ttatcggatg 2940

attagggtct acatcaataa aacttaacgc taatgcatta gcggtaaaag gatcggttga 3000

cgatgttttt ataatacact gattcttagt taaaattgcg cgtaatatag acatgatccc 3060

agataatgga acattacctg ccaacagatg tacagattta cctttcggaa aagcccgaac 3120

ataactttca tcctgaggta gccattcatc catgatatgg cgagaaccaa gttcattttc 3180

tacaacatca taaaggccgc ctttagaaca taaaatcata gatatccaat tggcctctag 3240

cttagccatt tcttctgaat atcccatata tttttttaag tcacgaatgt atgtcctgcg 3300

tcttgagtat tcttcatttt tccatctttg ccctaccgta tagagaaaat tgacaatgtt 3360

atgcaaccgt aattcgttat ttccattaca atcaataatg ttttttacat gagagtcatt 3420

caatattggc aggtaaacac tattatcacc aaaattaatg gattgcacta aatcatcact 3480

ttcgggaaag atttcaacct ggccgttaat aatgaatgaa atttttttag tcatatttgc 3540

cttcctcctg gtaccccatt atatcacatt atccattaaa aagcaaacaa attttcggta 3600

cctattaggt atattccatg tggtacttct taatattatc atcaacaata ttgattacat 3660

ttttttggct catcaaatca ttcattggtt caaaggacag caatacactt ttcgcaccac 3720

acttttcaat tgccaactta gccgcagtta tacactccgt ataatttccg acagcgtttt 3780

ctgcaattat ttcttcaagt ttattttcga aattttcatt agggtgcatt tcaagaacat 3840

aatcactaat aaatgcacgc gtctcttgtt tagctttatt actatcttcg ttatagttaa 3900

ctaatatcat taactgatgg tctatctctg ataggtcaac gtcatattta tccgcaacgg 3960

ctttatatct ttcagcatat tcatatctaa catcattaga atcatcccac ttaaagatga 4020

gaggaatacc ttttttggcc gcccactcaa caatatgatg actggttgct gttacatatt 4080

tccgaggtcc gcctggcgta taagcatggg gatttacaga tattttaggg aagctataaa 4140

aatcgttatc tggattacaa tagcctgttg ttaaagcatc gttaatgatt tcataacact 4200

cttcaaatag ttgctgttga tattcaaccg ggcgattaaa aaaatgcatt tcatcttttt 4260

tttcgcaatc actaaaccct aaaataaatc tcccttcact taactgatcc aataagcaag 4320

cttcctccgc tatggcgaca ggatgatgag ttgtaatgat gtgatttaat gaaccaattt 4380

taattttctc tgttaaaccg agcagaaaac cagaaacagt cagaggagcg ccgacaacac 4440

cattatctga aaaatgattt tcatacacta aaatctgttc aaaattcaac ttatcaacat 4500

actccgttat ttcctgcatg cgaactatac tttgttcttg aacagttgtt gaattgatga 4560

agttaaggaa gaacaatcca aatttcattt ctttctcctt agctaatata atagcgaacg 4620

ttgtttttct ttaagaaatg gcatgacatc agactggaag agcttcatgg aagcaataat 4680

ttcgtctact gttccattag cttcaaatcc acaacaaata tttgatattc ctgtagcatc 4740

aatgtctttt tgaattatgt caatacattc ctgcggcgtt cccacgggat tgatttcgta 4800

actgtaatca atacggcgat tagtatcttt atgtcctttt aatacaaagt cacgccactg 4860

ccctttattg aaatcataac ctcttgtttg gtctgaatca tcaaaaatag tcgtagcatt 4920

cacataagaa tcataccaat gccccagaaa tttccggcaa atctctttcg ctttaattga 4980

gtcatgatct acagatgtta tatatgataa gcaatggtcg atattatgaa tatcgtgccc 5040

atattcttga gccacttcat tataaagctc aagttgtgct ttcttttcgt tagtatttat 5100

aatccaactt aatatcatcg gtaggccaaa ttgagcagcc cactcagtcg tcgaagctga 5160

ttcagccacc acataaaccg gtgcgccacc tctgctatac gccgcggggt ttacttttac 5220

cttatggaac ttgatatgtt cattatcagc ttccatatat ccctctgtca tgccattctt 5280

tatcagcccg taccagcatt ccgctaaggc gcgactgtta ttcatatctg tgccgaatac 5340

gcgaaagtcc ttgttgtaaa gccctcggca aataccaaac cgaaatcgtc cttttgacat 5400

ttgatccaat aaattcacat cttcaagttg gcgtactgga tgggctgtgg gaagaacaat 5460

agcggcagtt cctacattca attttttagt cgcgccaagt aaatatgcag cagcgacata 5520

agggttacca agcaaaccaa actccgtgaa atgatgctcc agtaaccata cggtatcaaa 5580

accacactcc tcagagatgc gacctaattt aaccaaacgt ttcattacct ctgtttgaga 5640

aaattgggga ggttggtatg taagcaaaaa gtttccaaat ttcatagaga gtcctcctgt 5700

cgacggatcc tctttacgga ttgaaatttc ttttgttgta ccaaataccg cttcttcaaa 5760

tgttaatgtc attgtatact gaagatcatc acctttttgc ggtgcatttg gatctctttg 5820

tctgccgcca ccgaagaaag agctaaagat atcttcaaaa ccgccgccac cgaagccact 5880

aaaaccgcca aagtcagagc cattgaatcc ttgtccacca aaaccttgtg gaccatcatg 5940

tccaaattga tcatagcttg cgcgtttatt atcatcactt aaaacttcat aggcttcaga 6000

aatttcttta aacttttcat ctgcaccttc ttctttgtta atatctggat gatatttttt 6060

cgaaagcttt cgatacgctt ttttgatttc atcttttgaa gcatccttac taatgcctaa 6120

aacttcataa taatctcttt tggccacagc tatctctcct tttcttaatt aactcatata 6180

gtttaacgta atatgtcata ctatccaaat aaaaagccaa agccaatgtt ctattgactt 6240

tgacttttca gatcatgaca acattctaat tgtattgttt aattattttt tgtcgtcgtc 6300

ttttacttct ttaaattcag catcttctac agtactatca ttgttttgac cagcattagc 6360

accttgtgct tgttgttgct gttgagccgc ttgctcatat acttttgctg ataattcttg 6420

aatcactttt tcaagttctt cttttttaga tttaatatct tctatatctt gaccttctaa 6480

agcagtttta agagcgtctt ttttctcttc agcagatttt ttatcttctt caccgatatt 6540

ttcgcctaaa ggatccccgg gaattcctgg cagtttatgg cgggcgtcct gcccgccacc 6600

ctccgggccg ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc ctactcagga 6660

gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc 6720

gttttatttg atgcctgggg aattcttgaa gacgaaaggg cctcgtgata cgcctatttt 6780

tataggttaa tgtcatgata ataatggttt cttagacgtc ttcaactaaa gcacccatta 6840

gttcaacaaa cgaaaattgg ataaagtggg atatttttaa aatatatatt tatgttacag 6900

taatattgac ttttaaaaaa ggattgattc taatgaagaa agcagacaag taagcctcct 6960

aaattcactt tagataaaaa tttaggaggc atatcaaatg aactttaata aaattgattt 7020

agacaattgg aagagaaaag agatatttaa tcattatttg aaccaacaaa cgacttttag 7080

tataaccaca gaaattgata ttagtgtttt ataccgaaac ataaaacaag aaggatataa 7140

attttaccct gcatttattt tcttagtgac aagggtgata aactcaaata cagcttttag 7200

aactggttac aatagcgacg gagagttagg ttattgggat aagttagagc cactttatac 7260

aatttttgat ggtgtatcta aaacattctc tggtatttgg actcctgtaa agaatgactt 7320

caaagagttt tatgatttat acctttctga tgtagagaaa tataatggtt cggggaaatt 7380

gtttcccaaa acacctatac ctgaaaatgc tttttctctt tctattattc catggacttc 7440

atttactggg tttaacttaa atatcaataa taatagtaat taccttctac ccattattac 7500

agcaggaaaa ttcattaata aaggtaattc aatatattta ccgctatctt tacaggtaca 7560

tcattctgtt tgtgatggtt atcatgcagg attgtttatg aactctattc aggaattgtc 7620

agataggcct aatgactggc ttttataata tgagataatg ccgactgtac tttttacagt 7680

cggttttcta atgtcactaa cctgccccgt tagttgaaga aggtttttat attacagctc 7740

cagatccata tccttcagat ccctagataa ttcttctgat aatttagttt ttgttagagt 7800

ttaacataac tgatgattat cagaagtttc tttaatataa ataaaagcac tataagcgac 7860

ttaacttata gtgcttattt aatatatttt ttcaaacaat cattagccac caaacaaacg 7920

actccagaag ccttttttag tttgggtttc ttctatagca atatctttat cattttgatt 7980

aaatgaatca tcttgttgtt gtgatttttc atcttgttga ctctcagata tatctttagg 8040

ttgaacctct ggtttctttt cttctttctg atattgatca gtatttacag aatccgaagt 8100

atatgatgct tcttgtacat caaagttttc tcgatcatta gtagatttat caaaggaata 8160

acttaattgt ctctcttcct ctaattgatg ttccaatttt tgaatttttt tattgctttc 8220

taaagctaaa atttgttgat tctctaataa tttatttaat gtattaacat tgctattttg 8280

ttggtctagt tgtttagtta aattctcaat atacttttca tcatttttag ctctagtttc 8340

aaagatttct acctgctttt ctaattcgtt tacttttgtt tttaatgtct caaagccatc 8400

tgaattatta tatctattct tttcagtgtt ttcataagtt atttctgatt ttttattttc 8460

tttattttga gttgttcttt ctttcttagt gattttttct actattttca aataatcatt 8520

atcatcaata taattcactc cattttcttt ttcaaaagat atattcaaat ttttagcatt 8580

attaacaaca gtttgtttag ttacattcaa ttcatcagca accattttta tagttttcat 8640

atcatccacc cttaggcacc taatttacta cctaatttta ccacctaaca atttttcttt 8700

catctttttt accgcctaaa tatatcattt ttactaccta agtacctaat ttactaccta 8760

attagccact aaaataataa attttgttgt tttaaggtca taacatgatt ctgatctgta 8820

cttaaaacgc tttatataca cctttaaaaa tgttaatatt atcttatgaa ttttaaaaag 8880

ccaatgctat tggcgtagca tcggctctgg taattaaaac gatttgcgtt cgtttattta 8940

tatatttttt tgatacttgt attatatata tctactcgtc taagtgcaag cacaaaacat 9000

ataacttacg taaaaattgt tttattacct caactctaag taaaaggaaa tgaggttttt 9060

tattatgtct aaatttaaaa aaatatctgc aagtgaattc gaaacattac gtttttatca 9120

attacccaaa tttttatttg aagatgaata cttttctaaa atgcccacag atgcaaaagt 9180

tatgtatgct ttattaaaag atcgctttga actatcaaga ttaaataact gggtagattc 9240

agaaaataat atttatcttt tatataccaa taaacagtta tgctcaattt taaattatgc 9300

agaaccaaaa atcattaaat taaaaaaaga attagaaaaa tacaatttga ttataaacga 9360

aagacaaggt ttaaataaac ctaacaaaat ttatttactc gaacccacat atgacaagga 9420

actaataaat tctaagttcc agaacaaaga atttattagt tccagaacta ataaatcatc 9480

agttcaagaa ctaataaatt ctaagtcaag tgatactgat tttaataata ctgaatatat 9540

agagactaag aataatgata cgaattatac gaatgataca tctaacatga tttctaagaa 9600

ttctcattcg aatcatacaa atcatcaaca aaccgaattt aataatgatg ccttaaaatt 9660

ccaggcgctt gaagaattac cttcgcaaat caaatcttat gtaagtaatt ttgaaattaa 9720

agacatccgt attattaaaa gtatcttact caaggggaaa aagtcattta ataatacaca 9780

tgatacatat taccgtttag aagacgtcga atttgaactt gtaagtgttt taaaacgttt 9840

taaagccatg ttgctacaaa aaaatgaaac cgttgaaact atgcaaggct atttaatgca 9900

atcaattaaa gctgaacttg aagaaataca tgcattaaat atgcgtcgtc aaaacatacc 9960

tcaatacaat atctttaatc aataactcaa ataatcttac aacaatcaaa acaacatcaa 10020

aatttggaat taagtcaaca gaaaaggatc tccccaggtg gcacttttcg gggaaatgtg 10080

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 10140

caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 10200

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 10260

gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 10320

gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 10380

atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg 10440

caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 10500

gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 10560

accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 10620

ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 10680

gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca 10740

acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 10800

atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 10860

ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 10920

gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 10980

gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 11040

tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 11100

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 11160

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 11220

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 11280

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 11340

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 11400

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 11460

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 11520

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 11580

accgaactga gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc cgaagggaga 11640

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 11700

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 11760

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 11820

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 11880

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 11940

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 12000

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca 12060

atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg 12120

tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 12180

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 12240

tttcaccgtc atcaccgaaa cgcgcgaggc aggggttaaa attccttcat tacactcttg 12300

gcggtttcac ttatcaactt atcatttggc ttatcacttt tattgtcttt attcgtaaaa 12360

atgactaaaa caataggttc agattggccc ttaggataaa caaaagcaac atcatttcta 12420

g 12421

<210> 3

<211> 12240

<212> DNA

<213>artificial sequence

<221>pAmilux-dnaK-luxABCDE plasmid sequence

<400> 3

aattcgacgc tgcaggatat caactatcaa acgcttcggt taagcttaaa gcacaccctt 60

tctgcgtcct cgtattgacg cgacgtaaaa tttcaacgag cacgccggga tacttaccat 120

attctctgct aattatcccg acatcatcgg taacaataaa tgctggataa ctggttgctg 180

acgcatccat ataactcatc aaccccggcg ttccatcagg tacaggtttc aacgtttcag 240

gatcaagcgc tcgcgcatat acccacggcg gaacatgttt acgctgcatt tcatcctcaa 300

agaaacaagt gttgagttca acttgattaa atatatctcg gatctgacta atatcactga 360

gattgaaagt atcaaataaa agatgattga aatcatcacg tttcagagat tctttttcgt 420

aacttttcca gccgcctccg gttatgatat aaaggctttt atctccagaa aatgagattt 480

ttttatcttt catataatgg cagagtaaat aaataaagta tggcgaacca ataagacaaa 540

gatctttccc ttgatttttt attcgttcaa gactattcaa tgttttaaca aaatctattc 600

gttcttctgt tacggtaaat gtcgtaggat ataacaattc caccaaactc ataacatatt 660

taaaccaaat attatgagca ttaaatctat ctggtcccaa attgactaat tctatttgat 720

gatcaaacca actaccaaca tatttcatgc cataactcac agagcctaag agtctctcaa 780

tacttaatct gtcacgcgcc acctgacttt ttaaaccatt cgtgccgcta ctggtaaacc 840

aactttcaat ctcgttttcc tgagaagtta ataagcgagt aaacttaaaa accgatgttg 900

ggaatacagg tatgtcatca atttccgtaa tattgtcatc tactttgtgt gcctgacagt 960

agtgacgata ttctcgacaa tgtttataat gattacgaaa tgcatcaagc acaagtttct 1020

ttctgatttt ttcctgctcg tcgtaagacc acactaatgg atcgctcgaa aaaatcaaat 1080

catcaatttc tgagcttgct gtaatttctt gtttatcaac atatgaagtc atacctgttt 1140

tcctcctcaa gatcctttaa gacagagaaa ttgcttgatt ttcaatctca attctcattc 1200

ggcgttcatt gactgtcgca atagttaaat gttcaaatga cggttcagta atatcaacat 1260

caatatccag atgatcatta tccatcgcga tagcggcttt cgtaaccgat tgataaaaat 1320

tgcgcaggac cactaaattt tcactcaagt catgcgaact tcctaacaaa gaatatatct 1380

tgcatcgatt actacgaata tttgataaca atgtgataac ttcatcttgc ttgacccaat 1440

tatcgttatt tgcagtaaaa gcaataaacg gtatatcaag atacatcatg ttattaattg 1500

tagaagctaa atcttcccaa ccaaaatcaa gacaatctct cgcaaagact tcagcaccca 1560

atttatggcc ttcaaaatct agattatccg gcaattcatt aatgggtaga ctgagataat 1620

caaaccctaa agctctttca agagaatatc ttaagttaac aacaccgact gcggtgatta 1680

aaaacgaagc attgatttca gataggcttg cataagctat ccgcgcagat aagcttgaag 1740

ccaacatacc gaagttattt atttttcgtg tagttaacca atcaaccact gctaacaagc 1800

tctgctttcc tatagacatt gtaaattcat caattgtccc tgaactcaat ccaacgtggt 1860

gaagcgaatc atagcggatc acatgaaatc cattccgcga taaatattcc gccagaccag 1920

caaaatgatc catcctgcgg gcaaaaccag acgcaataat aatggcattc tttctctttg 1980

ggctgttttc ttctggcagc gtttcccaaa catgaatttt tttatttcct tcaacacaaa 2040

taacgtggtc gatggtttta tattttgatt cattttccat acttttacct attatgggac 2100

aaatacaagg aacttatctt cttccaggaa tcgagtctgt tctatttcaa ccgcaacatc 2160

cttagccgta tagttagatg gcctttcatg agaaatatat gtcactaatc gttgcaacgg 2220

tctcattccg tcatgagatc caccaactcg aaatatgtta ttcattcctg cttctacaat 2280

cctttccgca ccttttaatg ctaacgcatc tcgatattta aatgatgact cccaaggaaa 2340

aatagatatg gtttgcgtct tatttttttg aacataaggc aatatttgct caatattatc 2400

gacgtgatga aggtacacac atctgccaag tggttgatta aattccacac ctgcatttga 2460

ctcaataatc atccaacgtt gatgaatatc cacctctact tttaatccag caaacaagct 2520

ttctttttga actaaagaat aggccgcctt ttcatcaaaa tcttttttgg cattcggtaa 2580

tatatgcgca tatagattaa gtttttctat caacgctaac ttaaattcct cataatgatt 2640

tcccatgtaa tatatgtttt gggcagaaaa acaagctcgc tgatcgtaaa aacaaacatc 2700

atgagccgca cctgtcgctg cggacgtcaa atcaacagga ttatcgataa tgcaaagact 2760

ctttttagaa ccaaatttaa tcacatcagc ataagatggc gcatgctcta ccgcccaatt 2820

aatcgcatct ggccctcccc aagcgacaat aacatccgca tgtcgcataa tttcttttgc 2880

gagtgatgta tcaccttggt ggggccaata tataacagat aaagagcgcg ttatcggatg 2940

attagggtct acatcaataa aacttaacgc taatgcatta gcggtaaaag gatcggttga 3000

cgatgttttt ataatacact gattcttagt taaaattgcg cgtaatatag acatgatccc 3060

agataatgga acattacctg ccaacagatg tacagattta cctttcggaa aagcccgaac 3120

ataactttca tcctgaggta gccattcatc catgatatgg cgagaaccaa gttcattttc 3180

tacaacatca taaaggccgc ctttagaaca taaaatcata gatatccaat tggcctctag 3240

cttagccatt tcttctgaat atcccatata tttttttaag tcacgaatgt atgtcctgcg 3300

tcttgagtat tcttcatttt tccatctttg ccctaccgta tagagaaaat tgacaatgtt 3360

atgcaaccgt aattcgttat ttccattaca atcaataatg ttttttacat gagagtcatt 3420

caatattggc aggtaaacac tattatcacc aaaattaatg gattgcacta aatcatcact 3480

ttcgggaaag atttcaacct ggccgttaat aatgaatgaa atttttttag tcatatttgc 3540

cttcctcctg gtaccccatt atatcacatt atccattaaa aagcaaacaa attttcggta 3600

cctattaggt atattccatg tggtacttct taatattatc atcaacaata ttgattacat 3660

ttttttggct catcaaatca ttcattggtt caaaggacag caatacactt ttcgcaccac 3720

acttttcaat tgccaactta gccgcagtta tacactccgt ataatttccg acagcgtttt 3780

ctgcaattat ttcttcaagt ttattttcga aattttcatt agggtgcatt tcaagaacat 3840

aatcactaat aaatgcacgc gtctcttgtt tagctttatt actatcttcg ttatagttaa 3900

ctaatatcat taactgatgg tctatctctg ataggtcaac gtcatattta tccgcaacgg 3960

ctttatatct ttcagcatat tcatatctaa catcattaga atcatcccac ttaaagatga 4020

gaggaatacc ttttttggcc gcccactcaa caatatgatg actggttgct gttacatatt 4080

tccgaggtcc gcctggcgta taagcatggg gatttacaga tattttaggg aagctataaa 4140

aatcgttatc tggattacaa tagcctgttg ttaaagcatc gttaatgatt tcataacact 4200

cttcaaatag ttgctgttga tattcaaccg ggcgattaaa aaaatgcatt tcatcttttt 4260

tttcgcaatc actaaaccct aaaataaatc tcccttcact taactgatcc aataagcaag 4320

cttcctccgc tatggcgaca ggatgatgag ttgtaatgat gtgatttaat gaaccaattt 4380

taattttctc tgttaaaccg agcagaaaac cagaaacagt cagaggagcg ccgacaacac 4440

cattatctga aaaatgattt tcatacacta aaatctgttc aaaattcaac ttatcaacat 4500

actccgttat ttcctgcatg cgaactatac tttgttcttg aacagttgtt gaattgatga 4560

agttaaggaa gaacaatcca aatttcattt ctttctcctt agctaatata atagcgaacg 4620

ttgtttttct ttaagaaatg gcatgacatc agactggaag agcttcatgg aagcaataat 4680

ttcgtctact gttccattag cttcaaatcc acaacaaata tttgatattc ctgtagcatc 4740

aatgtctttt tgaattatgt caatacattc ctgcggcgtt cccacgggat tgatttcgta 4800

actgtaatca atacggcgat tagtatcttt atgtcctttt aatacaaagt cacgccactg 4860

ccctttattg aaatcataac ctcttgtttg gtctgaatca tcaaaaatag tcgtagcatt 4920

cacataagaa tcataccaat gccccagaaa tttccggcaa atctctttcg ctttaattga 4980

gtcatgatct acagatgtta tatatgataa gcaatggtcg atattatgaa tatcgtgccc 5040

atattcttga gccacttcat tataaagctc aagttgtgct ttcttttcgt tagtatttat 5100

aatccaactt aatatcatcg gtaggccaaa ttgagcagcc cactcagtcg tcgaagctga 5160

ttcagccacc acataaaccg gtgcgccacc tctgctatac gccgcggggt ttacttttac 5220

cttatggaac ttgatatgtt cattatcagc ttccatatat ccctctgtca tgccattctt 5280

tatcagcccg taccagcatt ccgctaaggc gcgactgtta ttcatatctg tgccgaatac 5340

gcgaaagtcc ttgttgtaaa gccctcggca aataccaaac cgaaatcgtc cttttgacat 5400

ttgatccaat aaattcacat cttcaagttg gcgtactgga tgggctgtgg gaagaacaat 5460

agcggcagtt cctacattca attttttagt cgcgccaagt aaatatgcag cagcgacata 5520

agggttacca agcaaaccaa actccgtgaa atgatgctcc agtaaccata cggtatcaaa 5580

accacactcc tcagagatgc gacctaattt aaccaaacgt ttcattacct ctgtttgaga 5640

aaattgggga ggttggtatg taagcaaaaa gtttccaaat ttcatagaga gtcctcctgt 5700

cgacggatcc tcaagctgtt tcatctcatt tctaattgta gcaggactaa cattcaagtt 5760

atgtcgctca attagtgttt tagaaccaac gggttgtcca aaatcaacat aatcctcaac 5820

aattgcgttt aatatactca attgcctatc tgtaatcatg ttttcacctc attagcactc 5880

acttatctca agtgctaatt ataatttatc aaattggtca agtaagtcaa tgttaaagac 5940

tcgaaatttc aatttttttt aatcatttat taggaaagct tcaaaaacct cattacctat 6000

gactttccct ctatttgtaa gtgcaatcac atcgttcttt tctacaatta attccttctc 6060

ttttaaatta tttattgttt gaccaaagac actttcaata gattggtcaa acttcttttt 6120

gaacctacta ctactcacac cttcatttaa acgcaaccca agaaacattt cttcttccat 6180

tctctcagtc aaagaaggtt tatttgatac taaaattgct ttactttctt tatttatagc 6240

tttgatataa tgattcactg gattgatatt cgtataacgc acaccatcta cataaccact 6300

tgcacctgct ccaaatccat aatattcctc attaaaccag taaaccttat tatgttctga 6360

ttcatggccg gatccccggg aattcctggc agtttatggc gggcgtcctg cccgccaccc 6420

tccgggccgt tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag 6480

agcgttcacc gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg 6540

ttttatttga tgcctgggga attcttgaag acgaaagggc ctcgtgatac gcctattttt 6600

ataggttaat gtcatgataa taatggtttc ttagacgtct tcaactaaag cacccattag 6660

ttcaacaaac gaaaattgga taaagtggga tatttttaaa atatatattt atgttacagt 6720

aatattgact tttaaaaaag gattgattct aatgaagaaa gcagacaagt aagcctccta 6780

aattcacttt agataaaaat ttaggaggca tatcaaatga actttaataa aattgattta 6840

gacaattgga agagaaaaga gatatttaat cattatttga accaacaaac gacttttagt 6900

ataaccacag aaattgatat tagtgtttta taccgaaaca taaaacaaga aggatataaa 6960

ttttaccctg catttatttt cttagtgaca agggtgataa actcaaatac agcttttaga 7020

actggttaca atagcgacgg agagttaggt tattgggata agttagagcc actttataca 7080

atttttgatg gtgtatctaa aacattctct ggtatttgga ctcctgtaaa gaatgacttc 7140

aaagagtttt atgatttata cctttctgat gtagagaaat ataatggttc ggggaaattg 7200

tttcccaaaa cacctatacc tgaaaatgct ttttctcttt ctattattcc atggacttca 7260

tttactgggt ttaacttaaa tatcaataat aatagtaatt accttctacc cattattaca 7320

gcaggaaaat tcattaataa aggtaattca atatatttac cgctatcttt acaggtacat 7380

cattctgttt gtgatggtta tcatgcagga ttgtttatga actctattca ggaattgtca 7440

gataggccta atgactggct tttataatat gagataatgc cgactgtact ttttacagtc 7500

ggttttctaa tgtcactaac ctgccccgtt agttgaagaa ggtttttata ttacagctcc 7560

agatccatat ccttcagatc cctagataat tcttctgata atttagtttt tgttagagtt 7620

taacataact gatgattatc agaagtttct ttaatataaa taaaagcact ataagcgact 7680

taacttatag tgcttattta atatattttt tcaaacaatc attagccacc aaacaaacga 7740

ctccagaagc cttttttagt ttgggtttct tctatagcaa tatctttatc attttgatta 7800

aatgaatcat cttgttgttg tgatttttca tcttgttgac tctcagatat atctttaggt 7860

tgaacctctg gtttcttttc ttctttctga tattgatcag tatttacaga atccgaagta 7920

tatgatgctt cttgtacatc aaagttttct cgatcattag tagatttatc aaaggaataa 7980

cttaattgtc tctcttcctc taattgatgt tccaattttt gaattttttt attgctttct 8040

aaagctaaaa tttgttgatt ctctaataat ttatttaatg tattaacatt gctattttgt 8100

tggtctagtt gtttagttaa attctcaata tacttttcat catttttagc tctagtttca 8160

aagatttcta cctgcttttc taattcgttt acttttgttt ttaatgtctc aaagccatct 8220

gaattattat atctattctt ttcagtgttt tcataagtta tttctgattt tttattttct 8280

ttattttgag ttgttctttc tttcttagtg attttttcta ctattttcaa ataatcatta 8340

tcatcaatat aattcactcc attttctttt tcaaaagata tattcaaatt tttagcatta 8400

ttaacaacag tttgtttagt tacattcaat tcatcagcaa ccatttttat agttttcata 8460

tcatccaccc ttaggcacct aatttactac ctaattttac cacctaacaa tttttctttc 8520

atctttttta ccgcctaaat atatcatttt tactacctaa gtacctaatt tactacctaa 8580

ttagccacta aaataataaa ttttgttgtt ttaaggtcat aacatgattc tgatctgtac 8640

ttaaaacgct ttatatacac ctttaaaaat gttaatatta tcttatgaat tttaaaaagc 8700

caatgctatt ggcgtagcat cggctctggt aattaaaacg atttgcgttc gtttatttat 8760

atattttttt gatacttgta ttatatatat ctactcgtct aagtgcaagc acaaaacata 8820

taacttacgt aaaaattgtt ttattacctc aactctaagt aaaaggaaat gaggtttttt 8880

attatgtcta aatttaaaaa aatatctgca agtgaattcg aaacattacg tttttatcaa 8940

ttacccaaat ttttatttga agatgaatac ttttctaaaa tgcccacaga tgcaaaagtt 9000

atgtatgctt tattaaaaga tcgctttgaa ctatcaagat taaataactg ggtagattca 9060

gaaaataata tttatctttt atataccaat aaacagttat gctcaatttt aaattatgca 9120

gaaccaaaaa tcattaaatt aaaaaaagaa ttagaaaaat acaatttgat tataaacgaa 9180

agacaaggtt taaataaacc taacaaaatt tatttactcg aacccacata tgacaaggaa 9240

ctaataaatt ctaagttcca gaacaaagaa tttattagtt ccagaactaa taaatcatca 9300

gttcaagaac taataaattc taagtcaagt gatactgatt ttaataatac tgaatatata 9360

gagactaaga ataatgatac gaattatacg aatgatacat ctaacatgat ttctaagaat 9420

tctcattcga atcatacaaa tcatcaacaa accgaattta ataatgatgc cttaaaattc 9480

caggcgcttg aagaattacc ttcgcaaatc aaatcttatg taagtaattt tgaaattaaa 9540

gacatccgta ttattaaaag tatcttactc aaggggaaaa agtcatttaa taatacacat 9600

gatacatatt accgtttaga agacgtcgaa tttgaacttg taagtgtttt aaaacgtttt 9660

aaagccatgt tgctacaaaa aaatgaaacc gttgaaacta tgcaaggcta tttaatgcaa 9720

tcaattaaag ctgaacttga agaaatacat gcattaaata tgcgtcgtca aaacatacct 9780

caatacaata tctttaatca ataactcaaa taatcttaca acaatcaaaa caacatcaaa 9840

atttggaatt aagtcaacag aaaaggatct ccccaggtgg cacttttcgg ggaaatgtgc 9900

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 9960

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 10020

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 10080

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 10140

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 10200

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc 10260

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 10320

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 10380

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 10440

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 10500

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa 10560

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 10620

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 10680

gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 10740

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 10800

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 10860

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 10920

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 10980

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 11040

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 11100

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 11160

gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 11220

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 11280

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 11340

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 11400

ccgaactgag atacctacag cgtgagcatt gagaaagcgc cacgcttccc gaagggagaa 11460

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 11520

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 11580

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 11640

cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 11700

cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 11760

gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt 11820

attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 11880

tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt 11940

catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct 12000

cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt 12060

ttcaccgtca tcaccgaaac gcgcgaggca ggggttaaaa ttccttcatt acactcttgg 12120

cggtttcact tatcaactta tcatttggct tatcactttt attgtcttta ttcgtaaaaa 12180

tgactaaaac aataggttca gattggccct taggataaac aaaagcaaca tcatttctag 12240

<210> 4

<211> 12601

<212> DNA

<213>artificial sequence

<221>pAmilux-groE-luxABCDE plasmid sequence

<400> 4

aattcgacgc tgcaggatat caactatcaa acgcttcggt taagcttaaa gcacaccctt 60

tctgcgtcct cgtattgacg cgacgtaaaa tttcaacgag cacgccggga tacttaccat 120

attctctgct aattatcccg acatcatcgg taacaataaa tgctggataa ctggttgctg 180

acgcatccat ataactcatc aaccccggcg ttccatcagg tacaggtttc aacgtttcag 240

gatcaagcgc tcgcgcatat acccacggcg gaacatgttt acgctgcatt tcatcctcaa 300

agaaacaagt gttgagttca acttgattaa atatatctcg gatctgacta atatcactga 360

gattgaaagt atcaaataaa agatgattga aatcatcacg tttcagagat tctttttcgt 420

aacttttcca gccgcctccg gttatgatat aaaggctttt atctccagaa aatgagattt 480

ttttatcttt catataatgg cagagtaaat aaataaagta tggcgaacca ataagacaaa 540

gatctttccc ttgatttttt attcgttcaa gactattcaa tgttttaaca aaatctattc 600

gttcttctgt tacggtaaat gtcgtaggat ataacaattc caccaaactc ataacatatt 660

taaaccaaat attatgagca ttaaatctat ctggtcccaa attgactaat tctatttgat 720

gatcaaacca actaccaaca tatttcatgc cataactcac agagcctaag agtctctcaa 780

tacttaatct gtcacgcgcc acctgacttt ttaaaccatt cgtgccgcta ctggtaaacc 840

aactttcaat ctcgttttcc tgagaagtta ataagcgagt aaacttaaaa accgatgttg 900

ggaatacagg tatgtcatca atttccgtaa tattgtcatc tactttgtgt gcctgacagt 960

agtgacgata ttctcgacaa tgtttataat gattacgaaa tgcatcaagc acaagtttct 1020

ttctgatttt ttcctgctcg tcgtaagacc acactaatgg atcgctcgaa aaaatcaaat 1080

catcaatttc tgagcttgct gtaatttctt gtttatcaac atatgaagtc atacctgttt 1140

tcctcctcaa gatcctttaa gacagagaaa ttgcttgatt ttcaatctca attctcattc 1200

ggcgttcatt gactgtcgca atagttaaat gttcaaatga cggttcagta atatcaacat 1260

caatatccag atgatcatta tccatcgcga tagcggcttt cgtaaccgat tgataaaaat 1320

tgcgcaggac cactaaattt tcactcaagt catgcgaact tcctaacaaa gaatatatct 1380

tgcatcgatt actacgaata tttgataaca atgtgataac ttcatcttgc ttgacccaat 1440

tatcgttatt tgcagtaaaa gcaataaacg gtatatcaag atacatcatg ttattaattg 1500

tagaagctaa atcttcccaa ccaaaatcaa gacaatctct cgcaaagact tcagcaccca 1560

atttatggcc ttcaaaatct agattatccg gcaattcatt aatgggtaga ctgagataat 1620

caaaccctaa agctctttca agagaatatc ttaagttaac aacaccgact gcggtgatta 1680

aaaacgaagc attgatttca gataggcttg cataagctat ccgcgcagat aagcttgaag 1740

ccaacatacc gaagttattt atttttcgtg tagttaacca atcaaccact gctaacaagc 1800

tctgctttcc tatagacatt gtaaattcat caattgtccc tgaactcaat ccaacgtggt 1860

gaagcgaatc atagcggatc acatgaaatc cattccgcga taaatattcc gccagaccag 1920

caaaatgatc catcctgcgg gcaaaaccag acgcaataat aatggcattc tttctctttg 1980

ggctgttttc ttctggcagc gtttcccaaa catgaatttt tttatttcct tcaacacaaa 2040

taacgtggtc gatggtttta tattttgatt cattttccat acttttacct attatgggac 2100

aaatacaagg aacttatctt cttccaggaa tcgagtctgt tctatttcaa ccgcaacatc 2160

cttagccgta tagttagatg gcctttcatg agaaatatat gtcactaatc gttgcaacgg 2220

tctcattccg tcatgagatc caccaactcg aaatatgtta ttcattcctg cttctacaat 2280

cctttccgca ccttttaatg ctaacgcatc tcgatattta aatgatgact cccaaggaaa 2340

aatagatatg gtttgcgtct tatttttttg aacataaggc aatatttgct caatattatc 2400

gacgtgatga aggtacacac atctgccaag tggttgatta aattccacac ctgcatttga 2460

ctcaataatc atccaacgtt gatgaatatc cacctctact tttaatccag caaacaagct 2520

ttctttttga actaaagaat aggccgcctt ttcatcaaaa tcttttttgg cattcggtaa 2580

tatatgcgca tatagattaa gtttttctat caacgctaac ttaaattcct cataatgatt 2640

tcccatgtaa tatatgtttt gggcagaaaa acaagctcgc tgatcgtaaa aacaaacatc 2700

atgagccgca cctgtcgctg cggacgtcaa atcaacagga ttatcgataa tgcaaagact 2760

ctttttagaa ccaaatttaa tcacatcagc ataagatggc gcatgctcta ccgcccaatt 2820

aatcgcatct ggccctcccc aagcgacaat aacatccgca tgtcgcataa tttcttttgc 2880

gagtgatgta tcaccttggt ggggccaata tataacagat aaagagcgcg ttatcggatg 2940

attagggtct acatcaataa aacttaacgc taatgcatta gcggtaaaag gatcggttga 3000

cgatgttttt ataatacact gattcttagt taaaattgcg cgtaatatag acatgatccc 3060

agataatgga acattacctg ccaacagatg tacagattta cctttcggaa aagcccgaac 3120

ataactttca tcctgaggta gccattcatc catgatatgg cgagaaccaa gttcattttc 3180

tacaacatca taaaggccgc ctttagaaca taaaatcata gatatccaat tggcctctag 3240

cttagccatt tcttctgaat atcccatata tttttttaag tcacgaatgt atgtcctgcg 3300

tcttgagtat tcttcatttt tccatctttg ccctaccgta tagagaaaat tgacaatgtt 3360

atgcaaccgt aattcgttat ttccattaca atcaataatg ttttttacat gagagtcatt 3420

caatattggc aggtaaacac tattatcacc aaaattaatg gattgcacta aatcatcact 3480

ttcgggaaag atttcaacct ggccgttaat aatgaatgaa atttttttag tcatatttgc 3540

cttcctcctg gtaccccatt atatcacatt atccattaaa aagcaaacaa attttcggta 3600

cctattaggt atattccatg tggtacttct taatattatc atcaacaata ttgattacat 3660

ttttttggct catcaaatca ttcattggtt caaaggacag caatacactt ttcgcaccac 3720

acttttcaat tgccaactta gccgcagtta tacactccgt ataatttccg acagcgtttt 3780

ctgcaattat ttcttcaagt ttattttcga aattttcatt agggtgcatt tcaagaacat 3840

aatcactaat aaatgcacgc gtctcttgtt tagctttatt actatcttcg ttatagttaa 3900

ctaatatcat taactgatgg tctatctctg ataggtcaac gtcatattta tccgcaacgg 3960

ctttatatct ttcagcatat tcatatctaa catcattaga atcatcccac ttaaagatga 4020

gaggaatacc ttttttggcc gcccactcaa caatatgatg actggttgct gttacatatt 4080

tccgaggtcc gcctggcgta taagcatggg gatttacaga tattttaggg aagctataaa 4140

aatcgttatc tggattacaa tagcctgttg ttaaagcatc gttaatgatt tcataacact 4200

cttcaaatag ttgctgttga tattcaaccg ggcgattaaa aaaatgcatt tcatcttttt 4260

tttcgcaatc actaaaccct aaaataaatc tcccttcact taactgatcc aataagcaag 4320

cttcctccgc tatggcgaca ggatgatgag ttgtaatgat gtgatttaat gaaccaattt 4380

taattttctc tgttaaaccg agcagaaaac cagaaacagt cagaggagcg ccgacaacac 4440

cattatctga aaaatgattt tcatacacta aaatctgttc aaaattcaac ttatcaacat 4500

actccgttat ttcctgcatg cgaactatac tttgttcttg aacagttgtt gaattgatga 4560

agttaaggaa gaacaatcca aatttcattt ctttctcctt agctaatata atagcgaacg 4620

ttgtttttct ttaagaaatg gcatgacatc agactggaag agcttcatgg aagcaataat 4680

ttcgtctact gttccattag cttcaaatcc acaacaaata tttgatattc ctgtagcatc 4740

aatgtctttt tgaattatgt caatacattc ctgcggcgtt cccacgggat tgatttcgta 4800

actgtaatca atacggcgat tagtatcttt atgtcctttt aatacaaagt cacgccactg 4860

ccctttattg aaatcataac ctcttgtttg gtctgaatca tcaaaaatag tcgtagcatt 4920

cacataagaa tcataccaat gccccagaaa tttccggcaa atctctttcg ctttaattga 4980

gtcatgatct acagatgtta tatatgataa gcaatggtcg atattatgaa tatcgtgccc 5040

atattcttga gccacttcat tataaagctc aagttgtgct ttcttttcgt tagtatttat 5100

aatccaactt aatatcatcg gtaggccaaa ttgagcagcc cactcagtcg tcgaagctga 5160

ttcagccacc acataaaccg gtgcgccacc tctgctatac gccgcggggt ttacttttac 5220

cttatggaac ttgatatgtt cattatcagc ttccatatat ccctctgtca tgccattctt 5280

tatcagcccg taccagcatt ccgctaaggc gcgactgtta ttcatatctg tgccgaatac 5340

gcgaaagtcc ttgttgtaaa gccctcggca aataccaaac cgaaatcgtc cttttgacat 5400

ttgatccaat aaattcacat cttcaagttg gcgtactgga tgggctgtgg gaagaacaat 5460

agcggcagtt cctacattca attttttagt cgcgccaagt aaatatgcag cagcgacata 5520

agggttacca agcaaaccaa actccgtgaa atgatgctcc agtaaccata cggtatcaaa 5580

accacactcc tcagagatgc gacctaattt aaccaaacgt ttcattacct ctgtttgaga 5640

aaattgggga ggttggtatg taagcaaaaa gtttccaaat ttcatagaga gtcctcctgt 5700

cgacggatcc tatttaattt atttatgaat taagttctgt attattcaat aactgctaaa 5760

atatcttctt catttaatac cagatatgtt tcattatctc gtttaacttc tgtaccagca 5820

tattgttgga acacgacacg gtccccttct ttcacttcag gagtcactct tgtaccatca 5880

tttaataggc gtccagttcc tactgcaacg ataacgcctt cgtttgattt ttctttagca 5940

ctatcagtta aaacaatacc acttttagtt gtttgttctt gttctttttt ctcaataatc 6000

acacgatttc caattggttt tagcatgatt gttcctcctt aaaaaaccta aagtttagca 6060

cttaacatta aagagtgcta acatacattt ataataatca aatttggtca aaatttcaag 6120

tcataacctt ttaattaagt tttgtaactt caagttattt acgataaaat aaattataaa 6180

caaatatttg gaggaaaatt atgacaagat tatgggcatc attgctaact gttattattt 6240

atatattgtc tcaattttta ccgcttctca ttgtaaaaaa attaccattt gtacaatata 6300

gtggcataga actgactaaa gcagtcattt acatacaact tgttctattt ttaatcgccg 6360

ccacgacgat tattttaatt aatttaaaaa ttaaaaatcc aacaaaatta gaattagaag 6420

ttaaagaacc taaaaaatat atcattccat gggcattgct tggatttgca ttggtaatga 6480

tttatcaaat ggtagtgagc attgtattaa cgcaaattta tggtggacaa caagtaagtc 6540

ctaatacaga aaagctaatt attattgctc gaaaaatacc tatatttatc ttctttgtat 6600

ctattattgg tcctttatta gaagaatatg tattcagaaa agtaatcttt ggagaattat 6660

ttaatgcgat taaaggtaat cgtatcgtgg catttattat tgctacaaca gtaagttcat 6720

taatatttgc ggatccccgg gaattcctgg cagtttatgg cgggcgtcct gcccgccacc 6780

ctccgggccg ttgcttcgca acgttcaaat ccgctcccgg cggatttgtc ctactcagga 6840

gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac tgagcctttc 6900

gttttatttg atgcctgggg aattcttgaa gacgaaaggg cctcgtgata cgcctatttt 6960

tataggttaa tgtcatgata ataatggttt cttagacgtc ttcaactaaa gcacccatta 7020

gttcaacaaa cgaaaattgg ataaagtggg atatttttaa aatatatatt tatgttacag 7080

taatattgac ttttaaaaaa ggattgattc taatgaagaa agcagacaag taagcctcct 7140

aaattcactt tagataaaaa tttaggaggc atatcaaatg aactttaata aaattgattt 7200

agacaattgg aagagaaaag agatatttaa tcattatttg aaccaacaaa cgacttttag 7260

tataaccaca gaaattgata ttagtgtttt ataccgaaac ataaaacaag aaggatataa 7320

attttaccct gcatttattt tcttagtgac aagggtgata aactcaaata cagcttttag 7380

aactggttac aatagcgacg gagagttagg ttattgggat aagttagagc cactttatac 7440

aatttttgat ggtgtatcta aaacattctc tggtatttgg actcctgtaa agaatgactt 7500

caaagagttt tatgatttat acctttctga tgtagagaaa tataatggtt cggggaaatt 7560

gtttcccaaa acacctatac ctgaaaatgc tttttctctt tctattattc catggacttc 7620

atttactggg tttaacttaa atatcaataa taatagtaat taccttctac ccattattac 7680

agcaggaaaa ttcattaata aaggtaattc aatatattta ccgctatctt tacaggtaca 7740

tcattctgtt tgtgatggtt atcatgcagg attgtttatg aactctattc aggaattgtc 7800

agataggcct aatgactggc ttttataata tgagataatg ccgactgtac tttttacagt 7860

cggttttcta atgtcactaa cctgccccgt tagttgaaga aggtttttat attacagctc 7920

cagatccata tccttcagat ccctagataa ttcttctgat aatttagttt ttgttagagt 7980

ttaacataac tgatgattat cagaagtttc tttaatataa ataaaagcac tataagcgac 8040

ttaacttata gtgcttattt aatatatttt ttcaaacaat cattagccac caaacaaacg 8100

actccagaag ccttttttag tttgggtttc ttctatagca atatctttat cattttgatt 8160

aaatgaatca tcttgttgtt gtgatttttc atcttgttga ctctcagata tatctttagg 8220

ttgaacctct ggtttctttt cttctttctg atattgatca gtatttacag aatccgaagt 8280

atatgatgct tcttgtacat caaagttttc tcgatcatta gtagatttat caaaggaata 8340

acttaattgt ctctcttcct ctaattgatg ttccaatttt tgaatttttt tattgctttc 8400

taaagctaaa atttgttgat tctctaataa tttatttaat gtattaacat tgctattttg 8460

ttggtctagt tgtttagtta aattctcaat atacttttca tcatttttag ctctagtttc 8520

aaagatttct acctgctttt ctaattcgtt tacttttgtt tttaatgtct caaagccatc 8580

tgaattatta tatctattct tttcagtgtt ttcataagtt atttctgatt ttttattttc 8640

tttattttga gttgttcttt ctttcttagt gattttttct actattttca aataatcatt 8700

atcatcaata taattcactc cattttcttt ttcaaaagat atattcaaat ttttagcatt 8760

attaacaaca gtttgtttag ttacattcaa ttcatcagca accattttta tagttttcat 8820

atcatccacc cttaggcacc taatttacta cctaatttta ccacctaaca atttttcttt 8880

catctttttt accgcctaaa tatatcattt ttactaccta agtacctaat ttactaccta 8940

attagccact aaaataataa attttgttgt tttaaggtca taacatgatt ctgatctgta 9000

cttaaaacgc tttatataca cctttaaaaa tgttaatatt atcttatgaa ttttaaaaag 9060

ccaatgctat tggcgtagca tcggctctgg taattaaaac gatttgcgtt cgtttattta 9120

tatatttttt tgatacttgt attatatata tctactcgtc taagtgcaag cacaaaacat 9180

ataacttacg taaaaattgt tttattacct caactctaag taaaaggaaa tgaggttttt 9240

tattatgtct aaatttaaaa aaatatctgc aagtgaattc gaaacattac gtttttatca 9300

attacccaaa tttttatttg aagatgaata cttttctaaa atgcccacag atgcaaaagt 9360

tatgtatgct ttattaaaag atcgctttga actatcaaga ttaaataact gggtagattc 9420

agaaaataat atttatcttt tatataccaa taaacagtta tgctcaattt taaattatgc 9480

agaaccaaaa atcattaaat taaaaaaaga attagaaaaa tacaatttga ttataaacga 9540

aagacaaggt ttaaataaac ctaacaaaat ttatttactc gaacccacat atgacaagga 9600

actaataaat tctaagttcc agaacaaaga atttattagt tccagaacta ataaatcatc 9660

agttcaagaa ctaataaatt ctaagtcaag tgatactgat tttaataata ctgaatatat 9720

agagactaag aataatgata cgaattatac gaatgataca tctaacatga tttctaagaa 9780

ttctcattcg aatcatacaa atcatcaaca aaccgaattt aataatgatg ccttaaaatt 9840

ccaggcgctt gaagaattac cttcgcaaat caaatcttat gtaagtaatt ttgaaattaa 9900

agacatccgt attattaaaa gtatcttact caaggggaaa aagtcattta ataatacaca 9960

tgatacatat taccgtttag aagacgtcga atttgaactt gtaagtgttt taaaacgttt 10020

taaagccatg ttgctacaaa aaaatgaaac cgttgaaact atgcaaggct atttaatgca 10080

atcaattaaa gctgaacttg aagaaataca tgcattaaat atgcgtcgtc aaaacatacc 10140

tcaatacaat atctttaatc aataactcaa ataatcttac aacaatcaaa acaacatcaa 10200

aatttggaat taagtcaaca gaaaaggatc tccccaggtg gcacttttcg gggaaatgtg 10260

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 10320

caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 10380

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 10440

gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 10500

gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 10560

atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg 10620

caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 10680

gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 10740

accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 10800

ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 10860

gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca 10920

acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 10980

atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 11040

ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 11100

gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 11160

gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 11220

tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 11280

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 11340

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 11400

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 11460

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 11520

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 11580

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 11640

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 11700

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 11760

accgaactga gatacctaca gcgtgagcat tgagaaagcg ccacgcttcc cgaagggaga 11820

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 11880

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 11940

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 12000

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 12060

tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 12120

agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 12180

tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca 12240

atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg 12300

tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 12360

tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 12420

tttcaccgtc atcaccgaaa cgcgcgaggc aggggttaaa attccttcat tacactcttg 12480

gcggtttcac ttatcaactt atcatttggc ttatcacttt tattgtcttt attcgtaaaa 12540

atgactaaaa caataggttc agattggccc ttaggataaa caaaagcaac atcatttcta 12600

g 12601

Claims (6)

1. a kind of method for determining antibiotic mechanism of action using bioluminescence reporting system, step is:

(1) strain is reported using bioluminescence reporting system preparation staphylococcus aureus lux；Wherein, the luminous reporting system By pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, pAmilux-dnaK-luxABCDE matter Grain, pAmilux-groE-luxABCDE plasmid composition；The nucleotide sequence of the pAmilux-clpX-luxABCDE plasmid is such as Shown in SEQ ID NO:1, the nucleotide sequence of the pAmilux-dnaJ-luxABCDE plasmid is as shown in SEQ ID NO:2, institute The nucleotide sequence of pAmilux-dnaK-luxABCDE plasmid is stated as shown in SEQ ID NO:3, the pAmilux-groE- The nucleotide sequence of luxABCDE plasmid is as shown in SEQ ID NO:4；The staphylococcus aureus lux report strain is by four kinds Plasmid pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, pAmilux-dnaK-luxABCDE Plasmid, pAmilux-groE-luxABCDE plasmid are transformed into staphylococcus aureus respectively, manufactured four plants golden yellow grapes Coccus reports strain, is respectively designated as: reporting bacterial strain S1, reporting bacterial strain S2, reporting bacterial strain S3, reporting bacterial strain S4；

(2) the agarose double-layer plate containing four plants of staphylococcus aureus report strains is prepared, by the filter paper of sterilizing in agar Sugared double-layer plate center or media surface ordered arrangement are simultaneously closely affixed with culture medium；

(3) solution to be checked containing antibiotic is added drop-wise on filter paper, continues to cultivate and shoot bacterial luminescence picture；

(4) luminosity curve is converted by luminous picture, extracts luminosity curve feature and is compared with known antibiotic profile luminosity curve Hierarchical clustering is carried out, if the luminosity curve of detected antibiotic luminosity curve and the antibiotic of known action mechanism cluster is one It rises, then determines that antibiotic mechanism of action to be checked is identical as the antibiotic of known action mechanism.

2. the method for antibiotic mechanism of action is determined using bioluminescence reporting system as described in claim 1, it is characterised in that: Step (2) described filter paper is the small scraps of paper of circle that No. 1 qualitative filter paper of Xinhua is broken into diameter 6mm with punch.

3. the method for antibiotic mechanism of action is determined using bioluminescence reporting system as described in claim 1, it is characterised in that: Step (3) is described the solution to be checked containing antibiotic is added drop-wise on filter paper after, plate is inverted in incubator, 37 DEG C Cultivate 20~25h.

4. the method for antibiotic mechanism of action is determined using bioluminescence reporting system as described in claim 1, which is characterized in that Step (4) method for drafting for converting luminosity curve or known antibiotic profile luminosity curve for luminous picture is:

1) using plate center as origin (x₀,y₀), plate radius is divided into one such as 300 or 1200 parts of specific number, gained To each annulus in average distance be denoted as its midpoint to the maximum distance of origin and the average value d of minimum range；

2) A is set as the average canbdle power of all the points in some annulus, then average canbdle power is the function of d: A=f (d)；

3) function of application settings is depicted as luminosity curve.

5. determining the method for antibiotic mechanism of action using bioluminescence reporting system as described in claim 1 or 4, feature exists In, the known antibiotic be Daptomycin (DAP), polymyxin B (PMB), Imipenem (IPM), benzyl penicillin (PEN), ten thousand Ancient mycin (VAN), erythromycin (ERY), kanamycins (KAN), spectinomycin (SPE), streptomysin (STR), tetracycline (TET), Mitomycin C (MMC), Ciprofloxacin (CIP), gatifloxacin (GAT), trimethoprim (TMP), rifampin (RIF), phleomycin (PHL)。

6. the method for antibiotic mechanism of action is determined using bioluminescence reporting system as described in claim 1, which is characterized in that Step (4) method for carrying out hierarchical clustering analysis according to characteristic luminescence curve is:

1) the characteristic luminescence curve by each from inhibition zone edge to plate edge part is denoted as is characterized by 300 luminous intensities A vector, each luminous intensity represent shine picture in from inhibition zone edge to each annulus in plate edge part i.e. press down Bacterium circle edge is to the average canbdle power in the annulus of plate edge part 1/300；

2) by the characteristic luminescence of four plants of staphylococcus aureus report strains in bio-luminescence system corresponding to each antibiotic Curve is merged into one and characterizes vector by the merging luminous intensity that 1200 luminous intensities are characterized；

3) Euclidean distance calculation method is used, the distance between merging luminous intensity characterization vector of antibiotic is calculated, and Construct the distance matrix of antibiotic；

4) the merging luminous intensity of antibiotic is characterized between vector with UPGMA algorithm using the hclust function in R language Carry out clustering, according to antibiotic to be checked whether with known action mechanism antibiotic cluster judge its antibiotic together The type of mechanism of action.