CN110286118B - Method for judging antibiotic action mechanism by using bioluminescence report system - Google Patents

Abstract

The invention discloses a method for judging an action mechanism of antibiotics by using a bioluminescence report system. The method comprises the steps of preparing a staphylococcus aureus lux report strain by using a bioluminescence report system, arranging a filter paper sheet of a solution to be detected containing antibiotics on an agarose double-layer flat plate containing the strain, culturing and obtaining a luminescence picture, converting the luminescence picture into a luminescence curve, extracting luminescence curve characteristics, comparing the luminescence curve characteristics with a known antibiotic characteristic luminescence curve, carrying out hierarchical clustering, and judging that the action mechanism of the antibiotics to be detected is the same as that of the antibiotics with known action mechanisms if the detected antibiotic luminescence curve and the luminescence curve of the antibiotics with known action mechanisms are clustered together. The method can judge the action mechanism of the antibiotic, reduce the cost of identifying the action mechanism of the antibiotic, improve the speed of identifying the action mechanism of the antibiotic, improve the screening capacity of the antibiotic and have wide application prospect.

Description

Method for judging antibiotic action mechanism by using bioluminescence report system

Technical Field

The invention relates to a method for judging an action mechanism of an antibiotic by using a bioluminescent report system. Belongs to the field of biotechnology.

Background

Antibiotics are the primary weapons of humans against bacterial infections. The widespread use of antibiotics poses a serious problem of antibiotic resistance, so that existing antibiotics rapidly lose efficacy and bacterial infections become a serious threat to human health again. Statistically, about 70 million people die globally due to antibiotic resistance. In 2050, 1000 million people are expected to die every year in the world due to antibiotic resistance, and become the first cause of death of human beings. Because of the threat, the discovery of new antibiotics is increasingly important, and the discovery of new antibiotics is also the focus of research and work in the scientific and medical communities.

Antibiotics can achieve bacterial kill by a variety of mechanisms, such as preventing cell wall synthesis, preventing protein synthesis, preventing DNA replication, and the like. Different sterilization mechanisms result in different sterilization effects. Therefore, the judgment of antibiotic sterilization mechanism is one of the important indexes for screening antibiotics. However, the identification of antibiotic bactericidal mechanisms requires complex and time-consuming molecular mechanism studies, which often cannot be completely determined after the antibiotics are marketed, which reduces the efficiency of antibiotic screening and also reduces the effectiveness of antibiotic usage.

Bacteria can use luciferase encoded by the lux gene to achieve bioluminescence. In the bacterial lux gene, luxA and luxB function to encode catalytic luminescent subunits, while luxC, luxD and luxE encode proteins involved in substrate synthesis. Bacterial luciferases are widely used in a variety of reporter systems, including systems that detect antibiotics.

To date, existing methods for identifying the mechanism of action of antibiotics rely heavily on complex biochemical, molecular biological, genetic studies, are time-consuming and labor-intensive, and rely on high-level scientific studies, and there is no method for rapidly determining the mechanism of action of antibiotics. This situation seriously affects the rapid screening, discovery, identification and research of action mechanism of antibiotics. Through retrieval, no report exists for judging the action mechanism of the antibiotic by using a luminescence report system constructed by promoter fragments of molecular chaperone genes clpX, groE, dnaJ and dnaK and a luxABCDE gene fragment connected with the molecular chaperone gene promoter.

Disclosure of Invention

The invention aims to provide a method for judging the action mechanism of antibiotics by using a bioluminescent report system.

The invention discloses a method for judging an action mechanism of antibiotics by using a bioluminescence report system, which comprises the following steps:

(1) preparing a staphylococcus aureus lux reporter strain by using a bioluminescence reporter system; wherein the luminescence reporting system consists of pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, pAmilux-dnaK-luxABCDE plasmid and pAmilux-groE-luxABCDE plasmid; the nucleotide sequence of the pAmilux-clpX-luxABCDE plasmid is shown as SEQ ID NO: 1, the nucleotide sequence of the pAmilux-dnaJ-luxABCDE plasmid is shown as SEQ ID NO: 2, the nucleotide sequence of the pAmilux-dnaK-luxABCDE plasmid is shown as SEQ ID NO: 3, the nucleotide sequence of the pAmilux-groE-luxABCDE plasmid is shown as SEQ ID NO: 4 is shown in the specification; the staphylococcus aureus lux report strain is prepared by respectively transforming four plasmids, namely pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, pAmilux-dnaK-luxABCDE plasmid and pAmilux-groE-luxABCDE plasmid into staphylococcus aureus, and the four staphylococcus aureus lux report strains are respectively named as: reporter strain S1, reporter strain S2, reporter strain S3, reporter strain S4;

(2) preparing an agarose double-layer plate containing four staphylococcus aureus reporter strains, and orderly arranging sterilized filter paper sheets in the center of the agarose double-layer plate or on the surface of a culture medium and closely attaching the sterilized filter paper sheets to the culture medium;

(3) dripping a solution to be detected to contain antibiotics onto a filter paper sheet, continuously culturing and shooting a bacterial luminescence picture;

(4) and converting the luminescence picture into a luminescence curve, extracting luminescence curve characteristics, comparing the luminescence curve characteristics with known antibiotic characteristic luminescence curves, and carrying out hierarchical clustering, wherein if the luminescence curve of the detected antibiotic is clustered with the luminescence curve of the antibiotic with a known action mechanism, the action mechanism of the antibiotic to be detected is judged to be the same as that of the antibiotic with the known action mechanism.

In the method for determining the action mechanism of the antibiotic by using the bioluminescence report system, the following steps are carried out: the filter paper sheet in the step (2) is preferably a small circular paper sheet with the diameter of 6mm made of Xinhua No. 1 qualitative filter paper by a puncher.

In the method for determining the action mechanism of the antibiotic by using the bioluminescence report system, the following steps are carried out: and (3) after the solution to be detected containing the antibiotics is dripped on the filter paper, preferably, the flat plate is placed in an incubator upside down and cultured for 20-25 h at 37 ℃.

In the method for determining the antibiotic action mechanism by using the bioluminescence report system, the method for converting the luminescence picture into the luminescence curve or the known antibiotic characteristic luminescence curve in the step (4) comprises the following steps:

1) with the center of the plate as the origin (x)₀,y₀) Equally dividing the radius of the flat plate into a specific number of parts (such as 300 or 1200 parts), and recording the obtained average distance in each circular ring as the average value d of the maximum distance and the minimum distance from the middle point to the origin;

2) let A be the average luminous intensity of all points in a certain circle, the average luminous intensity is a function of d: a ═ f (d);

3) and drawing a luminous curve by using the set function.

In the method for determining the action mechanism of the antibiotic by using the bioluminescence report system, the following steps are carried out: the known antibiotics are Daptomycin (DAP), polymyxin b (pmb), Imipenem (IPM), penicillin g (pen), Vancomycin (VAN), Erythromycin (ERY), Kanamycin (KAN), Spectinomycin (SPE), Streptomycin (STR), tetracycline (TET), mitomycin c (mmc), Ciprofloxacin (CIP), Gatifloxacin (GAT), Trimethoprim (TMP), Rifampicin (RIF), Phleomycin (PHL).

In the method for determining the action mechanism of the antibiotic by using the bioluminescence report system, the following steps are carried out: the method for carrying out hierarchical clustering analysis according to the characteristic light-emitting curve in the step (4) comprises the following steps:

1) recording each characteristic light-emitting curve from the edge of the inhibition zone to the edge part of the flat plate as a vector (light-emitting intensity characteristic vector) represented by 300 light-emitting intensities, wherein each light-emitting intensity represents the average light-emitting intensity in each circular ring from the edge of the inhibition zone to the edge part of the flat plate, namely the circular ring from the edge of the inhibition zone to the edge part of the flat plate 1/300 in the light-emitting picture;

2) combining the characteristic luminescence curves of four staphylococcus aureus report strains in a bioluminescent system corresponding to each antibiotic into a combined luminescence intensity characteristic vector characterized by 1200 luminescence intensities;

3) calculating the distance between the combined luminous intensity characteristic vectors of the antibiotics by using an Euclidean distance calculation method, and constructing a distance matrix of the antibiotics;

4) and (3) performing cluster analysis on the combined luminous intensity characteristic vectors of the antibiotics by using an hclust function in the R language and using a UPGMA algorithm, and judging the type of the action mechanism of the antibiotics according to whether the to-be-detected antibiotics are clustered with the antibiotics with known action mechanisms.

The method disclosed by the invention can be used for quickly and simply judging the action mechanism type of the antibiotic, and has higher clinical application value. Has important significance in the aspects of antibiotic discovery, antibiotic mechanism identification, further guidance of bacterial infection treatment schemes and the like.

The invention utilizes a bioluminescence report system and a data analysis method, converts the bioluminescence report system into staphylococcus aureus, shoots a luminescence picture according to the phenomenon that the staphylococcus aureus containing the bioluminescence report system emits light on a double-layer flat plate loaded with a paper sheet containing antibiotics, converts the luminescence picture into a luminescence curve, calculates the luminescence characteristic curve, and performs cluster analysis on the characteristic curve. Analysis of the luminescence profiles for the different antibiotics showed that: the method can cluster the light-emitting characteristic curves of the antibiotics with the same action mechanism through the cluster analysis of the light-emitting characteristic curves, so that the action mechanism of the antibiotics is judged by judging the light-emitting characteristic curves of the antibiotics and which light-emitting characteristic curves of the antibiotics with known action mechanisms are clustered together, the sensitivity and specificity of detection are both over 0.89, and the action of the method is proved.

Compared with the prior art, the invention has the beneficial effects that:

the method avoids a complex basic mechanism research method, can quickly judge the action mechanism of the antibiotic, thereby improving the discovery speed of the new antibiotic and the identification speed of the action mechanism of the new antibiotic.

Drawings

FIG. 1: the antibiotic (ciprofloxacin) caused the bioluminescent reporter system (pAmilux-clpX-luxABCDE) to emit light.

FIG. 2: characteristic luminescence curves of different antibiotics.

Wherein: clpX, dnaJ, dnaK, groE refer to four plasmids in the luminescent reporter system of the invention. Three lines (from left to right) indicate the radius of the paper sheet, the radius of the zone of inhibition, and the radius of the flat plate, respectively. RLU, relative luminescence units. CIP, ciprofloxacin; DAP, daptomycin; ERY, erythromycin; GAT, gatifloxacin; IPM, imipenem; KAN, kanamycin; MMC, mitomycin C; PEN, penicillin; PHL, phleomycin; PMB, polymyxin B; RIF, rifampin; SPE, spectinomycin; STR, streptomycin; TET, tetracycline; TMP, trimethoprim; VAN, vancomycin.

FIG. 3: clustering analysis for antibiotic action mechanism determination.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting in any way, and any simple modifications, equivalent changes and modifications of the embodiments according to the technical spirit of the present invention are within the scope of the present invention.

Unless otherwise specified, the contents of the methods described in the following examples are all conventional experimental methods. The related reagents, vectors and strains are obtained from known sales channels unless specified otherwise.

Example 1

Experimental strains and plasmids:

escherichia coli DH5 α: used for competent preparation, and is obtained by China general microbiological culture Collection center (CGMCC 1.12873).

Escherichia coli DH 10B: for the preparation of competence, available from ThermoFisher scientific Inc. (Cat. No. 18297010)

Staphylococcus aureus RN 4220: used for competence preparation and luminescence detection, BEI Resources acquisition.

pMD19-T available from Dalibao Bio (TaKaRa)

pAmilux: from the university of Columbia, the genus British, Canada (documents: Mesak LR, Yim G, Davies J, Improved lux reporters for use in Staphylococcus aureus, Plasmid,2009,61(3):182-

Culture medium and preparation required by experiment:

LB culture medium: each liter of the medium was prepared by adding tryptone (10g), sodium chloride (10g), and yeast extract (5g) to 950ml of deionized water, and stirring with a magnetic stirrer until dissolved. 5mol/L sodium hydroxide is dripped to adjust the pH value to 7.0, deionized water is used for constant volume to 1L, and split charging and high pressure steam sterilization is carried out for 20min at the temperature of 121 ℃ for standby. If a solid culture medium is needed, agar powder is added according to the proportion of 1.5 percent.

NYE culture medium: 1 liter of the medium was prepared, and casein hydrolysate (10g), yeast extract (5g), and sodium chloride (5g) were added to 950ml of deionized water, and stirred with a magnetic stirrer until dissolved. Dropwise adding 5mol/L sodium hydroxide to adjust pH to about 7.2, diluting to constant volume with deionized water to 1L, packaging, and sterilizing with high pressure steam at 121 deg.C for 20 min. If a solid culture medium is needed, agar powder is added according to the proportion of 1.5 percent.

Antibiotics required by experiments and preparation thereof:

ampicillin (AMP) solution 100mg of ampicillin was weighed, dissolved in 1ml of sterile ultrapure water and vortexed, shaken and mixed until the final concentration was 100mg/ml, and stored in a refrigerator at-20 ℃ for further use. When in use, the mixture is added into a culture medium according to the proportion of 1: 1000.

Chloramphenicol (CHL) solution: weighing 10mg of chloramphenicol, dissolving in 1ml of absolute ethanol, vortex, shaking, mixing to a final concentration of 10mg/ml, and storing in a refrigerator at-20 deg.C for use. When in use, the mixture is added into a culture medium according to the proportion of 1: 1000.

Solution required by experiment and preparation thereof:

and (3) weighing 0.333g of CaCl2, 0.105g of 3-propanesulfonic acid sodium salt (MOPS) and 0.148g of MnCl 2.4H2O by using an electronic balance, dissolving the materials in 40ml of deionized water, weighing 7.5ml of glycerol by using a liquid transfer gun, adding the glycerol into the solution, metering the volume to 50ml by using the deionized water, and adjusting the pH to about 7 by using NaOH. The buffer was sterilized by filtration through a 0.22 μm filter and transferred to a 50ml sterile centrifuge tube for precooling on ice. The buffer solution is prepared as it is used.

80% (v/v) glycerol 80ml of glycerol was dissolved in 20ml of ultrapure water and autoclaved at 121 ℃ for 20min for further use.

50 × TAE electrophoresis buffer: 242g of Tris and 18.612g of EDTA were weighed into a beaker, and 700ml of ultrapure water and 57.1ml of glacial acetic acid were added thereto and dissolved by stirring well. And then, adding NaOH to adjust the pH value to 8.3, finally fixing the volume to 1L, storing at normal temperature for later use, and diluting by 50 times when in use.

Reagents and instruments required for the experiment:

reagents required by the experiment are an Ezup column type bacterial genome DNA extraction kit (Sangong biotech, Shanghai, China), an EZ-10 column type plasmid small-amount extraction kit (Sangong biotech, Shanghai, China), an EZ-10 column type DNA glue recovery kit (Sangong biotech, Shanghai, China), and a pMD^TM19-T Vector Cloning Kit (Takara, Dalian, China), FlyCut BamHI quick endonuclease (TransGene Biotech, Beijing, China), T4DNA Ligase (Takara, Dalian, China), 4S Green Plus nontoxic nucleic acid dye (Sangong Biotech, Shanghai, China), NormalRunTM stabilized 250bp-I DNA ladder (Generay Biotech, Shanghai, China), 1 XT 3 SuperPCR Mix (Tsinge, Beijing, China), sterile filter (0.22 μm).

Instruments required for the experiment:

the experimental apparatus includes a DNP-9082 type constant temperature incubator (shanghai essence macro experimental facility, ltd), a constant temperature shaking incubator (jiangsu taicang laboratory instrument plant), an HH-S constant temperature water bath (jiangsu jintan city medical instrument plant), a microwave oven (glanshi), an ultraclean bench (jiangsu purification laboratory equipment plant), an analytical balance (sydoris scientific instruments ltd), a vortex oscillator (jiangsu linbeier instruments manufacturing ltd), a centrifuge (this china ltd), a PCR instrument (berle life medical products (shanghai) ltd), and an DYY-6C type electrophoresis instrument (beijing siyinji instrument plant).

The experimental steps are as follows:

step 1. Strain activation and purification

Cryopreserved glycerol-preserved e.coli (15% glycerol) was grown at 1: 300 into LB medium, and cultured overnight at 37 ℃ with shaking.

Escherichia coli was cultured in LB medium and Staphylococcus aureus was cultured in NYE medium. And (3) sucking 10 mu l of activated bacterium liquid, dropwise adding the bacterium liquid onto a culture dish, then burning a red inoculating loop, and dipping the bacterium liquid for scribing after cooling. Care is taken not to streak too sparsely or densely, otherwise it is difficult to isolate a single colony. Culturing at 37 deg.C for 1 hr, and culturing overnight in inverted state after the bacteria liquid is completely absorbed.

Step 2, extracting bacterial genome DNA

The experiment adopts an Ezup column type bacterial genome DNA extraction kit, and comprises the following steps:

and (3) collecting thalli:

staphylococcus aureus: 1ml of bacterial liquid cultured overnight is taken into a 1.5ml centrifuge tube and centrifuged for 1min at 8000-12000rpm, and the supernatant is removed as clean as possible. Adding 180. mu.l of lysozyme solution (adding lysozyme into enzymic lysis buffer before use to prepare 30mg/ml lysozyme solution), repeatedly blowing and beating the resuspended thallus by using a pipette gun, and carrying out water bath at 37 ℃ for at least 3 h. Subsequently, 20. mu.l of protease K is added, mixed by inversion or shaking, and incubated in a water bath at 56 ℃ for at least 1h until the liquid in the EP tube is clear like egg white. This step is critical because the wall thickness of staphylococcus aureus cells is difficult to extract its genomic DNA if the lysis is incomplete.

Add 200. mu.l Buffer BD and mix well.

Adding 200 mul of absolute ethyl alcohol and mixing evenly.

And (3) placing an adsorption column in the kit into a collection tube, transferring all liquid added with BufferBD and absolute ethyl alcohol into the adsorption column by using a pipette, standing for 2-3min, centrifuging at 12000rpm for 3min, and discarding waste liquid in the collection tube.

The adsorption column was returned to the collection tube, 500. mu.l of PW Solution was added dropwise, centrifuged at 12000rpm for 1min, and the waste liquid in the collection tube was discarded.

The adsorption column was returned to the collection tube and centrifuged at 12000rpm for 1min with 500. mu.l of Wssh solution added.

The column was returned to the collection tube and centrifuged at 12000rpm for 5min to remove residual rinse.

The adsorption column was taken out, placed in a new 1.5ml EP tube, added with 50-100. mu.l of sterile ultrapure water, allowed to stand for 2min, and centrifuged at 12000rpm for 2min to obtain a DNA solution.

Step 3. construction of promoter-T vector

Amplification of the promoter fragments of the genes groES-EL, dnaK, dnaJ, clpX:

with staphylococcus aureus genome DNA as a template, after designing corresponding primers (aiming at clpX, dnaK, dnaJ and groE promoter sequences) by using Primer Premier 5.0 software, adding BamH I enzyme cutting sites at the 5' ends of an upstream Primer and a downstream Primer respectively: GGATCC. In addition, since many restriction endonucleases have difficulty cleaving the recognition sequence near the end of the DNA molecule, in order to solve this problem, an additional protective base (e.g., CGC) should be designed at the 5' end of the oligonucleotide primer, the primer sequence is shown in Table 1.

TABLE 1 primers for recombinant plasmid construction and validation

The PCR reaction system was referenced to the Super PCR Mix instructions (Table 2):

TABLE 2 PCR reaction System

The annealing temperature in the PCR process can be adjusted according to the amplification conditions, and if the number of bands is large, the annealing temperature is increased, and if the number of bands is difficult to amplify, the annealing temperature is decreased. The extension time is related to the size of the target gene fragment, and the normal amplification speed is 1 Kb/min.

TABLE 3 PCR reaction procedure

Detection of PCR products by Sepharose electrophoresis of Staphylococcus aureus RN 4220:

(1) 0.8% agarose gel is prepared, 0.8g agarose is weighed, mixed with 100ml 1 XTAE electrophoresis buffer solution and heated in a microwave oven until the agarose is completely melted until clear and transparent solution is obtained.

(2) Cooling to about 50 ℃ at room temperature, and adding 1: adding nucleic acid dye according to the proportion of 1000, uniformly mixing, pouring into a mould, inserting a proper comb, and waiting for coagulation. The distance between the bottom of the comb teeth and the tray surface is 0.5-1mm, and if the comb teeth are too close to the tray surface, gel sample dispensing holes are easily damaged when the comb is pulled out, so that sample leakage is caused.

(3) After the gel is solidified, the gel is placed into an electrophoresis tank filled with 1 XTAE electrophoresis buffer, and a PCR product and a DNA marker are respectively spotted in a gel hole by using a pipette. Closing the electrophoresis tank cover, connecting an electrode plug, and carrying out electrophoresis at a voltage of 120V.

The electrophoresis solution used for gel preparation and the electrophoresis solution placed in the electrophoresis tank are the same batch as much as possible, because even small ionic strength changes can cause gel performance abnormity and influence the migration rate of DNA.

(4) And (4) placing the agarose gel after the electrophoresis into an imager, observing under an ultraviolet lamp of 300nm, and taking a picture.

And (3) PCR product purification:

(1) the agarose gel was observed using a gel imager and the gel containing the band of interest was cut with a clean razor blade and the edges cut to minimize the gel volume. Subsequently, the slab was weighed and transferred to a 1.5ml EP tube.

(2) Add 300. mu.l Binding buffer II per 100mg gel, incubate in a 50-60 ℃ water bath for 10min, while gently shaking the EP tube until the gel melts.

(3) The collection tube was placed in a 2ml EP tube, the mixture obtained in the previous step was transferred to the collection tube, allowed to stand at room temperature for 2min, centrifuged at 12000rpm for 1min, and the waste liquid in the EP tube was discarded.

(4) Add 500. mu.l of Wash Solution to the collection tube, centrifuge at 12000rpm for 1min, discard the waste Solution from the EP tube.

(5) And (4) repeating the step.

(6) The collection tube was placed back into the EP tube and centrifuged at 12000rpm for 1min to remove residual Wash Solution.

(7) The collection tube was placed in a new 1.5ml EP tube, 30-40. mu.l of sterile ultrapure water was added to the filter membrane, incubated in a water bath at 50 ℃ for 10min, and centrifuged at 12000rpm for 1min to obtain the target DNA solution.

Linkage of promoter to T-vector:

the intermediate vector is constructed in order to completely cleave the promoter sequence. Using pMD^TM19-T Vector Cloning Kit, ligation system as follows (10. mu.l):

TABLE 4 connection system

The above linked system was gently mixed and placed in a refrigerator at 4 ℃ overnight.

Preparation of competent cells of Escherichia coli:

(1) mu.l of glycerol tube cells were inoculated into a fresh 3ml LB tube and cultured overnight at 37 ℃.

(2) Inoculating the activated thallus into a fresh 100ml LB culture medium according to the proportion of 1% -5%, carrying out shake culture at the rotating speed of 150r at the temperature of 37 ℃ until the OD value is 0.3-0.6, then placing on ice for at least 30min, and stopping the rapid growth of bacteria, wherein the step is the key for successful competence preparation and cannot be omitted.

(3) The bacterial solution was transferred to a 50ml sterile EP tube, centrifuged at 5000rpm using a 4 ℃ low temperature centrifuge for 10min, and the supernatant was discarded.

(4) 5ml of precooled competent buffer was added, and the cells were gently resuspended with a pipette tip and ice-cooled for 10 min.

(5) Centrifuge at 5000rpm using 4 deg.C low temperature centrifuge for 10min, and discard the supernatant. 1ml of a precooled competent buffer was added, the cells were gently resuspended with a pipette tip, and then divided into sterile EP tubes at a volume of 100. mu.l per tube and stored frozen in a freezer at-80 ℃ for future use.

(6) And (3) detection: 10. mu.l of the liquid of the allelochemicals was inoculated into 3ml of fresh LB medium containing AMP and cultured overnight at 37 ℃. If no thallus grows in the culture medium on the next day, the culture medium is competent and free of infected bacteria, and can be normally used. If the medium becomes turbid, the batch should be discarded and reconstituted.

Conversion (heat shock method):

(1) the flask containing 100ml of LB agar medium was placed in a microwave oven and heated to completely melt, cooled to about 60 ℃ at room temperature, and 100. mu.l of ampicillin (100mg/ml) was added to the medium. The flask was gently shaken to take care of absence of air bubbles, and then poured into a sterilized petri dish to prepare an ampicillin-resistant plate.

(2) The ice box was filled with ice and placed next to a freezer at-80 ℃. The preserved competent cells were removed from the refrigerator and rapidly thawed using the temperature of the palm. Immediately upon the competence just melted, it was inserted into ice.

(3) And adding 10 mu l of the reaction system after the connection reaction of the promoter and the T vector into 100 mu l of competent cells, flicking an EP tube, uniformly mixing, and carrying out ice bath for 30 min.

(4) The mixture after ice-bath is heated and shocked for 90s in a water bath kettle at 42 ℃, the temperature and the time of the step are very critical, and the EP tube cannot be shaken when the temperature and the time of the step are concerned.

(5) After the heat shock, the EP tube was quickly transferred to ice and ice-cooled for 5 min.

(6) To the EP tube, 800. mu.l of fresh LB medium without antibiotics was added, and the culture was resumed at 37 ℃ for 1 hour at 150 r. Subsequently, centrifugation was carried out at 3000r, part of the supernatant was discarded and mixed well, and 150. mu.l of the mixture was applied to LB plates containing the corresponding antibiotics. Culturing at 37 deg.C for 1 hr, and culturing under inverted condition overnight after the bacterial solution is completely absorbed by the culture medium.

Identification of positive clones:

add 10. mu.l of sterile ultrapure water to the EP tube and pick up the monoclonal on the plate and mix well in water. 1 μ l was used as PCR template for colony PCR, and the remaining 9 μ l was used for inoculation (Table 5).

Table 5 PCR reaction procedure:

carrying out electrophoresis on the PCR product obtained by amplification to obtain a target strip, carrying out sequencing, carrying out sequence comparison on a sequencing result by using Bioedit software, and screening out correct positive clones so as to prepare a successful bioluminescence report system, wherein the successful bioluminescence report system consists of a pAmilux-clpX-luxABCDE plasmid, a pAmilux-dnaJ-luxABCDE plasmid, a pAmilux-dnaK-luxABCDE plasmid and a pAmilux-groE-luxABCDE plasmid; the nucleotide sequence of the pAmilux-clpX-luxABCDE plasmid is shown as SEQ ID NO: 1, the nucleotide sequence of the pAmilux-dnaJ-luxABCDE plasmid is shown as SEQ ID NO: 2, the nucleotide sequence of the pAmilux-dnaK-luxABCDE plasmid is shown as SEQ ID NO: 3, the nucleotide sequence of the pAmilux-groE-luxABCDE plasmid is shown as SEQ ID NO: 4, respectively.

Table 6 construction of the T plasmid

Step 4. construction of luminescent reporting System

Enzyme digestion: plasmids pAmilux, PM1, PM2, PM3 and PM4 were digested with BamH I. The reaction system is as follows (table 7):

TABLE 7 enzyme digestion System

The mixture is incubated at 37 deg.C for 1h, and then at 80 deg.C for 20min to inactivate the endonuclease, which can not be omitted to avoid the occurrence of star activity. It is recommended to use a PCR instrument for temperature control.

And (3) dephosphorizing the carrier: after the plasmid pAmilux is subjected to single enzyme digestion, the 5' phosphate group is removed, so that the self-ligation and cyclization of plasmid DNA can be inhibited. In an in vitro ligation reaction, the formation of phosphodiester bonds is catalyzed by DNA ligase only when one nucleotide contains a 5 '-phosphate group and the other bears a 3' -hydroxyl terminus. Therefore, treatment of plasmid DNA with alkaline phosphatase to remove the phosphate group at the 5' -end can effectively prevent the occurrence of self-cyclization. The reaction system is as follows (table 8):

TABLE 8 Carrier dephosphorylation systems

The alkaline phosphatase was inactivated by reaction at 37 ℃ for 1 hour, followed by heating at 75 ℃ for 5 min. The promoter fragment does not need to be dephosphorylated, and the dephosphorylation time of the vector is not too long so as to avoid reducing the connection efficiency.

DNA purification: reference kit purification procedure.

Connecting: the digested and purified promoter fragment and plasmid pAmilux were ligated with T4 ligase in the following system (Table 9):

TABLE 9 connection System

The ligation system was mixed well and placed in a refrigerator at 4 ℃ overnight for ligation.

Transformation and screening of positive clones: because the staphylococcus aureus has thick cell wall and the plasmid is difficult to transform, the ligation product is transformed into E.coli DH5 alpha by a heat shock method, which is convenient for screening positive clones. Primers (LuxF, LuxR) were then designed from both sides of the BamHI site to allow verification of the direction of insertion of the fragment.

Preparation of electrotransformation competence of staphylococcus aureus:

the staphylococcus aureus competence is prepared at normal temperature in the whole process.

(1) Overnight grown S.aureus RN4220 was inoculated into fresh 100ml NYE liquid medium at a ratio of 2% and cultured with shaking at 37 ℃ and 150r to OD 0.2-0.3.

(2) The cells were collected in a 50ml sterile EP tube and centrifuged at 5000rpm for 10 min.

(3) The supernatant was discarded, 25ml of sterile ultrapure water was added thereto, the cells were resuspended by gentle pipetting with a pipette tip, and centrifuged at 5000rpm for ten minutes.

(4) The supernatant was discarded, 10ml of sterile ultrapure water was added, the cells were resuspended, and centrifuged at 5000rpm for 10 min.

(5) The supernatant was discarded, 5ml of 10% glycerol was added, the cells were resuspended, and centrifuged at 5000rpm for 10 min.

(6) And (5) repeating the step.

(7) 1ml of 10% glycerol was added to the cells, resuspended, and dispensed into sterile EP tubes at a volume of 70. mu.l per tube, and stored at-80 ℃ for further use.

In the process of making electrotransformation competence, the cells must be washed thoroughly with deionized water to remove the salt ions contained in the culture medium and prevent the cup from bursting upon electric shock.

And (3) electrically converting staphylococcus aureus:

(1) erlenmeyer flasks containing 100ml NYE agar medium were placed in a microwave oven and heated to complete melting, cooled to about 60 ℃ at room temperature, and 100. mu.l chloramphenicol (10mg/ml) was added to the medium. The flask was gently shaken to take care of absence of air bubbles and then poured into sterilized petri dishes to make chloramphenicol resistant plates.

(2) The appropriate amount of frozen competent cells were taken out of the refrigerator at-80 ℃ and placed at room temperature, when they had just thawed, 10. mu.l of pAmilux-clpX-luxABCDE plasmid, or pAmilux-groE-luxABCDE plasmid, or pAmilux-dnaJ-luxABCDE plasmid, or pAmilux-dnaK-luxABCDE plasmid was added thereto, and the tube walls of the flick EP were mixed and incubated at room temperature for 30 min. It is reported in literature that the incubation time is preferably 30min, and that exceeding or falling short of 30min will reduce the transformation efficiency.

(3) The mixture is transferred to a 2mm electric rotor, preferably by inserting a pipette tip along the wall of the electric rotor, applying the cells to one end of the well, and tapping the electric rotor on a flat table to shake out the air bubbles contained in the liquid. Then, covering the electric rotating cup with a cover, loading the electric rotating cup into an electric rotating instrument, and selecting a preset program: StA, press Pulse button, hear the beep and quickly take out the electric rotor, add 800. mu.l of fresh NYE culture medium to the electric rotor within 30 s.

Ions contained in the mixed solution of competent cells and DNA increase the conductivity of the solution, resulting in the generation of electric arcs, at which time the electric rotor bursts, greatly reducing the efficiency of electric conversion. If this occurs, it is necessary to consider whether the competent cells are not completely washed during preparation, or whether the solution for dissolving plasmid DNA contains some ions, and the examination and solution should be performed one by one.

(4) The liquid was transferred to a sterile EP tube and cultured at 37 ℃ for 1.5h at 100r recovery. After electric shock, the bacterial thallus is very fragile and cannot be shaken violently.

(5) Centrifuging the recovered bacteria liquid at 3000rpm for 5min, discarding part of supernatant, and coating the residual supernatant with the thalli after resuspending the residual supernatant with 450 ul of thalli, wherein each plate is coated with 150 ul of thalli. Culturing at 37 deg.C for 1 hr, and culturing for 24-48 hr after the bacteria solution is absorbed to obtain visible colony. It is not advisable to apply the entire centrifugally concentrated bacterial suspension to a single plate, since electrotransformation would produce a large number of dead cells, which attached to the surface of the medium could inhibit the growth of the otherwise rare transformants.

Four staphylococcus aureus lux reporter strains obtained by respectively transforming the four plasmids pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, pAmilux-dnaK-luxABCDE plasmid and pAmilux-groE-luxABCDE plasmid into staphylococcus aureus are named as follows: reporter strain S1, reporter strain S2, reporter strain S3 and reporter strain S4.

Example 2 plotting of characteristic luminescence curves

Experimental strains: the strains used in the experiment were four staphylococcus aureus lux reporter strains: s1, S2, S3 and S4.

Culture medium for experiment and its preparation

NYE agar Medium

2.0.7% agar: 0.7g of agar is weighed by balance and added into a conical flask, then 100ml of deionized water is added, and the mixture is sterilized by high-pressure steam for 20min at the temperature of 121 ℃ for standby.

Antibiotic for experiment and its preparation

1. Daptomycin (DAP) solution: dissolving 30mg of daptomycin in 1ml of ultrapure water to ensure that the final concentration is 30mg/ml, and filtering and sterilizing by using a 0.22-micron filter membrane;

2. polymyxin b (pmb) solution: dissolving 100mg of polymyxin B in 1ml of ultrapure water to make the final concentration of the polymyxin B100 mg/ml, and filtering and sterilizing by using a 0.22-micron filter membrane;

3. imipenem (IPM) solution: dissolving imipenem 10mg in ultrapure water 1ml to make its final concentration 10mg/ml, filtering with 0.22 μm filter membrane for sterilization;

4. penicillin g (pen) solution: dissolving 10mg of penicillin G in 1ml of ultrapure water to a final concentration of 10mg/ml, and filtering and sterilizing by using a 0.22-micron filter membrane;

5. vancomycin (VAN) solution: dissolving 30mg of vancomycin in 1ml of ultrapure water to make the final concentration of the vancomycin be 30mg/ml, and filtering and sterilizing the vancomycin by using a 0.22-micron filter membrane;

6. erythromycin (ERY) solution: dissolving 15mg of erythromycin in 1ml of absolute ethanol to obtain a final concentration of 30mg/ml, and filtering with 0.22 μm filter membrane for sterilization;

7. kanamycin (KAN) solution: dissolving 30mg kanamycin in 1ml ultrapure water to make the final concentration 30mg/ml, and filtering and sterilizing by using a 0.22-micron filter membrane;

8. spectinomycin (SPE) solution: dissolving 100mg of spectinomycin in 1ml of ultrapure water to make the final concentration of the spectinomycin be 100mg/ml, and filtering and sterilizing by using a 0.22-micron filter membrane;

9. streptomycin (STR) solution: dissolving 10mg streptomycin in 1ml ultrapure water to make the final concentration 10mg/ml, filtering with 0.22 μm filter membrane for sterilization;

10. tetracycline (TET) solution: dissolving 30mg of tetracycline in 1ml of ultrapure water to a final concentration of 30mg/ml, and filtering with a 0.22 μm filter membrane for sterilization;

11. mitomycin c (mmc) solution: dissolving 5mg of mitomycin C in 1ml of ultrapure water to make the final concentration of the mitomycin C be 5mg/ml, and filtering and sterilizing by using a 0.22-micron filter membrane;

12. ciprofloxacin (CIP) solution: dissolving 5mg of ciprofloxacin in 1ml of ultrapure water to ensure that the final concentration is 5mg/ml, and filtering and sterilizing by using a 0.22-micron filter membrane;

13. gatifloxacin (GAT) solution: dissolving 5mg of gatifloxacin in 1ml of ultrapure water to a final concentration of 5mg/ml, and filtering and sterilizing with a 0.22-micron filter membrane;

14. trimethoprim (TMP) solution: taking 5mg trimethoprim in 500ml ultrapure water, slowly dropwise adding dilute hydrochloric acid until the trimethoprim is completely dissolved, using the ultrapure water to fix the volume to 1ml, leading the final concentration to be 5mg/ml, and filtering and sterilizing by using a 0.22 mu m filter membrane;

15. rifampicin (RIF) solution: dissolving 10mg rifampicin in 1ml DMSO to make its final concentration 10mg/ml, filtering with 0.22 μm filter membrane for sterilization;

16. phleomycin (PHL) solution: 5mg of phleomycin is dissolved in 1ml of ultrapure water to a final concentration of 5mg/ml, and the solution is subjected to filtration sterilization by a 0.22 μm filter membrane.

Instruments required for the experiment: an ultra-clean workbench (Jiangsu purification laboratory plant), an electric heating constant temperature incubator (Shanghai sperm macro laboratory equipment Co., Ltd.), and a Lumazone PyLoN 2048B plant living body imaging system (Boyi Wei apparatus Co., Ltd.).

Experimental procedure

Step 1, plate preparation

1. The reporter strain S1, the reporter strain S2, the reporter strain S3 and the reporter strain S4 are streaked on an NYE plate and cultured overnight at 37 ℃, and then the plate is wrapped by a sealing film and put into a refrigerator at 4 ℃ for standby.

2. Taking Xinhua No. 1 qualitative filter paper, beating into round small paper sheets with the diameter of 6mm by using a puncher, placing the round small paper sheets in a glass culture dish, wrapping the round small paper sheets by using kraft paper, sterilizing at 121 ℃ for 20min, and drying in an oven for later use.

3. Heating NYE agar culture medium with microwave oven until agar melts completely, cooling at room temperature to about 60 deg.C, and pouring out flat plate to obtain uniform plate thickness.

4. Single colonies of reporter strain S1, or reporter strain S2, or reporter strain S3, or reporter strain S4 were picked up and dissolved in 200 μ l of sterile ultrapure water, and then mixed as 1:1000, adding the bacterial suspension into 0.7 percent agar, uniformly mixing, pouring the mixture on the upper layer of an NYE plate, and correspondingly preparing 16 double-layer plates containing a strain S0, a reporter strain S1, a reporter strain S2, a reporter strain S3 and a reporter strain S4.

5. After the double-layer flat plate is solidified, the tweezers are fired on the flame, and the filter paper piece is clamped and lightly stuck to the surface of the culture medium after the tweezers are cooled. To bring the disc into close proximity with the medium, the disc was gently pressed with forceps. In order to be able to observe the result accurately, the position of the paper sheets on the surface of the culture medium should be distributed orderly.

6. During the cooling of the culture medium, condensation water drops may appear on the surface. In order to prevent the condensed water from dispersing the medicine to influence the shape of the bacteriostatic circle, the cover of the culture medium can be opened before the paper sheets are added with the medicine, and the excess water is evaporated by utilizing the sterile air in the super clean bench.

7. In the prepared double-layer plates (16 in total) containing the strain S1, 1. mu.l of the antibiotic solution was dropped onto a filter paper sheet placed in advance in each plate using a pipette gun, and the plates were placed upside down in an incubator and incubated at 37 ℃ for 20 hours. Wherein the antibiotic is CIP, DAP, ERY, GAT, IPM, KAN, MMC, PEN, PHL, PMB, RIF, SPE, STR, TET, TMP, or VAN, and is added dropwise to each plate in an amount of 1. mu.l.

8. In the prepared double-layer plates (16 in total) containing the strain S2, 1. mu.l of the antibiotic solution was dropped onto a filter paper sheet placed in advance in each plate using a pipette gun, and the plates were placed upside down in an incubator and incubated at 37 ℃ for 20 hours. Wherein the antibiotic is CIP, DAP, ERY, GAT, IPM, KAN, MMC, PEN, PHL, PMB, RIF, SPE, STR, TET, TMP, or VAN, and is added dropwise to each plate in an amount of 1. mu.l.

9. In the prepared double-layer plates (16 in total) containing the strain S3, 1. mu.l of the antibiotic solution was dropped onto a filter paper sheet placed in advance in each plate using a pipette gun, and the plates were placed upside down in an incubator and incubated at 37 ℃ for 20 hours. Wherein the antibiotic is CIP, DAP, ERY, GAT, IPM, KAN, MMC, PEN, PHL, PMB, RIF, SPE, STR, TET, TMP, or VAN, and is added dropwise to each plate in an amount of 1. mu.l.

10. In the prepared double-layer plates (16 in total) containing the strain S4, 1. mu.l of the antibiotic solution was dropped onto a filter paper sheet placed in advance in each plate using a pipette gun, and the plates were placed upside down in an incubator and incubated at 37 ℃ for 20 hours. Wherein the antibiotic is CIP, DAP, ERY, GAT, IPM, KAN, MMC, PEN, PHL, PMB, RIF, SPE, STR, TET, TMP, or VAN, and is added dropwise to each plate in an amount of 1. mu.l.

Step 2, flat panel imaging

The Lumazone imaging system consists of three parts, namely a CCD camera, a dark box and a light source. The specific operation steps are as follows:

1. and turning on a power switch of the CCD camera, a switch of the camera bellows and a power switch, and turning on a computer and software.

2. And opening a liquid nitrogen switch, starting to fill liquid nitrogen into the CCD camera at the moment, and reducing the temperature to-110 ℃ after about 2 hours.

3. Placing a culture dish needing imaging in a dark box, opening an Acquire option of software, selecting a bright field, setting exposure time to be 100ms and sampling frequency to be 1MHz, then clicking the Acquire to Acquire a bright field image, and storing the image in a TIF format.

4. When the bioluminescence image is acquired, the Acquire option of the software is turned on, Luc is selected, the exposure time is 10min, the sampling frequency is 50KHz, and then the Acquire is clicked to Acquire the image and the image is stored in a TIF format.

5. After the use, the analysis software is firstly closed, and then the power supply of the camera bellows and the power supply of the light source are closed. Note that: the power supply of the CCD camera cannot be immediately turned off, and the temperature of the CCD camera is turned off after the temperature is raised to the room temperature after waiting for 24 hours.

An example of the luminescent response of a luminescent reporter system to an antibiotic is shown in FIG. 1.

Step 3, drawing a characteristic luminescence curve

1. With the center of the plate as the origin (x)₀,y₀) The radius of the plate is divided equally into a specific number of portions (e.g., 300 or 1200 portions), and the resulting average distance in each circle is expressed as the average d of the maximum and minimum distances from the midpoint thereof to the origin.

2. Let A be the average luminous intensity of all points in a certain circle, the average luminous intensity is a function of d: a ═ f (d).

3. The set function is applied to draw a characteristic light-emitting curve.

The resulting characteristic luminescence curve was plotted as shown in fig. 2.

Example 3 Cluster analysis of characteristic luminescence curves

Experimental procedure

Step 1, clustering analysis

1. Each characteristic luminescence curve (portion from the edge of the zone of inhibition to the edge of the panel) was recorded as a vector (luminescence intensity characteristic vector) characterized by 300 luminescence intensities, each of which represents the average luminescence intensity in each circle (1/300 spanning from the edge of the zone of inhibition to the edge of the panel) in the luminescence picture of example 2.

2. The characteristic luminescence curves of the four reporter strains for each antibiotic were combined into a combined luminescence intensity characterization vector characterized by 1200 luminescence intensities.

3. Using the euclidean distance calculation method, the distances between the combined luminous intensity characterization vectors of the antibiotics are calculated, and a distance matrix of the antibiotics is constructed.

4. And (4) performing cluster analysis between the combined luminous intensity characteristic vectors of the antibiotics by using an hclust function in the R language and using a UPGMA algorithm.

5. A combined light intensity characterization vector was obtained for 16 antibiotics (3 replicates each): CIP, ciprofloxacin; DAP, daptomycin; ERY, erythromycin; GAT, gatifloxacin; IPM, imipenem; KAN, kanamycin; MMC, mitomycin C; PEN, penicillin; PHL, phleomycin; PMB, polymyxin B; RIF, rifampin; SPE, spectinomycin; STR, streptomycin; TET, tetracycline; TMP, trimethoprim; VAN, vancomycin.

6. And (3) carrying out cluster analysis on the combined luminous intensity characteristic vectors of the 16 antibiotics, and finding that the antibiotics with the same action mechanism are clustered together for different report strains. The clustering results are shown in fig. 3.

7. The cluster analysis results of antibiotics with the same mechanism of action were analyzed (Table 10), which shows that the sensitivity and specificity of antibiotic mechanism of action judgment by cluster analysis were both 0.89 or higher.

TABLE 10 sensitivity and specificity of antibiotic mechanism of action

Step 2, determination of antibiotic action mechanism

The same antibiotic solution was tested using four reporter strains S1, S2, S3, S4.

Agarose bi-plates containing four staphylococcus aureus reporter strains were prepared using the methods as described in examples 2 and 3, and sterilized filter paper sheets were arranged in order in the center of the agarose bi-plate or on the surface of the medium and closely attached to the medium; dripping a solution to be detected to contain antibiotics onto a filter paper sheet, continuously culturing and shooting a bacterial luminescence picture; and converting the luminescence picture into a luminescence curve, extracting luminescence curve characteristics, comparing the luminescence curve characteristics with known antibiotic characteristic luminescence curves, and carrying out hierarchical clustering, wherein if the luminescence curve of the detected antibiotic is clustered with the luminescence curve of the antibiotic with a known action mechanism, the action mechanism of the antibiotic to be detected is judged to be the same as that of the antibiotic with the known action mechanism.

Wherein, the filter paper sheet is a circular small paper sheet with the diameter of 6mm which is made by punching Xinhua No. 1 qualitative filter paper by a puncher. After the solution to be detected to contain the antibiotic is dripped on the filter paper, the flat plate is placed in an incubator upside down and cultured for 25h at 37 ℃.

The drawing method for converting the luminescence picture into the luminescence curve comprises the following steps:

1) with the center of the plate as the origin (x)₀,y₀) Equally dividing the radius of the flat plate into a specific number of parts such as 300 or 1200 parts, and recording the obtained average distance in each circular ring as the average value d of the maximum distance and the minimum distance from the middle point to the origin;

2) let A be the average luminous intensity of all points in a certain circle, the average luminous intensity is a function of d: a ═ f (d);

3) and drawing a luminous curve by using the set function.

The hierarchical clustering analysis method based on the characteristic luminescence curve comprises the following steps:

1) recording each characteristic luminous curve from the edge of the inhibition zone to the edge part of the flat plate as a vector represented by 300 luminous intensities, wherein each luminous intensity represents the average luminous intensity in each circular ring from the edge of the inhibition zone to the edge part of the flat plate, namely the circular ring from the edge of the inhibition zone to the edge part of the flat plate 1/300 in the luminous picture;

2) combining the characteristic luminescence curves of four staphylococcus aureus report strains in a bioluminescent system corresponding to each antibiotic into a combined luminescence intensity characteristic vector characterized by 1200 luminescence intensities;

3) calculating the distance between the combined luminous intensity characteristic vectors of the antibiotics by using an Euclidean distance calculation method, and constructing a distance matrix of the antibiotics;

4) and (3) performing cluster analysis on the combined luminous intensity characteristic vectors of the antibiotics by using an hclust function in the R language and using a UPGMA algorithm, and judging the type of the action mechanism of the antibiotics according to whether the to-be-detected antibiotics are clustered with the antibiotics with known action mechanisms.

Sequence listing

<110> Shandong university

<120> a method for determining antibiotic action mechanism using bioluminescence reporter 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

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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

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ttctgatttt ttcctgctcg tcgtaagacc acactaatgg atcgctcgaa aaaatcaaat 1080

catcaatttc tgagcttgct gtaatttctt gtttatcaac atatgaagtc atacctgttt 1140

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ggcgttcatt gactgtcgca atagttaaat gttcaaatga cggttcagta atatcaacat 1260

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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

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aatagatatg gtttgcgtct tatttttttg aacataaggc aatatttgct caatattatc 2400

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acttttcaat tgccaactta gccgcagtta tacactccgt ataatttccg acagcgtttt 3780

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aatcactaat aaatgcacgc gtctcttgtt tagctttatt actatcttcg ttatagttaa 3900

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gaggaatacc ttttttggcc gcccactcaa caatatgatg actggttgct gttacatatt 4080

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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> plasmid sequence of pAmilux-dnaJ-luxABCDE

<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> plasmid sequence of pAmilux-dnaK-luxABCDE

<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 method for determining the action mechanism of antibiotics by using a bioluminescent report system comprises the following steps:

(1) preparing a staphylococcus aureus lux reporter strain by using a bioluminescence reporter system; wherein the luminescence reporting system consists of pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, pAmilux-dnaK-luxABCDE plasmid and pAmilux-groE-luxABCDE plasmid; the nucleotide sequence of the pAmilux-clpX-luxABCDE plasmid is shown as SEQ ID NO: 1, the nucleotide sequence of the pAmilux-dnaJ-luxABCDE plasmid is shown as SEQ ID NO: 2, the nucleotide sequence of the pAmilux-dnaK-luxABCDE plasmid is shown as SEQ ID NO: 3, the nucleotide sequence of the pAmilux-groE-luxABCDE plasmid is shown as SEQ ID NO: 4 is shown in the specification; the staphylococcus aureus lux report strain is prepared by respectively transforming four plasmids, namely pAmilux-clpX-luxABCDE plasmid, pAmilux-dnaJ-luxABCDE plasmid, pAmilux-dnaK-luxABCDE plasmid and pAmilux-groE-luxABCDE plasmid into staphylococcus aureus, and the four staphylococcus aureus lux report strains are respectively named as: reporter strain S1, reporter strain S2, reporter strain S3, reporter strain S4;

(2) preparing an agarose double-layer plate containing four staphylococcus aureus reporter strains, and orderly arranging sterilized filter paper sheets in the center of the agarose double-layer plate or on the surface of a culture medium and closely attaching the sterilized filter paper sheets to the culture medium;

(3) dripping a solution to be detected to contain antibiotics onto a filter paper sheet, continuously culturing and shooting a bacterial luminescence picture;

(4) and converting the luminescence picture into a luminescence curve, extracting luminescence curve characteristics, comparing the luminescence curve characteristics with known antibiotic characteristic luminescence curves, and carrying out hierarchical clustering, wherein if the luminescence curve of the detected antibiotic is clustered with the luminescence curve of the antibiotic with a known action mechanism, the action mechanism of the antibiotic to be detected is judged to be the same as that of the antibiotic with the known action mechanism.

2. The method of claim 1 for determining the mechanism of action of an antibiotic using a bioluminescent reporter system, wherein: and (3) the filter paper sheet in the step (2) is a round small paper sheet with the diameter of 6mm which is made of Xinhua No. 1 qualitative filter paper by a puncher.

3. The method of claim 1 for determining the mechanism of action of an antibiotic using a bioluminescent reporter system, wherein: and (3) after the solution to be detected containing the antibiotics is dripped onto the filter paper, inverting the flat plate in an incubator, and culturing for 20-25 h at 37 ℃.

4. The method for determining the action mechanism of antibiotics by using a bioluminescent reporting system as claimed in claim 1, wherein the step (4) of converting the luminescence picture into a luminescence curve or a known characteristic luminescence curve of antibiotics is performed by the following steps:

1) equally dividing the radius of the flat plate into specific parts of 300 or 1200 parts by taking the center of the flat plate as an origin (x0, y0), and recording the obtained average distance in each circular ring as the average value d of the maximum distance and the minimum distance from the midpoint of the circular ring to the origin;

2) let A be the average luminous intensity of all points in a certain circle, the average luminous intensity is a function of d: a ═ f (d);

3) and drawing a luminous curve by using the set function.

5. The method of determining the mechanism of action of an antibiotic using a bioluminescent reporter system of claim 1 or 4, wherein the known antibiotic is Daptomycin (DAP), polymyxin b (pmb), Imipenem (IPM), penicillin g (pen), Vancomycin (VAN), Erythromycin (ERY), Kanamycin (KAN), Spectinomycin (SPE), Streptomycin (STR), tetracycline (TET), mitomycin c (mmc), Ciprofloxacin (CIP), Gatifloxacin (GAT), Trimethoprim (TMP), Rifampin (RIF), Phleomycin (PHL).

6. The method for determining the mechanism of action of an antibiotic using a bioluminescent reporter system according to claim 1, wherein the hierarchical clustering analysis based on the characteristic luminescence curve in step (4) is performed by:

1) recording each characteristic luminous curve from the edge of the inhibition zone to the edge part of the flat plate as a vector represented by 300 luminous intensities, wherein each luminous intensity represents the average luminous intensity in each circular ring from the edge of the inhibition zone to the edge part of the flat plate, namely the circular ring from the edge of the inhibition zone to the edge part of the flat plate 1/300 in the luminous picture;

2) combining the characteristic luminescence curves of four staphylococcus aureus report strains in a bioluminescent system corresponding to each antibiotic into a combined luminescence intensity characteristic vector characterized by 1200 luminescence intensities;

3) calculating the distance between the combined luminous intensity characteristic vectors of the antibiotics by using an Euclidean distance calculation method, and constructing a distance matrix of the antibiotics;

4) and (3) performing cluster analysis on the combined luminous intensity characteristic vectors of the antibiotics by using an hclust function in the R language and using a UPGMA algorithm, and judging the type of the action mechanism of the antibiotics according to whether the to-be-detected antibiotics are clustered with the antibiotics with known action mechanisms.

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