CN110241019A - A kind of light-operated steady gene oscillatory system - Google Patents

Abstract

It include light-operated gene oscillator and regulation light source the invention discloses a kind of light-operated steady gene oscillatory system；The light-operated gene oscillator includes light-dependent control element and gene oscillating element；The regulation light source includes calculating regulation component and light source, calculates regulation component according to the oscillation observation of light-operated gene oscillator and issues adjustment signal in real time, adjusts the exposure intensity and irradiation time of light source；Light-operated gene oscillator adjusts its oscillation behavior according to the light source exposure intensity and irradiation time of adjusting.Present invention introduces external source illumination regulatory mechanisms to carry out accuracy controlling to gene oscillation behavior, keeps it more steady.The present invention helps to deepen the understanding to gene oscillation circuit, has certain theory directive significance to the steady large-scale synthetic biology system of the regulation and building vibrated in life system.In terms of practical application, steady light-operated gene oscillatory system can be integrated into cell, it is made periodically to express certain gene according to demand, save production cost, improve production efficiency.

Description

A kind of light-operated steady gene oscillatory system

Technical field

The present invention relates to gene regulation technology fields, more particularly to a kind of light-operated steady gene oscillatory system.

Background technique

Now with many types of gene oscillator and light-operated gene expression system, but as far as we know, utilization is light-operated System controls the research of gene oscillator not yet.

Due to the complexity inside biosystem, it is relatively difficult for directly researching biological oscillator, so in recent years, section Scholars construct gene oscillator, often through simpler Gene circuits are artificially designed to study life oscillation behavior. It is improved but if only starting with from biological elements itself, controllability is poor, is difficult to accomplish accurately to adjust.In addition, current people The quantification of these elements is understood deep not enough, cannot achieve flexible modulation.

Summary of the invention

A kind of light-operated steady gene oscillation is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place System.The present invention utilizes light genetic method, by integrating light science of heredity gene expression control module and applying light-operated increase The controllability and robustness of gene oscillator.

The present invention is demonstrated by taking two sets of light-operated steady gene oscillatory systems as an example.Firstly, in Escherichia coli respectively Construct two sets of group synchronous gene oscillator --- the light-operated gene oscillator of blue light and light-operated bases of feux rouges regulated and controled by illumination Because of oscillator.Meanwhile it also writing in a computer and can adjust in real time light according to the oscillation observation of light-operated gene oscillator The computer program of source exposure intensity and irradiation time, with " the synchronous gene oscillator analog program of group " (name are as follows: virtual Oscillator) for.Regulation of two kinds of light-operated gene oscillators (feux rouges and blue light) by virtual oscillator, constitutes two kinds of light Control steady oscillatory system.In the light-operated steady oscillatory system of every set, light-operated gene oscillator is carried out using virtual oscillator real When regulate and control, that is, make intensity of illumination and time that stable oscillation be presented by virtual oscillator, by the illumination tune of stable oscillation stationary vibration The expression for controlling related gene in light-operated gene oscillator, finally makes the oscillation behavior of gene oscillator more steady and can Control.

To achieve the above object, the technical scheme adopted by the invention is as follows:

A kind of light-operated steady gene oscillatory system, including light-operated gene oscillator and regulation light source；The light-operated gene vibration Swinging device is integrated in cell by light-dependent control element and gene oscillating element；The regulation light source includes to calculate regulation component And light source, wherein the calculating regulation component can issue in real time tune based on the oscillation observation of the light-operated gene oscillator Signal is controlled, the exposure intensity and irradiation time of the light source are adjusted；The light-operated gene oscillator is irradiated according to the light source of adjusting Intensity and irradiation time adjust its oscillation behavior.

Further, the light-dependent control element is light science of heredity gene expression control module.

Further, the light source is the light source that can cause the specific wavelength of light science of heredity molecules in response.

Further, calculating regulation component include the computer program with logical operation and logical process function, In Intelligent mobile equipment, embedded hardware system, digital circuit, digital optical path, analog operational circuit or simulation trial optical path Any one or combinations thereof.

Further, the light-operated gene oscillator includes but is not limited to the light-operated gene oscillator of blue light and the light-operated base of feux rouges Because of oscillator, regulation of the light-operated gene oscillator of blue light by blue light, the light-operated gene oscillator of feux rouges is by feux rouges Regulation, wherein the fluorescent reporter protein of the light-operated gene oscillator of blue light be mTagBFP2, the light-operated gene oscillator of feux rouges it is glimmering Light reporter protein is sfGFP.

Further, the light-dependent control element of the light-operated gene oscillator of the blue light is the plasmid as shown in sequence SEQ NO:1.

Further, the gene oscillating element of the light-operated gene oscillator of the blue light is the matter as shown in sequence SEQ NO:2 Grain.

Further, the light-dependent control element of the light-operated gene oscillator of the feux rouges is the plasmid as shown in sequence SEQ NO:3.

Further, the gene oscillating element of the light-operated gene oscillator of the feux rouges is the matter as shown in sequence SEQ NO:4 Grain.

Further, the light-dependent control element of the light-operated gene oscillator of the blue light includes EL222 gene and luxI promoter；Institute State -10 area downstreams that EL222 gene order is located at promoter P luxI；Or EL222 gene order replaces promoter P luxI - 35th area and -10th area intervening sequence, while being also inserted into EL222 gene order in the downstream in -10th area；Or EL222 gene sequence Column are positioned at -35th area of promoter P luxI and the intervening sequence in -10th area.

Beneficial effects of the present invention: the present invention accurately adjusts gene oscillation behavior by addition light source regulatory mechanism Control keeps it more steady and controllable.The present invention helps to deepen the understanding to gene oscillation circuit, to vibrating in life system Regulation and the steady large-scale synthetic biology system of building have certain theory directive significance.In terms of practical application, steadily and surely Light-operated gene oscillatory system can be integrated into cell, so that it is periodically expressed certain gene according to demand, save production Cost improves production efficiency.

Detailed description of the invention

Fig. 1 is that (wherein A is illumination to regulatory mechanism schematic diagram of the virtual oscillator to light-operated gene oscillator in embodiment 4 Intensity function；B is the mathematical model of virtual oscillator；C is virtual oscillator oscillation behavior ideograph).

Fig. 2 is that (wherein A is fluorescence vibration before regulating and controlling to the light-operated gene oscillator Robustness Analysis schematic diagram of blue light in embodiment 4 Swing phenomenon and each period of oscillation, amplitude variations analysis schematic diagram；B is fluorescence oscillatory occurences and each oscillation after regulation Amplitude, mechanical periodicity analyze schematic diagram).

Fig. 3 is that (wherein A is fluorescence vibration before regulating and controlling to the light-operated gene oscillator Robustness Analysis schematic diagram of feux rouges in embodiment 4 Swing phenomenon and each period of oscillation, amplitude variations analysis schematic diagram；B is fluorescence oscillatory occurences and each oscillation after regulation Amplitude, mechanical periodicity analyze schematic diagram).

Specific embodiment

In order to more concisely show technical solution of the present invention, objects and advantages, combined with specific embodiments below And its attached drawing is described in further detail the present invention.

The building of the light-operated gene oscillator of 1 blue light of embodiment

1. the building of blue light light-dependent control element

The building of the light-operated plasmid of 1.1 blue lights

(1) it by the EL222 genetic fragment of synthesis, is inserted into pTD103aiiA (Cm) plasmid, obtains glycerol stock and plasmid Dry powder (pTD103aiiA_EL222).

(2) bacterium activation and the preservation of bacterial strain

1. taking the glycerol stock containing pTD103aiiA_EL222 plasmid in carrying out plate on the LB solid culture ware of Cm resistance Scribing line activation (streaking inoculation), is incubated overnight.

2. the picking single bacterium colony on plate is inoculated in the LB liquid medium of 5mL, Cm resistance, and in constant-temperature table In, 37 DEG C, 180r/min is incubated overnight.

3. protecting bacterium: 30 μ l of switching stay overnight bacterium solution and are cultivated in the LB liquid medium of 3mL, Cm resistance to mid-log phase (greatly General 3-4h), in glycerol tube, the sterile glycerol of bacterium solution and 50% is uniformly mixed according to the ratio of 1:1, respectively at -20 DEG C With -80 DEG C of refrigerator middle or short terms and long-term preservation.

(3) plasmid extraction (small amount plasmid extraction kit)

1. being enriched with.2mL centrifuge tube is taken, 2mL is added thereto and contains the bacterium solution of pTD103aiiA_EL222 plasmid, 12000 × g is centrifuged 1min, after abandoning supernatant, 2mL bacterium solution is added and is centrifuged abandoning supernatant again, is repeated with this.

2. being resuspended.200 μ l Solution I are added into centrifuge tube, is placed in vortex instrument and mixes well to no fungus block.

3. cracking.200 μ l Solution II are added, it is soft to mix 5 times or so, so that solution becomes clear state.

4. neutralizing.200 μ l Solution III are added, gentle inversion mixes 5 times or so, occurs a large amount of white waddings at this time Shape precipitating.

5. centrifugation.Centrifuge tube is placed in a centrifuge, 12000 × g is centrifuged 8-10min, until white flock precipitate Sink to tube bottom or tube wall.

6. taking in adsorption column insertion collecting pipe, the supernatant (not be drawn onto white precipitate) other than white precipitate is drawn in suction In attached column, 12000 × g is centrifuged 1min.

7. using up filtrate, Wash the Solution A, 12000 × g that 500 μ l are added into adsorption column are centrifuged 1min.

8. using up filtrate, Wash the Solution B, 12000 × g that 700 μ l are added into adsorption column are centrifuged 1min, abandon filter It is primary to repeat the step for liquid.

9. using up filtrate, centrifuge tube is put back into centrifuge, 12000 × g sky gets rid of 5min.

10. taking a clean 1.5mL centrifuge tube, adsorption column is inserted, 50-100 μ l Elution is added Solution, after standing 1-2min, 12000 × g is centrifuged 1min, can be obtained plasmid.Finally surveyed with ultramicrospectrophotometer It measures the concentration of pTD103aiiA_EL222 plasmid and is recorded.

(4) digestion

After extracting pTD103aiiA_EL222 plasmid, digestion verification is carried out with I restriction endonuclease of Spe.It is expected that mesh Mark plasmid will form two linear fragments after I endonuclease digestion of Spe, and length is respectively 1005bp and 3581bp.According to table 1 Reaction system mixes after preparing digestion system, centrifugation, 37 DEG C of digestion 30min.

Table 1: endonuclease reaction system

(5) agarose gel electrophoresis is verified

1. taking wide type organic glass plastic plate, level is put into plastic tank, is inserted into comb.

2. take 3g agarose and 30mL concentration be 1 × TAE solution be added in 250mL conical flask, be placed in micro-wave oven plus Heat is melted to complete, and 1% separation gel is made.

3. the Golden View that 1 μ l is added is uniformly mixed when glue is cooled to 60 DEG C or so, after pouring into plastic plate, place It is cooling in horizontal table top room temperature, form the glue surface of even level.

4. carefully pulling up comb after being gelled admittedly, after the gel around plastic plate is cleaned out, being put into electrophoresis tank In, and pour into electrophoresis tank 1 × TAE solution to covering glue surface.

5. being injected in well, and injected into other well after loading buffer mixing is added in sample The Marker of 5000bp is as reference.

6. connecting electrophoresis apparatus and electrophoresis tank, 80V electrophoresis about 30-40min, the band (blue) to bromophenol blue goes to three points Two when close electrophoresis apparatus.

(6) after agarose gel electrophoresis identification is positive, plasmid is sent to sequencing company sequencing identification.

The building of 1.2 blue light light-dependent control element promoters

This experiment is constructed using Gibson Assembly method.

(1) primer is designed and synthesized, to realize between -10 area downstreams, -35th area and -10th area of P luxI and the two Region be inserted into EL222 simultaneously and target sequence.This experiment template plasmid used is pTD103aiiA_EL222.PCR reaction is drawn Object, amplified fragments, reaction system and response procedures are shown in Table 2-5 respectively.

Table 2:PCR reacts primer

Table 3:PCR amplified fragments

Table 4:PCR reaction system

Table 5:PCR response procedures

(2) agarose gel electrophoresis separates target DNA fragments

(3) target DNA fragments are recycled

1. cutting the Ago-Gel containing target DNA fragments in the UV lamp, it is placed in 2mL centrifuge tube.Calculated for gel Weight and using the weight as a gel volume.

2. the Buffer DE-A of 3 gel volumes is added, after mixing 75 DEG C of heating water baths, interruption mixing is until solidifying Blob of viscose melts completely.

3. the Buffer DE-B of 0.5 Buffer DE-A volume is added, mix.

4. drawing the mixed liquor of previous step, it is transferred to DNA and prepares in pipe, 12000 × g is centrifuged 1min, abandons filtrate.

5. will prepare pipe puts back into centrifuge tube, the Buffer W1,12000 × g that 500 μ l are added are centrifuged 30s, abandon filtrate.

6. will prepare pipe puts back into centrifuge tube, the Buffer W2,12000 × g that 700 μ l are added are centrifuged 30s, abandon filtrate.It weighs again It is multiple primary.

7. will prepare pipe to be placed in 2mL centrifuge tube, 12000 × g is centrifuged 1min.

8. being placed in pipe is prepared in clean 1.5mL centrifuge tube, film centre is prepared in DNA and adds 30 μ l Eluent, room temperature Stand 2min.12000 × g is centrifuged 1min, and eluted dna obtains DNA sample.

(4) recombining reaction

It is loaded (reaction system is shown in Table 6) by Gibson Assembly reaction system, 50 DEG C of warm bath 30min.

Table 6:Gibson Assembly reaction system

(5) it converts

1. the T1 competent cell that -80 DEG C save is placed in ice and melts 10min.

2. plasmid obtained in the previous step being added in the T1 competence of 100 μ l, being mixed gently in rear ice in super-clean bench Place 30min.

3. placing 2min in ice immediately after 42 DEG C of water-bath heat shock 90sec, pay attention to not shaking centrifugation acutely during this Pipe.

4. the LB culture medium of 37 DEG C of pre-temperatures of 500 μ l is added, suction is mixed.

5. recovery bacterium solution, 37 DEG C, 180r/min cultivates 45min.

6. taking 100 μ l bacterium solutions to be added in the LB solid medium tablets of Cm resistance, after being smeared uniformly with spreading rod, by plate It is inverted in 37 DEG C of constant incubators and is incubated overnight.

(6) recombinant receptor bacterium, preservation strain are selected

The promoter plasmid of this experimental construction is pTD103aiiA_EL222_10EB, pTD103aiiA_EL222_ 35EB10, pTD103a iiA_EL222_35EB10EB plasmid.

(7) digestion is examined, send survey

With Pst I endonuclease digestion, the length of three kinds of plasmids is about 4600bp, agarose gel electrophoresis detection after digestion After being positive, plasmid is sent to sequencing company sequencing identification.

The building of 1.3 blue light light controlling effects inspection plasmid

For the light controlling effects of three kinds of light-operated promoters of visual test, this experiment is needed the subsequent aiiA of light-operated promoter Gene replacement is sfGFP fluorescent reporter protein gene.

(1) upgrading grain (pTD103aiiA_EL222_10EB, pTD103aiiA_EL222_35EB10, pTD103a iiA_ EL222_35EB10EB、pTD103luxI_sfGFP)。

(2) design and synthesize primer, carry out PCR reaction, then by recombining reaction by pTD103aiiA_EL222_10EB, The aiiA element of tri- kinds of plasmids of pTD103aiiA_EL222_35EB10, pTD103aiiA_EL222_35EB10EB changes sfGFP into Element.PCR reaction primer and amplified fragments are shown in Table 7, table 8.

Table 7:PCR reacts primer

Table 8:PCR amplified fragments

(3) agarose gel electrophoresis separation target DNA fragments, recycling target DNA fragments

Step is same as above.

(4) recombining reaction

It is loaded by 9 reaction system of table, 50 DEG C of water-baths, 30min.

Table 9: recombining reaction system

(5) it converts:

It is converted using MG1655 competent cell, step is same as above.

(6) recombinant receptor bacterium, preservation strain are selected.

(7) after PCR, agarose gel electrophoresis tests positive, plasmid is sent to sequencing company sequencing identification.It builds Plasmid be pTD103aiiA_EL222_10EB_sfGFP, pTD103aiiA_EL222_35EB10_sfGFP, pTD103aiiA_EL222_35EB10EB_sfGFP。

The improvement of the synchronous gene oscillator of 1.4 groups

1.4.1 the acquisition of fluorescin

(1) acquisition of mTagBFP fluorescin

1. adding the sterile ddH2O of 10 μ l in 1 hole 19I iGEM 2015kit plate, solution after the dissolution of plasmid dry powder Become red.(element source: iGEM 2015kit plate 1 19I, plasmid backbone pSB1C3, resistance Cm.)

2. take 5 μ l plasmids to be transformed into T1 competence, shake bacterium culture, preservation strain it is spare.

3. plasmid or bacterium solution are sent to sequencing company sequencing identification, determine that sequence correctly carries out subsequent operation afterwards.

(2) acquisition of mTagBFP2

1. designing and synthesizing primer carries out PCR reaction, to change mTagBFP sequence as mTagBFP2 (the of mTagBFP One, two amino acid are changed to six amino acid of MVSKGE, and the 174th amino acid I is changed to A).The template plasmid of PCR is pSB1C3-mTagBFP.PCR reaction primer and amplified fragments are shown in Table 10-11.

Table 10:PCR reacts primer

Table 11:PCR amplified fragments

2. agarose gel electrophoresis separates target DNA fragments

3. recycling target DNA fragments

4. recombining reaction

It is loaded according to 12 reaction system of table, 50 DEG C of water-baths, 30min.

Table 12: recombining reaction system

5. converting

It is converted using T1 competent cell, step is same as above.

6. selecting recombinant receptor bacterium, preservation strain

7. after PCR, agarose gel electrophoresis tests positive, plasmid is sent to sequencing company sequencing identification.

1.4.2 fluorescin performance compares

In order to compare the albumen brightness of mTagBFP fluorescin, mTagBFP2 fluorescin, more suitable fluorescence is chosen Promoter before fluorescence protein gene is changed to pBAD, enables protein expression by arabinose concentrations by albumen, this experiment Control.Meanwhile this experiment is using sfGFP fluorescin as control.

(1) fluorescin performance detection plasmid construction

It first passes through after PCR amplification obtains junction fragment, then agarose gel electrophoresis separation target DNA fragments, recycling and carries out Then Gibson Assembly reaction, the operation such as is converted, applies plate, experimental procedure is substantially same as above, and finally obtains pBAD_ SfGFP, pBAD_mTagBFP, pBAD_mTagBFP2 plasmid.PCR reaction primer and amplified fragments are shown in Table 13-14.

Table 13:PCR reacts primer

Table 14:PCR amplified fragments

(2) fluorescin performance compares

1. being added to three kinds of performance detection thallus in the LB liquid medium containing 1% arabinose solution, culture is extremely Logarithmic phase.

2. carrying out the observation of E. coli population fluorescence microscopy, compare the brightness case of three kinds of fluorescins.

2. the building of gene oscillating element

The replacement of the synchronous gene oscillating element fluorescin of 2.1 groups

Using mTagBFP2 as synchronous gene oscillating element plasmid (pTD103luxI_mTagBFP2) structure of the group of reporter protein It builds and needs three steps, need to first construct pTD_luxR_mTagBFP2 plasmid, then pTD 103luxI_ is constructed by insertion luxI segment Fast degradation label is finally added in mTagBFP2V plasmid behind the area CDS of mTagBFP2 element.

Experimental method is Gibson Assembly, and experimental procedure is substantially same as above.Drawn after the completion of plasmid construction with P1, m2-PV After object amplification and EcoR I endonuclease digestion (digestion post-fragment length is 1864bp, 3675bp), agarose gel electrophoresis is carried out It examines.Pcr amplified fragment and reaction primer are shown in Table 15-16.

Table 15:PCR amplified fragments

Table 16:PCR reacts primer

3. the integration of the light-operated gene oscillator of blue light

PTD103luxI_mTagBFP2 plasmid and pTD103aiiA_EL222_35EB10 (Cm) plasmid cotransformation are arrived In MG1655 bacterial strain, the light-operated gene oscillator of blue light is formed.

(1) culture of plasmid donor bacterium, upgrading grain

Bacterial strain containing pTD103luxI_mTagBFP2, pTD103aiiA_EL222_35EB10 (Cm) plasmid is shaken overnight Upgrading grain after bacterium.

(2) it converts

By above-mentioned two plasmids cotransformation into MG1655 competent cell.

(3) target bacterium colony, preservation strain are selected

Experimental procedure is same as above.

(4) digestion, sequencing are examined

1. digestion is examined: II digestion with restriction enzyme of Bgl is used, in pTD103luxI_mTagBFP2 and pTD All it is single endonuclease digestion site in 103aiiA_EL222_35EB10 plasmid, the linear piece of 5539bp and 4589bp is respectively formed after digestion Section.

2. sequencing is examined

Sequencing reaction primer sequence is shown in Table 17.

Table 17: sequencing reaction primer

The building of the light-operated gene oscillator of 2 feux rouges of embodiment

1. the building of feux rouges light-dependent control element

The building of 1.1pTD103aiiA_ho1_pcyA_aiiA plasmid

By on different plasmids light-dependent control element and oscillator element integrate, be mainly carried out in two steps.

1.1.1pEO100c_aiiA the building of plasmid

(1) bacterial strain of pEO100c plasmid and pTD103aiiA (Cm) plasmid the culture of plasmid donor bacterium, upgrading grain: will be contained Upgrading grain after bacterium is shaken overnight.

(2) design primer carries out PCR amplification, Insert Fragment and carrier segments is obtained, to carry out subsequent Gibson Assembly reaction.Pcr amplified fragment and reaction primer are shown in Table 18-19.

Table 18:PCR amplified fragments

Table 19:PCR reacts primer

(3) agarose gel electrophoresis separates target DNA fragments and is recycled

Experimental procedure is same as above.

(4)Gibson Assembly

It is loaded according to 20 reaction system of table, 50 DEG C of warm bath 30min.

Table 20:Gibson Assembly reaction system

(5) it converts

The obtained plasmid of Gibson Assembly is transferred in T1 competence.

(6) recombinant receptor bacterium, preservation strain are selected

(7) digestion, sequencing identification

The linear fragment that length is 5372bp will be obtained after pEO100c_aiiA plasmid I digestion with restriction enzyme of Pst, After agarose gel electrophoresis tests positive, plasmid is sent to sequencing company sequencing identification.

1.1.2 the building of target plasmid

Above-mentioned pEO100c_aiiA plasmid is integrated with pTD103aiiA_ho1_pcyA plasmid.

(1) Insert Fragment PCR

Template DNA is pEO100c_aiiA, forms PompC+aiiA segment after PCR, length 2193bp, PCR used is anti- Primer is answered to be shown in Table 21.

Table 21:PCR reacts primer

(2) carrier segments digestion

Digestion vector plasmid is pTD103aiiA_ho1_pcyA, obtains length after I digestion with restriction enzyme of Spe and is The linear fragment of 5372bp.

Table 22: endonuclease reaction system

Into PCR pipe, adds the principle of small system again according to first increasing system, is separately added into reaction system shown in table 24, Bubble removing, low-speed centrifugal are flicked after mixing.In 37 DEG C of warm bath 1h digestions.

(3) agarose gel electrophoresis separation target DNA fragments, recycling target DNA fragments

After Insert Fragment and carrier segments to be carried out to agarose gel electrophoresis separation target DNA fragments respectively, target is recycled DNA segment.

(4) recombining reaction

Reaction system shown in table 25, low-speed centrifugal after mixing are added in PCR pipe.50 DEG C, warm bath 30min.

Table 23: recombining reaction system

(5) it converts, apply plate

It is transformed into T1 competence, step is same as above.

(6) recombinant receptor bacterium, preservation strain are selected

(7) digestion is examined, send survey

Target plasmid is subjected to digestion with Pst1 restriction endonuclease, it is contemplated that plasmid is cut into two parts, and length is respectively 4625bp And 2918bp, after agarose gel electrophoresis tests positive, plasmid is sent to sequencing company sequencing identification.

The building of 2 gene oscillating elements

The building of pTD103luxI_sfGFP_Cph8 (Amp) plasmid:

Because the resistance of pTD103luxI_sfGFP_Cph8 (Kan) plasmid is identical as the resistance that host strain JT2 is carried, Plasmid loss is be easy to cause in incubation, so needing Kan gene being changed to Amp gene.

(1) using pEO100c_aiiA as template, using AMP-fwd, AMP-rev as primer, target fragment amplification is carried out, is Add I two restriction enzyme sites of Bgl II and Spe in Amp genetic fragment both ends.Target fragment length 1172bp, PCR reaction system and Response procedures are same as above, and reaction primer is shown in Table 24.

Table 24:PCR reacts primer

(2) PCR product purifying (EasyPure PCR Purification Kit)

1. taking the Amp fragment PCR product of 50 μ l, 250 μ l solution B B are added, centrifugal column is added after mixing, stands After 1min, 10000 × g is centrifuged 1min, discards efflux.

2. 650 μ l solution W B, 10000 × g centrifugation 1min are added, efflux is discarded.

3. 10000 × g is centrifuged 2min, remaining WB is completely removed.

4. centrifugal column is placed in clean centrifuge tube, 30 μ l solution E B are added in the center of column.1min is stored at room temperature, 10000 × g is centrifuged 1min, and eluted dna obtains PCR purified product.

(3) endonuclease reaction

PCR purified product and pTD103luxI_sfGFP_Cph8 (Kan) plasmid of Amp genetic fragment is all subjected to Spe I With II double digestion of Bgl.Because the most suitable buffer of two enzyme work is different, first with after II digestion of Bgl, it is pure to carry out product Change, then with I digestion of Spe.

II digestion of Bgl: into PCR pipe, add the principle of small system again according to first increasing system, be separately added into anti-shown in table 25 System is answered, bubble removing, low-speed centrifugal are flicked after mixing.In 37 DEG C of warm bath 15min digestions.

Table 25: endonuclease reaction system

2. digestion products purifying (step is purified with PCR product)

I digestion of Spe: reaction system shown in table 26 is added in PCR pipe, in 37 DEG C of warm bath 1h digestions.

Table 26: endonuclease reaction system

(4) agarose gel electrophoresis separation target DNA fragments, recycling target DNA fragments

Step is same as above.Length is 1155bp, pTD103luxI_sfGFP_Cph8 (Kan) carrier after Amp genetic fragment digestion Length is 7129bp after segment digestion.

(5) enzyme even reaction (DNA Ligation Kit Ver.2.1)

It is loaded in centrifuge tube according to reaction system shown in table 27,16 DEG C of reaction 30min.

Table 27:DNA coupled reaction system

(6) it converts, apply plate

It is transformed into T1 competence, step is same as above.

(7) aimed strain, preservation strain are selected

(8) PCR, send survey examine

PCR reaction is carried out respectively by primer of AMP-fwd, AMP-rev and CPH8-fwd shown in table 26, CPH8-rev, Form Amp and CPH6 segment, it is contemplated that be respectively 1197,2235bp.Then agarose gel electrophoresis detection is carried out, detection is in sun Property after, by plasmid be sent to sequencing company sequencing identification.

The integration of the light-operated gene oscillator of 3 feux rouges

(1) by pTD103luxI_sfGFP_Cph8 (Amp) and pTD103aiiA_ho1_pcyA_aiiA plasmid cotransformation Into JT2 coli strain, experimental procedure is same as above.

(2) target bacterium colony, preservation strain are selected

(3) digestion, agarose gel electrophoresis verifying

In PCR pipe, it is loaded according to system shown in table 30.37 DEG C, warm bath 1h.

After I digestion of Pst, pTD103LuxI_sfGFP_Cph8 (Amp) plasmid has 3 restriction enzyme sites, pTD103 aiiA_ Ho1_pcyA_aiiA plasmid has 2 restriction enzyme sites, and 5 segments are obtained with I digestion of Pst in two plasmids, and length is respectively as follows: 1867bp、2379bp、2920bp、3948bp、4609bp。

Table 28: endonuclease reaction system

(4) agarose gel electrophoresis tests positive then shows that target plasmid has been transferred in JT2.

3 Escherichia coli Fluirescence observation of embodiment

The observation of 1 Escherichia coli smear

The production of 1.1 Escherichia coli smears

(1) the MG1655 Escherichia coli containing pBAD_sfGFP, pBAD_mTagBFP, pBAD_mTagBFP2 plasmid are taken, Respectively at cultivating 3-4h in the LB culture solution containing arabinose solution, Cm antibiotic, until logarithmic phase mid-term.(above-mentioned culture is The selection of oscillatory system reporter protein is tested, and carries out group's fluorescent brightness detection；The comparative experiments of blue light light control system effect is then PTD103aiiA_EL222_10EB_sfGFP, pTD103aiiA_EL222_35EB10_sfGFP, pTD103aiiA_ will be contained The MG1655 Escherichia coli of EL222_35EB10EB_sfGFP plasmid are cultivated respectively in 3-oxo-C6-HSL solution, carry out single Cell fluorescence brightness detection.)

(2) 1.5mL sterile centrifugation tube is taken, 1.5mL bacterium solution is added, removes supernatant after 12000 × g centrifugation.Utilize remnants A little supernatant, Escherichia coli are deposited in high speed vortex instrument and are vortexed, is allowed to form uniform thick bacterium solution, be used as The detection of E. coli population fluorescent brightness.(Escherichia coli single-cell fluorescence brightness detection: taking 0.9% physiological saline, according to E. coli broth is diluted by centesimal ratio.)

(3) it takes clean glass slide, draws the 5 above-mentioned bacterium solutions of μ l, after glass slide even spread, gently covered, It has been careful not to bubble.Finally, liquid extra around coverslip is cleaned out with blotting paper.

The observation of 1.2 fluorescence microscopies

(1) smear made is placed on microscope carrier, is pushed down with piece pressing clip, at this time sample face light passing Hole.

(2) from low power lens (10 ×), focusing rotates rough quasi-coil, slowly increase objective table, until object Mirror is close to smear, and eyes look at object lens in order to avoid object lens bump against smear at this time.Then binocular fixation eyepiece rotates backward thick quasi- burnt spiral shell Rotation, declines objective table, until observing image, is rotated further by thin quasi- burnt spiral and slightly adjusts, be more clear the image seen.

(3) after finding corresponding observation point, low power objective is converted to high power objective (40 ×), then fine rotation is thin quasi- burnt Spiral can see clearly image.

(4) microscope is transformed into fluorescence observation mode, selects corresponding exciting light, carry out fluorescence observation.

The observation of 1.3 micro-fluidic chips

1.3.1 the culture and concentration of bacterium solution

(1) 30 μ l bacterium solutions after taking recovery add to 3mL and contain in the LB culture solution of corresponding antibiotic, are placed in constant-temperature table, 37 DEG C culture 3-4 hours, to reach logarithmic phase mid-term.

(2) centrifuge tube of 1.5mL is taken, 1.5mL bacterium solution is added, 2000r/min is centrifuged 5min, removes supernatant, leaves behind Remaining a little culture solution, then be vortexed in vortex instrument and mix bacterial precipitation, form uniform thick bacterium solution.

1.3.2 loading

(1) prepare the capillary of three suitable lengths, and the materials such as itself and micro-fluidic chip are placed in super-clean bench, it is purple Outer sterilizing 20min.

(2) micro-fluidic chip is placed in vacuum (-tight) housing and is vacuumized, need 10min or so.

(3) in super-clean bench, after drawing partial concentration bacterium solution with 10 μ l liquid-transfering guns, pipette tips are inserted into the liquid inlet of chip, Bacterium solution is injected since liquid inlet.

(4) syringe needle for choosing corresponding specification, is placed on syringe needle for capillary, the two is closely coupled.It is inhaled with syringe The culture solution containing corresponding antibiotic is taken, syringe is connected with syringe needle, presses piston handle, makes culture solution full of entire capillary Pipe, then the other end of capillary is inserted at the liquid inlet of chip.Meanwhile other two capillary is inserted into going out for chip At liquid mouth.

1.3.3 upper mirror observation

Micro-fluidic chip is fixed on objective table, syringe is fixed on syringe pump, just starts injection, observation.Pay attention to observation It is preceding first to press piston handle, make culture solution quickly through avoiding bacterium raised growth in the channel, cause to rinse chip channel Blocking.

The virtual oscillator of embodiment 4 regulates and controls light-operated gene oscillator and realizes steady oscillation

The present invention has write the synchronous gene of group according to the mathematical model (as shown in Figure 1B) of the synchronous gene oscillator of group Oscillator analog program (that is, virtual oscillator).The cycle of oscillation of virtual oscillator and amplitude are respectively 50min and 201, Cycle of oscillation and amplitude are all relatively more steady, and oscillation mode is as shown in Figure 1 C.Virtual oscillator can be according to its gene oscillator Oscillation observation control microscopical irradiation radiant, to influence the expression of aiiA gene.When virtual shake number is less than When 100, the intensity of illumination that microscope issues is changed linearly；When shake number is more than 100, microscope is then with 100% maximum Intensity is irradiated (as shown in Figure 1A).Stable change in oscillation is presented in the intensity of illumination that this regulatory mechanism applies us, To make the expression of this element of aiiA in gene oscillator that stable mode be presented, finally make the oscillation of whole gene oscillator Behavior is more steady.

In a computer, virtual oscillator is an independently operated program, the amplitude virtually vibrated can be written one A file, for the reading of other programs.In observation, shooting process, by writing program in microscope software, read virtual Shake number makes microscope light source issue the irradiation light of respective intensities, and carries out fluorescence shooting according to the interval of 15min.

By the regulation of the above virtual oscillator, the oscillation result of light-operated gene oscillator is as follows:

1. the light-operated steady oscillatory system of blue light

The light-operated gene oscillator of blue light in micro-fluidic chip is carried out to continue microscopic observation, records mTagBFP2 fluorescence egg White fluorescent brightness variation.As shown in Figure 2 A, when not applying regulation, the oscillatory occurences of the light-operated gene oscillator of blue light is inadequate Stablize, period of oscillation and amplitude variations are larger (period=0.195, σ amplitude=0.048 σ)；As shown in Figure 2 B, apply virtual After oscillation regulation, the robustness of oscillator is greatly increased, and period and amplitude gradually become more stable (σ period=0.083, σ Amplitude=0.038).When just having carried out blue light regulation, the amplitude variations respectively vibrated are bigger, it may be possible to due to the hysteresis quality of regulation The synthetic quantity of reporter protein is caused biggish fluctuation occur.In conclusion in the light-operated steady oscillatory system of blue light, virtually Oscillator is effective to the regulating and controlling effect of the light-operated gene oscillator of blue light.(note: standard deviation sigma=sqrt (((x1-x) 2+ (x2- X) 2+...... (xn-x) 2)/n), wherein x1, x2......xn are each data value, and x is statistical average, and n is data volume.)

2. the light-operated steady oscillatory system of feux rouges

The same cell of micro-fluidic chip is carried out to continue microscopic observation, as shown in Figure 3A, when not applying any regulation, The oscillatory occurences of the light-operated gene oscillator of feux rouges is more unstable, each period of oscillation and amplitude variations it is larger (the σ period= 0.291, σ amplitude=0.068)；As shown in Figure 3B, after applying virtual oscillator regulation, the robustness of oscillator is greatly increased, Period and amplitude become stable (period=0.079, σ amplitude=0.013 σ).This result shows that, the regulation of virtual oscillator is made With enhancing the robustness of the light-operated gene oscillator of feux rouges.(note: standard deviation sigma=sqrt (((x₁-x)²+(x₂-x)²+...... (x_n-x)²)/n), wherein x₁、 x₂......x_nFor each data value, x is statistical average, and n is data volume.)

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art It says, without departing from the inventive concept of the premise, several replacements, modification and improvement can also be made, these belong to the present invention Protection scope.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

SEQUENCE LISTING

<110>Hua Zhong Agriculture University

<120>a kind of light-operated steady gene oscillatory system

<130> 5.6

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 4589

<212> DNA

<213>artificial synthesized

<400> 1

gcctaatatt tttgaaatat cccaagagct ttttccttcg catgcccacg ctaaacattc 60

tttttctctt ttggttaaat cgttgtttga tttattattt gctatattta tttttcgata 120

attatcaact agagaaggaa caattaatgg tatgttcata cacgcatgta aaaataaact 180

atctatatag ttgtcttttt ctgaatgtgc aaaactaagc attccgaagc cattgttagc 240

cgtatgaata gggaaactaa acccagtgat aagacctgat gttttcgctt ctttaattac 300

atttggagat tttttattta cagcattgtt ttcaaatata ttccaattaa ttggtgaatg 360

attggagtta gaataatcta ctataggatc atattttatt aaattagcgt catcataata 420

ttgcctccat tttttagggt aattatctag aattgaaata tcagatttaa ccatagaatg 480

aggataaatg atcgcgagta aataatattc acaatgtacc attttagtca tatcagataa 540

gcattgatta atatcattat tgcttctaca agctttaatt ttattaatta ttctgtatgt 600

gtcgtcggca tttatgtttt tcatacccat ctctttatcc ttacctattg tttgtcgcaa 660

gttttgcgtg ttatatatca ttaaaacggt aatggattga catttgattc taataaattg 720

gatttttgtc acactattgt atcgctggga atacaattac ttaacataag cacctgtagg 780

atcgtacagg tttacgcggt agcctttagt ccatgtatag tcgaatgaat tcattaaaga 840

ggagaaaggt accatgacag taaagaaact ttatttcatc ccagcaggtc gttgcatgtt 900

ggatcattcg tctgttaaca gtgcgttaac accggggaaa ctattaaact tgccggtgtg 960

gtgttatctt ttggagacgg aagaaggtcc tattttagta gacacaggta tgccagaaag 1020

tgcagttaat aatgaagggc tttttaacgg tacatttgtt gaaggacaga tcttaccgaa 1080

aatgactgaa gaagatagaa tcgtgaatat attaaagcgt gtagggtatg agccggacga 1140

ccttttatat attattagtt ctcacttaca ttttgatcat gcaggaggaa acggtgcttt 1200

tacaaataca ccaattattg tgcagcgaac ggaatatgag gcagcacttc atagagaaga 1260

atatatgaaa gaatgtatat taccgcattt gaactacaaa attattgaag gggattatga 1320

agtggtacca ggtgttcaat tattgtatac gccaggtcat tctccaggcc atcagtcgct 1380

attcattgag acggagcaat ccggttcagt tttattaacg attgatgcat cgtacacgaa 1440

agagaatttt gaagatgaag tgccgttcgc aggatttgat ccagaattag ctttatcttc 1500

aattaaacgt ttaaaagaag ttgtgaaaaa agagaaacca attattttct ttggtcatga 1560

tatagagcag gaaaagagtt gtagagtgtt cccggaatat atagcagcga acgacgaaaa 1620

ttacgccctt gcagcgtaaa cgcgtgctag aggcatcaaa taaaacgaaa ggctcagtcg 1680

aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca 1740

aatccgccgc cctagaccta gggatatatt ccgcttcctc gctcactgac tcgctacgct 1800

cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag 1860

atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag 1920

gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc 1980

gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt 2040

tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac 2100

gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc 2160

ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa 2220

gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg 2280

aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca 2340

agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc 2400

aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa 2460

gatcatctta ttaatcagat aaaatatttc tagatttcag tgcaatttat ctcttcaaat 2520

gtagcacctg aagtcagccc catacgatat aagttgttac tagtgcttgg attctcacca 2580

ataaaaaacg cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc 2640

attactggat ctatcaacag gagtccaagc gagctcgata tcaaattacg ccccgccctg 2700

ccactcatcg cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca 2760

gacggcatga tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata 2820

tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa 2880

actggtgaaa ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt 2940

agggaaatag gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa 3000

ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg 3060

gaaaacggtg taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc 3120

catacggaat tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata 3180

aaacttgtgc ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt 3240

ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca 3300

ttgggatata tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc 3360

tcctgaaaat ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg 3420

aaagttggaa cctcttacgt gccgatcaac gtctcatttt cgccagatat cgacgtctaa 3480

gaaaccatta ttatcatgac attaacctat aaaggccgca tctagagttt acggctagct 3540

cagtcctagg tatagtgcta gctactagtg aaagaggaga aatactagat gttggatatg 3600

ggacaagatc ggccgatcga tggaagtggg gcacccgggg cagacgacac acgcgttgag 3660

gtgcaaccgc cggcgcagtg ggtcctcgac ctgatcgagg ccagcccgat cgcatcggtc 3720

gtgtccgatc cgcgtctcgc cgacaatccg ctgatcgcca tcaaccaggc cttcaccgac 3780

ctgaccggct attccgaaga agaatgcgtc ggccgcaatt gccgattcct ggcaggttcc 3840

ggcaccgagc cgtggctgac cgacaagatc cgccaaggcg tgcgcgagca caagccggtg 3900

ctggtcgaga tcctgaacta caagaaggac ggcacgccgt tccgcaatgc cgtgctcgtt 3960

gcaccgatct acgatgacga cgacgagctt ctctatttcc tcggcagcca ggtcgaagtc 4020

gacgacgacc agcccaacat gggcatggcg cgccgcgaac gcgccgcgga aatgctcaag 4080

acgctgtcgc cgcgccagct cgaggttacg acgctggtgg catcgggctt gcgcaacaag 4140

gaagtggcgg cccggctcgg cctgtcggag aaaaccgtca agatgcaccg cgggctggtg 4200

atggaaaagc tcaacctgaa gaccagcgcc gatctggtgc gcattgccgt cgaagccgga 4260

atctaataac gctgatagtg ctagtgtaga tcgctactag agccaggcat caaataaaac 4320

gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc 4380

tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat atactagaag 4440

cggcccgtct tcacctcgag ttaattttta aagtatgggc aatcaattgc tcctgttaaa 4500

attgctttag aaatactttg gcagcggttt gttgtattga gtttcatttg cgcattggtt 4560

aaatggaaag tgacagtacg ctcactgca 4589

<210> 2

<211> 5539

<212> DNA

<213>artificial synthesized

<400> 2

ctcgagttaa tttttaaagt atgggcaatc aattgctcct gttaaaattg ctttagaaat 60

actttggcag cggtttgttg tattgagttt catttgcgca ttggttaaat ggaaagtgac 120

agtacgctca ctgcagccta atatttttga aatatcccaa gagctttttc cttcgcatgc 180

ccacgctaaa cattcttttt ctcttttggt taaatcgttg tttgatttat tatttgctat 240

atttattttt cgataattat caactagaga aggaacaatt aatggtatgt tcatacacgc 300

atgtaaaaat aaactatcta tatagttgtc tttttctgaa tgtgcaaaac taagcattcc 360

gaagccattg ttagccgtat gaatagggaa actaaaccca gtgataagac ctgatgtttt 420

cgcttcttta attacatttg gagatttttt atttacagca ttgttttcaa atatattcca 480

attaattggt gaatgattgg agttagaata atctactata ggatcatatt ttattaaatt 540

agcgtcatca taatattgcc tccatttttt agggtaatta tctagaattg aaatatcaga 600

tttaaccata gaatgaggat aaatgatcgc gagtaaataa tattcacaat gtaccatttt 660

agtcatatca gataagcatt gattaatatc attattgctt ctacaagctt taattttatt 720

aattattctg tatgtgtcgt cggcatttat gtttttcata cccatctctt tatccttacc 780

tattgtttgt cgcaagtttt gcgtgttata tatcattaaa acggtaatgg attgacattt 840

gattctaata aattggattt ttgtcacact attgtatcgc tgggaataca attacttaac 900

ataagcacct gtaggatcgt acaggtttac gcaagaaaat ggtttgttat agtcgaatga 960

attcattaaa gaggagaaag gtaccatggt gtctaagggc gaagaactga tcaaagagaa 1020

catgcacatg aagctgtaca tggaaggcac cgttgacaac caccacttta agtgcacgtc 1080

tgagggtgag ggtaagccgt acgaaggcac ccaaaccatg cgtatcaaag ttgtggaggg 1140

cggtccactg ccgttcgctt ttgacattct ggcgaccagc ttcctgtacg gttccaaaac 1200

gttcattaac catactcagg gcattccgga tttcttcaaa cagagctttc cggaaggttt 1260

cacctgggag cgtgtcacca cgtatgaaga tggtggtgtg ttgaccgcca cccaagatac 1320

ctccctgcaa gatggctgtc tgatctataa cgtgaaaatt cgtggcgtca actttacgag 1380

caatggtccg gtgatgcaga agaaaaccct gggttgggag gcgtttacgg aaaccctgta 1440

tccggccgat ggtggcctgg agggccgtaa cgacatggca ctgaagctgg ttggtggcag 1500

ccatttgatc gcaaatgcaa agacgacgta ccgcagcaag aaaccggcga aaaatctgaa 1560

gatgccgggt gtttactatg tcgactaccg tctggaacgc attaaagaag cgaataatga 1620

gacttacgtg gagcagcacg aggttgcagt cgcgcgctat tgcgacttgc ctagcaagct 1680

gggtcataaa ctgaatgcag cgaacgacga aaattacgcc cttgcagcgt aaacgcgtgc 1740

tagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt 1800

tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgccctagac ctagccgtct 1860

tcacctcgag ttaattttta aagtatgggc aatcaattgc tcctgttaaa attgctttag 1920

aaatactttg gcagcggttt gttgtattga gtttcatttg cgcattggtt aaatggaaag 1980

tgacagtacg ctcactgcag cctaatattt ttgaaatatc ccaagagctt tttccttcgc 2040

atgcccacgc taaacattct ttttctcttt tggttaaatc gttgtttgat ttattatttg 2100

ctatatttat ttttcgataa ttatcaacta gagaaggaac aattaatggt atgttcatac 2160

acgcatgtaa aaataaacta tctatatagt tgtctttttc tgaatgtgca aaactaagca 2220

ttccgaagcc attgttagcc gtatgaatag ggaaactaaa cccagtgata agacctgatg 2280

ttttcgcttc tttaattaca tttggagatt ttttatttac agcattgttt tcaaatatat 2340

tccaattaat tggtgaatga ttggagttag aataatctac tataggatca tattttatta 2400

aattagcgtc atcataatat tgcctccatt ttttagggta attatctaga attgaaatat 2460

cagatttaac catagaatga ggataaatga tcgcgagtaa ataatattca caatgtacca 2520

ttttagtcat atcagataag cattgattaa tatcattatt gcttctacaa gctttaattt 2580

tattaattat tctgtatgtg tcgtcggcat ttatgttttt catacccatc tctttatcct 2640

tacctattgt ttgtcgcaag ttttgcgtgt tatatatcat taaaacggta atggattgac 2700

atttgattct aataaattgg atttttgtca cactattgta tcgctgggaa tacaattact 2760

taacataagc acctgtagga tcgtacaggt ttacgcaaga aaatggtttg ttatagtcga 2820

atgaattcat taaagaggag aaaggtacca tgactataat gataaaaaaa tcggattttt 2880

tggcaattcc atcggaggag tataaaggta ttctaagtct tcgttatcaa gtgtttaagc 2940

aaagacttga gtgggactta gttgtagaaa ataaccttga atcagatgag tatgataact 3000

caaatgcaga atatatttat gcttgtgatg atactgaaaa tgtaagtgga tgctggcgtt 3060

tattacctac aacaggtgat tatatgctga aaagtgtttt tcctgaattg cttggtcaac 3120

agagtgctcc caaagatcct aatatagtcg aattaagtcg ttttgctgta ggtaaaaata 3180

gctcaaagat aaataactct gctagtgaaa ttacaatgaa actatttgaa gctatatata 3240

aacacgctgt tagtcaaggt attacagaat atgtaacagt aacatcaaca gcaatagagc 3300

gatttttaaa gcgtattaaa gttccttgtc atcgtattgg agacaaagaa attcatgtat 3360

taggtgatac taaatcggtt gtattgtcta tgcctattaa tgaacagttt aaaaaagcag 3420

tcttaaatgc agcgaacgac gaaaattacg cccttgcagc gtaaacgcgt gctagaggca 3480

tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc 3540

ggtgaacgct ctcctgagta ggacaaatcc gccgccctag acctagggcg ttcggctgcg 3600

gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 3660

cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 3720

gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 3780

aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 3840

ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 3900

cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 3960

ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 4020

cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 4080

agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 4140

gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 4200

gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 4260

tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 4320

agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 4380

agggattttg gtcatgacta gtgcttggat tctcaccaat aaaaaacgcc cggcggcaac 4440

cgagcgttct gaacaaatcc agatggagtt ctgaggtcat tactggatct atcaacagga 4500

gtccaagcga gctctcgaac cccagagtcc cgctcagaag aactcgtcaa gaaggcgata 4560

gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga agcggtcagc 4620

ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt cctgatagcg 4680

gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat tttccaccat 4740

gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt cgggcatgcg 4800

cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt cgtccagatc 4860

atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc gatgtttcgc 4920

ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca ttgcatcagc 4980

catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct gccccggcac 5040

ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca cagctgcgca 5100

aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcctgca gttcattcag 5160

ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa 5220

cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga atagcctctc 5280

cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc 5340

tcatcctgtc tcttgatcag atcttgatcc cctgcgccat cagatccttg gcggcaagaa 5400

agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc cagctggcaa 5460

ttccgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 5520

gaggcccttt cgtcttcac 5539

<210> 3

<211> 7529

<212> DNA

<213>artificial synthesized

<400> 3

ctcgagttaa tttttaaagt atgggcaatc aattgctcct gttaaaattg ctttagaaat 60

actttggcag cggtttgttg tattgagttt catttgcgca ttggttaaat ggaaagtgac 120

agtacgctca ctgcagccta atatttttga aatatcccaa gagctttttc cttcgcatgc 180

ccacgctaaa cattcttttt ctcttttggt taaatcgttg tttgatttat tatttgctat 240

atttattttt cgataattat caactagaga aggaacaatt aatggtatgt tcatacacgc 300

atgtaaaaat aaactatcta tatagttgtc tttttctgaa tgtgcaaaac taagcattcc 360

gaagccattg ttagccgtat gaatagggaa actaaaccca gtgataagac ctgatgtttt 420

cgcttcttta attacatttg gagatttttt atttacagca ttgttttcaa atatattcca 480

attaattggt gaatgattgg agttagaata atctactata ggatcatatt ttattaaatt 540

agcgtcatca taatattgcc tccatttttt agggtaatta tctagaattg aaatatcaga 600

tttaaccata gaatgaggat aaatgatcgc gagtaaataa tattcacaat gtaccatttt 660

agtcatatca gataagcatt gattaatatc attattgctt ctacaagctt taattttatt 720

aattattctg tatgtgtcgt cggcatttat gtttttcata cccatctctt tatccttacc 780

tattgtttgt cgcaagtttt gcgtgttata tatcattaaa acggtaatgg attgacattt 840

gattctaata aattggattt ttgtcacact attgtatcgc tgggaataca attacttaac 900

ataagcacct gtaggatcgt acaggtttac gcaagaaaat ggtttgttat agtcgaatga 960

attcattaaa gaggagaaag gtaccatgac agtaaagaaa ctttatttca tcccagcagg 1020

tcgttgcatg ttggatcatt cgtctgttaa cagtgcgtta acaccgggga aactattaaa 1080

cttgccggtg tggtgttatc ttttggagac ggaagaaggt cctattttag tagacacagg 1140

tatgccagaa agtgcagtta ataatgaagg gctttttaac ggtacatttg ttgaaggaca 1200

gatcttaccg aaaatgactg aagaagatag aatcgtgaat atattaaagc gtgtagggta 1260

tgagccggac gaccttttat atattattag ttctcactta cattttgatc atgcaggagg 1320

aaacggtgct tttacaaata caccaattat tgtgcagcga acggaatatg aggcagcact 1380

tcatagagaa gaatatatga aagaatgtat attaccgcat ttgaactaca aaattattga 1440

aggggattat gaagtggtac caggtgttca attattgtat acgccaggtc attctccagg 1500

ccatcagtcg ctattcattg agacggagca atccggttca gttttattaa cgattgatgc 1560

atcgtacacg aaagagaatt ttgaagatga agtgccgttc gcaggatttg atccagaatt 1620

agctttatct tcaattaaac gtttaaaaga agttgtgaaa aaagagaaac caattatttt 1680

ctttggtcat gatatagagc aggaaaagag ttgtagagtg ttcccggaat atatagcagc 1740

gaacgacgaa aattacgccc ttgcagcgta aacgcgtgct agaggcatca aataaaacga 1800

aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 1860

ctgagtagga caaatccgcc gccctagacc tagggatata ttccgcttcc tcgctcactg 1920

actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag 1980

atttcctgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc 2040

gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg 2100

gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt 2160

gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt 2220

gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta 2280

tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt 2340

ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag 2400

gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac 2460

tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa 2520

aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa 2580

cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt 2640

atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgtt actagtgatg 2700

atttctggaa ttcgcggccg cttctagagt cccttgcatt tacattttga aacatctata 2760

gcgataaatg aaacatctta aaagttttag tatcatattc gtgttggatt attctgcatt 2820

tttggggaga atggacttgc cgactgatta atgagggtta atcagtatgc agtggcataa 2880

aaaagcaaat aaaggcatat aacagagggt taataacatg aaagttaaag tactgtccct 2940

cctggtccca gctctgctgg tagcaggcgc agcaaacgct gctgaagttt acaacaaaga 3000

cggcaacaaa ttagatctgt acggtaaagt agacggcctg cactatttct ctgacaacaa 3060

agatgtagat ggcgaccaga cctacatgcg tcttggcttc aaaggtgaaa ctcaggttac 3120

tgaccagctg accggttacg gccagtggga atatcagatc cagggcaaca gcgctgaaaa 3180

cgaaaacaac tcctggaccc gtgtggcatt cgcaggtctg aaattccagg atgtgggttc 3240

tttcgactac ggtcgtaact acggcgttgt ttatgacgta acttcctgga ccgacgtact 3300

gccagaattc ggtggtgaca cctacggttc tgacaacttc atgcagcagc gtggtaacgg 3360

cttcgcgacc taccgtaaca ctgacttctt cggtctggtt gacggcctga actttgctgt 3420

tcagtaccag ggtaaaaacg gcaacccatc tggtgaaggc tttactagtg gcgtaactaa 3480

caacggtcgt gacgcactgc gtcaaaacgg cgacggcgtc ggcggttcta tcacttatga 3540

ttacgaaggt ttcggtatcg gtggtgcgat ctccagctcc aaacgtactg atgctcagaa 3600

caccgctgct tacatcggta acggcgaccg tgctgaaacc tacactggtg gtctgaaata 3660

cgacgctaac aacatctacc tggctgctca gtacacccag acctactgaa gcggcgcacg 3720

aaaaacgcga aagctaaagt tttcgcattt atcgtgaaac gctttcgcgt ttttcgtgcg 3780

ccgcttcacc ggataaagag cagctactgg tggttaacct ttccggtcgc ggcgataaag 3840

acatcttcac cgttcacgat attttgaaag cacgagggga aatctgtcat atataaataa 3900

aataaggtag gtcaatatat gacagtaaag aaactttatt tcatcccagc aggtcgttgc 3960

atgttggatc attcgtctgt taacagtgcg ttaacaccgg ggaaactatt aaacttgccg 4020

gtgtggtgtt atcttttgga gacggaagaa ggtcctattt tagtagacac aggtatgcca 4080

gaaagtgcag ttaataatga agggcttttt aacggtacat ttgttgaagg acagatctta 4140

ccgaaaatga ctgaagaaga tagaatcgtg aatatattaa agcgtgtagg gtatgagccg 4200

gacgaccttt tatatattat tagttctcac ttacattttg atcatgcagg aggaaacggt 4260

gcttttacaa atacaccaat tattgtgcag cgaacggaat atgaggcagc acttcataga 4320

gaagaatata tgaaagaatg tatattaccg catttgaact acaaaattat tgaaggggat 4380

tatgaagtgg taccaggtgt tcaattattg tatacgccag gtcattctcc aggccatcag 4440

tcgctattca ttgagacgga gcaatccggt tcagttttat taacgattga tgcatcgtac 4500

acgaaagaga attttgaaga tgaagtgccg ttcgcaggat ttgatccaga attagcttta 4560

tcttcaatta aacgtttaaa agaagttgtg aaaaaagaga aaccaattat tttctttggt 4620

catgatatag agcaggaaaa gagttgtaga gtgttcccgg aatatatagc agcgaacgac 4680

gaaaattacg cccttgcagc gtaagcccgt atttcgcgta aggatactag tagcggccgc 4740

tgcagtccgg caaaaaaggg caaggtgtca ccaccctgcc ctttttcttt aaaaccgaaa 4800

agattacttc gcgttatgca ggcttcctcg ctcactgact cgctgactag tgcttggatt 4860

ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc 4920

tgaggtcatt actggatcta tcaacaggag tccaagcgag ctcgatatca aattacgccc 4980

cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc 5040

catcacagac ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg 5100

tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg gccacgttta 5160

aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata ttctcaataa 5220

accctttagg gaaataggcc aggttttcac cgtaacacgc cacatcttgc gaatatatgt 5280

gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac gtttcagttt 5340

gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc tcaccgtctt 5400

tcattgccat acggaattcc ggatgagcat tcatcaggcg ggcaagaatg tgaataaagg 5460

ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta atatccagct 5520

gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa tgttctttac 5580

gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc attttagctt 5640

ccttagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat cttatttcat 5700

tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc cagatatcga 5760

cgtcctgaca gctagctcag tcctaggtat aatgctagct cacacagaat tcattaaaga 5820

ggagaaaggt accatgagtg tcaacttagc ttcccagttg cgggaaggga cgaaaaaatc 5880

ccactccatg gcggagaacg tcggctttgt caaatgcttc ctcaagggcg ttgtcgagaa 5940

aaattcctac cgtaagctgg ttggcaatct ctactttgtc tacagtgcca tggaagagga 6000

aatggcaaaa tttaaggacc atcccatcct cagccacatt tacttccccg aactcaaccg 6060

caaacaaagc ctagagcaag acctgcaatt ctattacggc tccaactggc ggcaagaagt 6120

gaaaatttct gccgctggcc aagcctatgt ggaccgagtc cggcaagtgg ccgctacggc 6180

ccctgaattg ttggtggccc attcctacac ccgttacctg ggggatcttt ccggcggtca 6240

aattctcaag aaaattgccc aaaatgccat gaatctccac gatggtggca cagctttcta 6300

tgaatttgcc gacattgatg acgaaaaggc ttttaaaaat acctaccgtc aagctatgaa 6360

tgatctgccc attgaccaag ccaccgccga acggattgtg gatgaagcca atgacgcctt 6420

tgccatgaac atgaaaatgt tcaacgaact tgaaggcaac ctgatcaagg cgatcggcat 6480

tatggtgttc aacagcctca cccgtcgccg cagtcaaggc agcaccgaag ttggcctcgc 6540

cacctccgaa ggctagttaa agaggagaaa ggatccatgg ccgtcactga tttaagtttg 6600

accaattctt ccctgatgcc cacgttgaac ccgatgattc aacagttggc cctggcgatc 6660

gccgctagtt ggcaaagttt acccctcaag ccctatcaat tgccggagga tttgggctac 6720

gtagaaggcc gcctggaagg ggaaaagtta gtgattgaaa atcggtgcta ccaaacgccc 6780

cagtttcgca aaatgcattt ggagttggcc aaggtgggca aagggttgga tattctccac 6840

tgtgtaatgt ttcctgagcc tttatacggt ctacctttgt ttggctgtga cattgtggcc 6900

ggccccggtg gagtaagtgc ggctattgcg gatctatccc ccacccaaag cgatcgccaa 6960

ttgcccgcag cgtaccaaaa atcattggca gagctaggcc agccagaatt tgagcaacaa 7020

cgggaattgc ccccctgggg agaaatattt tctgaatatt gtttattcat ccgtcccagc 7080

aatgtcactg aagaagaaag atttgtacaa agggtagtgg actttttgca aattcattgt 7140

caccaatcca tcgttgccga acccttgtct gaagctcaaa ctttggagca ccgtcagggg 7200

caaattcatt actgccaaca acaacagaaa aatgataaaa cccgtcgggt actggaaaaa 7260

gcttttgggg aagcttgggc ggaacggtat atgagccaag tcttatttga tgttatccaa 7320

taatctagac caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 7380

ttatctgttg tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg 7440

ggcctttctg cgtttatatc taagaaacca ttattatcat gacattaacc tataaaaata 7500

ggcgtatcac gaggcccttt cgtcttcac 7529

<210> 4

<211> 8194

<212> DNA

<213>artificial synthesized

<400> 4

ctcgagttaa tttttaaagt atgggcaatc aattgctcct gttaaaattg ctttagaaat 60

actttggcag cggtttgttg tattgagttt catttgcgca ttggttaaat ggaaagtgac 120

agtacgctca ctgcagccta atatttttga aatatcccaa gagctttttc cttcgcatgc 180

ccacgctaaa cattcttttt ctcttttggt taaatcgttg tttgatttat tatttgctat 240

atttattttt cgataattat caactagaga aggaacaatt aatggtatgt tcatacacgc 300

atgtaaaaat aaactatcta tatagttgtc tttttctgaa tgtgcaaaac taagcattcc 360

gaagccattg ttagccgtat gaatagggaa actaaaccca gtgataagac ctgatgtttt 420

cgcttcttta attacatttg gagatttttt atttacagca ttgttttcaa atatattcca 480

attaattggt gaatgattgg agttagaata atctactata ggatcatatt ttattaaatt 540

agcgtcatca taatattgcc tccatttttt agggtaatta tctagaattg aaatatcaga 600

tttaaccata gaatgaggat aaatgatcgc gagtaaataa tattcacaat gtaccatttt 660

agtcatatca gataagcatt gattaatatc attattgctt ctacaagctt taattttatt 720

aattattctg tatgtgtcgt cggcatttat gtttttcata cccatctctt tatccttacc 780

tattgtttgt cgcaagtttt gcgtgttata tatcattaaa acggtaatgg attgacattt 840

gattctaata aattggattt ttgtcacact attgtatcgc tgggaataca attacttaac 900

ataagcacct gtaggatcgt acaggtttac gcaagaaaat ggtttgttat agtcgaatga 960

attcattaaa gaggagaaag gtaccatgag caaaggagaa gaacttttca ctggagttgt 1020

cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg tccgtggaga 1080

gggtgaaggt gatgctacaa acggaaaact cacccttaaa tttatttgca ctactggaaa 1140

actacctgtt ccgtggccaa cacttgtcac tactctgacc tatggtgttc aatgcttttc 1200

ccgttatccg gatcacatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta 1260

tgtacaggaa cgcactatat ctttcaaaga tgacgggacc tacaagacgc gtgctgaagt 1320

caagtttgaa ggtgataccc ttgttaatcg tatcgagtta aagggtattg attttaaaga 1380

agatggaaac attcttggac acaaactcga gtacaacttt aactcacaca atgtatacat 1440

cacggcagac aaacaaaaga atggaatcaa agctaacttc aaaattcgcc acaacgttga 1500

agatggttcc gttcaactag cagaccatta tcaacaaaat actccaattg gcgatggccc 1560

tgtcctttta ccagacaacc attacctgtc gacacaatct gtcctttcga aagatcccaa 1620

cgaaaagcgt gaccacatgg tccttcttga gtttgtaact gctgctggga ttacacatgg 1680

catggatgag ctctacaaag cagcgaacga cgaaaattac gcccttgcag cgtaaacgcg 1740

tgctagaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc 1800

tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgcccta gacctagccg 1860

tcttcacctc gagttaattt ttaaagtatg ggcaatcaat tgctcctgtt aaaattgctt 1920

tagaaatact ttggcagcgg tttgttgtat tgagtttcat ttgcgcattg gttaaatgga 1980

aagtgacagt acgctcactg cagcctaata tttttgaaat atcccaagag ctttttcctt 2040

cgcatgccca cgctaaacat tctttttctc ttttggttaa atcgttgttt gatttattat 2100

ttgctatatt tatttttcga taattatcaa ctagagaagg aacaattaat ggtatgttca 2160

tacacgcatg taaaaataaa ctatctatat agttgtcttt ttctgaatgt gcaaaactaa 2220

gcattccgaa gccattgtta gccgtatgaa tagggaaact aaacccagtg ataagacctg 2280

atgttttcgc ttctttaatt acatttggag attttttatt tacagcattg ttttcaaata 2340

tattccaatt aattggtgaa tgattggagt tagaataatc tactatagga tcatatttta 2400

ttaaattagc gtcatcataa tattgcctcc attttttagg gtaattatct agaattgaaa 2460

tatcagattt aaccatagaa tgaggataaa tgatcgcgag taaataatat tcacaatgta 2520

ccattttagt catatcagat aagcattgat taatatcatt attgcttcta caagctttaa 2580

ttttattaat tattctgtat gtgtcgtcgg catttatgtt tttcataccc atctctttat 2640

ccttacctat tgtttgtcgc aagttttgcg tgttatatat cattaaaacg gtaatggatt 2700

gacatttgat tctaataaat tggatttttg tcacactatt gtatcgctgg gaatacaatt 2760

acttaacata agcacctgta ggatcgtaca ggtttacgca agaaaatggt ttgttatagt 2820

cgaatgaatt cattaaagag gagaaaggta ccatgactat aatgataaaa aaatcggatt 2880

ttttggcaat tccatcggag gagtataaag gtattctaag tcttcgttat caagtgttta 2940

agcaaagact tgagtgggac ttagttgtag aaaataacct tgaatcagat gagtatgata 3000

actcaaatgc agaatatatt tatgcttgtg atgatactga aaatgtaagt ggatgctggc 3060

gtttattacc tacaacaggt gattatatgc tgaaaagtgt ttttcctgaa ttgcttggtc 3120

aacagagtgc tcccaaagat cctaatatag tcgaattaag tcgttttgct gtaggtaaaa 3180

atagctcaaa gataaataac tctgctagtg aaattacaat gaaactattt gaagctatat 3240

ataaacacgc tgttagtcaa ggtattacag aatatgtaac agtaacatca acagcaatag 3300

agcgattttt aaagcgtatt aaagttcctt gtcatcgtat tggagacaaa gaaattcatg 3360

tattaggtga tactaaatcg gttgtattgt ctatgcctat taatgaacag tttaaaaaag 3420

cagtcttaaa tgcagcgaac gacgaaaatt acgcccttgc agcgtaaacg cgtgctagag 3480

gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt 3540

gtcggtgaac gctctcctga gtaggacaaa tccgccgccc tagacctagg gcgttcggct 3600

gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 3660

taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 3720

cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 3780

ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 3840

aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 3900

tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 3960

gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 4020

cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 4080

ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 4140

cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct 4200

gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 4260

cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 4320

tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg 4380

ttaagggatt ttggtcatga ctagtttact gtccctagtg cttggattct caccaataaa 4440

aaacgcccgg cggcaaccga gcgttctgaa caaatccaga tggagttctg aggtcattac 4500

tggatctatc aacaggagtc caagcgagct cgtaaacttg gtctgacagt taccaatgct 4560

taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 4620

tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 4680

tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 4740

gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 4800

gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 4860

ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 4920

cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 4980

tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 5040

cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 5100

agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 5160

cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 5220

aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 5280

aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 5340

gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 5400

gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 5460

tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 5520

ttcccatggt gccacctgac gtctaagaaa ccatagatct tgatcccctg cgccatcaga 5580

tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg 5640

gcgccccagc tggcaattcc gacgtcctca ttttcgccag atatcgacgt ctaagaaacc 5700

attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcac 5760

ctcgagtccc tatcagtgat agagatactg agcacatcag caggacgcac tgaccgaatt 5820

cattaaagag gagaaaggta cccatggcca ccaccgtaca actcagcgac caatccctcc 5880

gtcagctaga aaccctcgcc atccacaccg cccacctgat tcagccccac ggtttagtgg 5940

tggtcctgca ggaaccagac ctcaccatca gccaaattag cgccaactgc accggcattt 6000

tagggcgatc gccagaggat ttgttgggca gaaccctagg ggaagtgttt gatagctttc 6060

agattgatcc catccagagt cgcctaacgg ccggacaaat cagcagcctc aaccccagta 6120

aactttgggc gcgggtcatg ggggacgact ttgtcatttt tgacggggtt tttcatcgca 6180

acagtgacgg tttattggta tgtgaactcg agccagccta cacttccgat aatctgccct 6240

tcctcggttt ttatcacatg gccaacgctg ccctgaatcg gttgcgccaa caagctaatc 6300

tacgggattt ctacgatgtt attgtcgaag aagtccgccg tatgactggc tttgaccggg 6360

tgatgctata ccgctttgat gaaaataacc acggtgatgt cattgccgaa gataaacggg 6420

atgatatgga accctatttg ggcctgcact atcccgaatc ggatattccc caacccgccc 6480

gtcggctatt tatccacaac cccattcgag taattcccga tgtttatggt gtggcggtgc 6540

ccctgacccc agcggttaac cccagcacca accgagcggt ggatttaaca gaatccattc 6600

tgcgcagtgc gtaccattgc cacttgacct atctgaaaaa tatgggggta ggagcgtctt 6660

taaccatttc cctaattaag gacggccatc tctgggggct cattgcctgc caccatcaaa 6720

cccccaaagt aattcccttt gaactgcgta aagcctgcga attttttggt cgggtggtgt 6780

ttagcaacat ttccgcccag gaagatacgg aaaccttcga ttaccgggtg cagctggcgg 6840

agcatgaagc ggttttattg gacaaaatga ccacggcggc ggattttgtc gaaggattaa 6900

ctaatcatcc cgatcgcctg ttgggattaa cgggctccca gggggcggcc atttgctttg 6960

gggaaaaatt gattttagta ggggaaaccc cggacgagaa agcagtgcaa tatttactgc 7020

aatggttgga gaatcgggaa gtgcaagacg ttttcttcac ctcttccctc tcacaaattt 7080

atcctgatgc agtgaatttt aaatccgtgg ccagtggctt attggccatt cccattgccc 7140

gtcacaactt tttgctctgg tttcgccctg aagtgttgca aacggttaat tggggcggtg 7200

acccaaatca tgcttacgaa gctacccagg aagacggtaa aatcgagctc catccccgcc 7260

aatcctttga cctctggaaa gaaattgtcc gactccaatc tttgccctgg caatcggtgg 7320

aaatccaaag tgccctggcc ctgaaaaagg cgatcgtcaa cctcattttg cgccaggcag 7380

aagaattgca tatggcggct ggtgttaagc aactggcgga tgaccgcacg ctgctgatgg 7440

cgggggtaag tcacgacttg cgcacgccgc tgacgcgtat tcgcctggcg actgagatga 7500

tgagcgagca ggatggctat ctggcagaat cgatcaataa agatatcgaa gagtgcaacg 7560

ccatcattga gcagtttatc gactacctgc gcaccgggca ggagatgccg atggaaatgg 7620

cggatcttaa tgcagtactc ggtgaggtga ttgctgccga aagtggctat gagcgggaaa 7680

ttgaaaccgc gctttacccc ggcagcattg aagtgaaaat gcacccgctg tcgatcaaac 7740

gcgcggtggc gaatatggtg gtcaacgccg cccgttatgg caatggctgg gtcaaagtca 7800

gcagcggaac ggagccgaat cgcgcctggt tccaggtgga agatgacggt ccgggaattg 7860

cgccggaaca acgtaagcac ctgttccagc cgtttgtccg cggcgacagt gcgcgcacca 7920

ttagcggcac gggattaggg ctggcaattg tgcagcgtat cgtggataac cataacggga 7980

tgctggagct tggcaccagc gagcggggcg ggctttccat tcgcgcctgg ctgccagtgc 8040

cggtaacgcg ggcgcagggc atgacaaaag aagggtaatc tagaggcatc aaataaaacg 8100

aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct 8160

cctgagtagg acaaatccgc cgccctagac ctag 8194

Claims (9)

1. a kind of light-operated steady gene oscillatory system, which is characterized in that including light-operated gene oscillator and regulation light source；The light Controlling gene oscillator is integrated in cell by light-dependent control element and gene oscillating element；The regulation light source includes calculating to adjust Component and light source are controlled, the calculating regulation component can issue in real time tune based on the oscillation observation of the light-operated gene oscillator Signal is controlled, the exposure intensity and irradiation time of the light source are adjusted；The light-operated gene oscillator is irradiated according to the light source of adjusting Intensity and irradiation time adjust its oscillation behavior.

2. light-operated steady gene oscillatory system as described in claim 1, which is characterized in that the light-dependent control element is light science of heredity Gene expression control module.

3. light-operated steady gene oscillatory system as described in claim 1, which is characterized in that the light source is light can be caused to lose Pass the light source for learning the specific wavelength of molecules in response.

4. light-operated steady gene oscillatory system as described in claim 1, which is characterized in that the calculating regulation component includes tool There are computer program, Intelligent mobile equipment, embedded hardware system, the number electricity of digital logical operation and logical process function In road, digital optical path, analog operational circuit or simulation trial optical path any one or combinations thereof.

5. light-operated steady gene oscillatory system as described in claim 1, which is characterized in that the light-operated gene oscillator includes But it is not limited to the light-operated gene oscillator of blue light and the light-operated gene oscillator of feux rouges, the light-operated gene oscillator of blue light is by blue light Regulation, regulation of the light-operated gene oscillator of feux rouges by feux rouges, the wherein fluorescence report of the light-operated gene oscillator of blue light Albumen is mTagBFP2, and the fluorescent reporter protein of the light-operated gene oscillator of feux rouges is sfGFP.

6. light-operated steady gene oscillatory system as claimed in claim 5, which is characterized in that the light-operated gene oscillator of blue light Light-dependent control element be the plasmid as shown in sequence SEQ NO:1.

7. light-operated steady gene oscillatory system as claimed in claim 5, which is characterized in that the light-operated gene oscillator of blue light Gene oscillating element be the plasmid as shown in sequence SEQ NO:2.

8. light-operated steady gene oscillatory system as claimed in claim 5, which is characterized in that the light-operated gene oscillator of feux rouges Light-dependent control element be the plasmid as shown in sequence SEQ NO:3.

9. light-operated steady gene oscillatory system as claimed in claim 5, which is characterized in that the light-operated gene oscillator of feux rouges Gene oscillating element be the plasmid as shown in sequence SEQ NO:4.