CN110241019B - Light-operated steady gene oscillation system - Google Patents

Abstract

The invention discloses a light-controlled robust gene oscillation system including a light-controlled gene oscillator and a regulation light source; the light-controlled gene oscillator includes a light control element and a gene oscillation element; the regulation light source includes a calculation regulation component and a light source, and the calculation The regulation component sends out regulation signals in real time according to the oscillation observation value of the light-controlled gene oscillator, and adjusts the illumination intensity and illumination time of the light source; the light-controlled gene oscillator adjusts its oscillation behavior according to the adjusted illumination intensity and illumination time of the light source. The invention introduces an exogenous light regulation mechanism to precisely regulate the gene oscillation behavior, making it more robust. The invention helps to deepen the understanding of the gene oscillation circuit, and has certain theoretical guiding significance for the regulation of oscillation in the living system and the construction of a robust large-scale synthetic biology system. In practical applications, a robust light-controlled gene oscillation system can be integrated into cells to periodically express a certain gene according to demand, saving production costs and improving production efficiency.

Description

A Light-Controlled Robust Gene Oscillator System

technical field

The invention relates to the technical field of gene regulation, in particular to a light-controlled robust gene oscillation system.

Background technique

There are many types of gene oscillators and light-controlled gene expression systems, but to the best of our knowledge, there is no research on using light-controlled systems to control gene oscillators.

Due to the complexity of biological systems, it is difficult to directly study biological oscillators. Therefore, in recent years, scientists often study life oscillation behavior by artificially designing simpler gene circuits and constructing gene oscillators. However, if the improvement is only made from the biological element itself, the controllability is relatively poor, and it is difficult to achieve precise adjustment. In addition, the quantitative understanding of these elements is not yet deep enough to enable flexible regulation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide a light-controlled robust gene oscillation system. The present invention utilizes optogenetic methods to increase the controllability and robustness of gene oscillators by integrating optogenetic gene expression control modules and applying light control.

The present invention is demonstrated by taking two sets of light-controlled robust gene oscillation systems as examples. First, two sets of population-synchronized gene oscillators regulated by light, blue light-controlled gene oscillators and red light-controlled gene oscillators, were constructed in Escherichia coli respectively. At the same time, a computer program that can adjust the irradiation intensity and irradiation time of the light source in real time according to the oscillation observation value of the light-controlled genetic oscillator is also written in the computer. example. Two light-controlled genetic oscillators (red light and blue light) are regulated by virtual oscillators, constituting two light-controlled robust oscillatory systems. In each light-controlled stable oscillation system, the virtual oscillator is used to control the light-controlled gene oscillator in real time, that is, the light intensity and time are oscillated in a stable manner through the virtual oscillator, and the light-controlled gene oscillation is regulated by the stable oscillating light. The expression levels of related genes in the organ are ultimately made more robust and controllable in the oscillatory behavior of gene oscillators.

To achieve the above object, the technical scheme adopted in the present invention is:

A light-controlled robust gene oscillation system, comprising a light-controlled gene oscillator and a control light source; the light-controlled gene oscillator is formed by integrating a light control element and a gene oscillation element in a cell; the control light source includes a calculation control component and a light source, wherein the calculation and regulation component can send out a regulation signal in real time based on the oscillation observation value of the light-controlled gene oscillator to adjust the irradiation intensity and irradiation time of the light source; Irradiation intensity and irradiation time to adjust its oscillatory behavior.

Further, the light control element is an optogenetic gene expression control module.

Further, the light source is a light source of a specific wavelength capable of eliciting a response of optogenetic molecules.

Further, the calculation control component includes any one or a combination thereof in a computer program with logic operation and logic processing functions, an intelligent mobile device, an embedded hardware system, a digital circuit, a digital optical circuit, an analog operation circuit or an analog operation optical path. .

Further, the light-controlled gene oscillators include but are not limited to blue light-controlled gene oscillators and red light-controlled gene oscillators, the blue light-controlled gene oscillators are regulated by blue light, and the red light-controlled gene oscillators are regulated by blue light. The fluorescent reporter protein of the blue light-controlled gene oscillator is mTagBFP2, and the fluorescent reporter protein of the red light-controlled gene oscillator is sfGFP.

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

Further, the gene oscillation element of the blue light-controlled gene oscillator is the plasmid shown in the sequence SEQ NO: 2.

Further, the light control element of the red light light control gene oscillator is the plasmid shown in the sequence of SEQ NO: 3.

Further, the gene oscillation element of the red light light-controlled gene oscillator is the plasmid shown in the sequence SEQ NO:4.

Further, the light control element of the blue light light-controlled gene oscillator includes the EL222 gene and the luxI promoter; the EL222 gene sequence is located downstream of the -10 region of the promoter P luxI; or the EL222 gene sequence replaces the -10 region of the promoter P luxI The spacer sequence between the 35 region and the -10 region is also inserted in the downstream of the -10 region;

Beneficial effects of the present invention: The present invention precisely regulates gene oscillation behavior by adding a light source regulation mechanism, making it more robust and controllable. The invention helps to deepen the understanding of the gene oscillation circuit, and has certain theoretical guiding significance for the regulation of oscillation in the living system and the construction of a robust large-scale synthetic biology system. In practical applications, a robust light-controlled gene oscillation system can be integrated into cells to periodically express a certain gene according to demand, saving production costs and improving production efficiency.

Description of drawings

Fig. 1 is a schematic diagram of the regulation mechanism of a virtual oscillator to a light-controlled genetic oscillator in Example 4 (wherein A is a light intensity function; B is a mathematical model of a virtual oscillator; C is a virtual oscillator oscillation behavior pattern diagram).

Fig. 2 is the schematic diagram of the robustness analysis of the blue light-controlled gene oscillator in Example 4 (wherein A is the analysis schematic diagram of the fluorescence oscillation phenomenon before regulation and the period and amplitude of each oscillation; B is the fluorescence oscillation phenomenon after regulation and the variation of each oscillation Schematic diagram of amplitude and period variation analysis).

Fig. 3 is a schematic diagram of the robustness analysis of the red light-controlled gene oscillator in Example 4 (wherein A is the analysis schematic diagram of the fluorescence oscillation phenomenon before regulation and the period and amplitude changes of each oscillation; B is the fluorescence oscillation phenomenon after regulation and each oscillation Schematic diagram of the amplitude and period variation analysis).

Detailed ways

In order to show the technical solutions, objects and advantages of the present invention more concisely and clearly, the present invention will be further described in detail below with reference to specific embodiments and the accompanying drawings.

Example 1 Construction of blue light-controlled gene oscillator

1. Construction of blue light control elements

1.1 Construction of blue light control plasmid

(1) insert the synthetic EL222 gene fragment into pTD103aiiA (Cm) plasmid, obtain glycerol bacteria and plasmid dry powder (pTD103aiiA_EL222).

(2) Activation of bacteria and preservation of strains

① Take the glycerol bacteria containing pTD103aiiA_EL222 plasmid on the Cm-resistant LB solid petri dish, carry out plate streaking activation (partition streaking method), and cultivate overnight.

② Pick a single colony on the plate, inoculate it in 5 mL of Cm-resistant LB liquid culture medium, and incubate it in a constant temperature shaker at 37°C and 180 r/min overnight.

③Bacteria preservation: transfer 30 μl of the overnight bacterial solution to 3 mL of Cm-resistant LB liquid culture medium and cultivate it to the mid-logarithmic phase (about 3-4 hours), and then in a glycerol tube, mix the bacterial solution with 50 mL in a ratio of 1:1. % sterilized glycerol was mixed evenly, and stored at -20°C and -80°C for short-term and long-term storage, respectively.

(3) Plasmid extraction (plasmid mini-extraction kit)

① enrichment. Take a 2 mL centrifuge tube, add 2 mL of the bacterial solution containing the pTD103aiiA_EL222 plasmid to it, centrifuge at 12,000 × g for 1 min, discard the supernatant, add 2 mL of the bacterial solution and centrifuge again to discard the supernatant, and repeat.

②Resuspend. Add 200 μl of Solution I to the centrifuge tube, place it on a vortexer and mix thoroughly until it becomes a sterile block.

③ cracking. Add 200 μl of Solution II and mix gently for about 5 times to make the solution clear.

④ Neutralize. Add 200 μl of Solution III, and mix by gently inverting for about 5 times. At this time, a large number of white flocculent precipitates appear.

⑤ Centrifuge sedimentation. Place the centrifuge tube in a centrifuge and centrifuge at 12,000 × g for 8-10 min until the white flocculent precipitate sinks to the bottom or wall of the tube.

⑥ Insert the adsorption column into the collection tube, suck the supernatant other than the white precipitate (do not suck the white precipitate) into the adsorption column, and centrifuge at 12000×g for 1 min.

⑦ Pour off the filtrate, add 500 μl of Wash Solution A to the adsorption column, and centrifuge at 12,000 × g for 1 min.

⑧ Pour off the filtrate, add 700 μl of Wash Solution B to the adsorption column, centrifuge at 12,000 × g for 1 min, discard the filtrate, and repeat this step once.

⑨ Pour off the filtrate, put the centrifuge tube back into the centrifuge, and shake it at 12000×g for 5min.

⑩Take a clean 1.5mL centrifuge tube, insert the adsorption column into it, add 50-100μl ElutionSolution, let stand for 1-2min, and centrifuge at 12000×g for 1min to obtain the plasmid. Finally, the concentration of pTD103aiiA_EL222 plasmid was measured and recorded with an ultra-micro spectrophotometer.

(4) Enzyme cleavage

After the plasmid pTD103aiiA_EL222 was extracted, it was verified by digestion with Spe I restriction endonuclease. It is expected that the target plasmid will form two linear fragments with lengths of 1005bp and 3581bp after Spe I endonuclease digestion. The enzyme digestion system was prepared according to the reaction system in Table 1, mixed well, centrifuged, and digested at 37°C for 30 min.

Table 1: Enzymatic digestion reaction system

(5) Verification by agarose gel electrophoresis

①Take a wide plexiglass rubber plate, put it horizontally into the glue tank, and insert a comb.

②Put 3g of agarose and 30mL of TAE solution with a concentration of 1× into a 250mL conical flask, put it in a microwave oven and heat it until it is completely melted to obtain a 1% separating gel.

③When the glue liquid is cooled to about 60℃, add 1μl of Golden View and mix it evenly. After pouring it into the glue making plate, place it on a horizontal table and let it cool at room temperature to form a uniform and horizontal glue surface.

④ After the gel has solidified, carefully pull up the comb, clean the gel around the gel plate, put it into the electrophoresis tank, and pour 1× TAE solution into the electrophoresis tank to cover the gel surface.

⑤ Add the sample into the loading buffer and mix well, then inject it into the sample well, and inject 5000bp Marker into another sample well as a reference.

⑥Turn on the electrophoresis apparatus and electrophoresis tank, electrophoresis at 80V for about 30-40min, and turn off the electrophoresis apparatus when the band (blue) of bromophenol blue runs to two-thirds.

(6) After the positive identification by agarose gel electrophoresis, the plasmid is sent to a sequencing company for sequencing identification.

1.2 Construction of blue light control element promoter

This experiment was constructed using the Gibson Assembly method.

(1) Design and synthesis of primers to achieve simultaneous insertion of the EL222 targeting sequence in the regions downstream of the -10 region of PluxI, between the -35 and -10 regions, and both. The template plasmid used in this experiment was pTD103aiiA_EL222. PCR primers, amplified fragments, reaction systems and reaction procedures are shown in Table 2-5 respectively.

Table 2: PCR reaction primers

Table 3: PCR amplified fragments

Table 4: PCR reaction system

Table 5: PCR reaction program

(2) Separation of target DNA fragments by agarose gel electrophoresis

(3) Recovery of target DNA fragments

①Cut out the agarose gel containing the target DNA fragment under UV light and place it in a 2mL centrifuge tube. Calculate the gel weight and use this weight as a gel volume.

②Add 3 gel volumes of Buffer DE-A, mix well, heat in a water bath at 75°C, and mix intermittently until the gel pieces are completely melted.

③ Add 0.5 volume of Buffer DE-B of Buffer DE-A and mix well.

④Aspirate the mixture from the previous step, transfer it to a DNA preparation tube, centrifuge at 12,000 × g for 1 min, and discard the filtrate.

⑤ Put the preparation tube back into the centrifuge tube, add 500 μl of Buffer W1, centrifuge at 12,000 × g for 30 s, and discard the filtrate.

⑥ Put the preparation tube back into the centrifuge tube, add 700 μl of Buffer W2, centrifuge at 12000×g for 30s, and discard the filtrate. Repeat again.

⑦Put the preparation tube into a 2mL centrifuge tube and centrifuge at 12000×g for 1min.

⑧Put the preparation tube in a clean 1.5mL centrifuge tube, add 30μl Eluent to the center of the DNA preparation membrane, and let stand at room temperature for 2min. Centrifuge at 12,000 × g for 1 min to elute DNA to obtain DNA samples.

(4) Recombination reaction

Add samples according to the Gibson Assembly reaction system (see Table 6 for the reaction system), and bath at 50°C for 30 minutes.

Table 6: Gibson Assembly reaction system

(5) Conversion

① Thaw T1 competent cells stored at -80°C in ice for 10 minutes.

② Add the plasmid obtained in the previous step to 100 μl of T1 competent cells in a clean bench, mix gently and place on ice for 30 min.

③ After heat shock in a water bath at 42°C for 90 sec, immediately place it in ice for 2 min. Be careful not to shake the centrifuge tube vigorously during this process.

④ Add 500 μl of LB medium pre-warmed at 37°C, and mix by suction.

⑤Resuscitate the bacteria solution, incubate at 37℃, 180r/min for 45min.

⑥ Take 100 μl of the bacterial solution and add it to the Cm-resistant LB solid medium plate, spread it evenly with a coating stick, and place the plate upside down in a 37°C constant temperature incubator for overnight culture.

(6) Selection of recombinant recipient bacteria and preserved strains

The promoter plasmids constructed in this experiment are pTD103aiiA_EL222_10EB, pTD103aiiA_EL222_35EB10, pTD103aiiA_EL222_35EB10EB plasmids.

(7) Enzyme digestion test and delivery test

The three plasmids were digested with Pst I endonuclease, and the lengths of the three plasmids were about 4600 bp. After the positive detection by agarose gel electrophoresis, the plasmids were sent to a sequencing company for sequencing and identification.

1.3 Construction of plasmid for blue light control effect test

In order to visually test the light control effect of the three light control promoters, the aiiA gene behind the light control promoter needs to be replaced with the sfGFP fluorescent reporter gene in this experiment.

(1) Plasmid extraction (pTD103aiiA_EL222_10EB, pTD103aiiA_EL222_35EB10, pTD103a iiA_EL222_35EB10EB, pTD103luxI_sfGFP).

(2) Design and synthesize primers, carry out PCR reaction, and then replace the aiiA elements of the three plasmids pTD103aiiA_EL222_10EB, pTD103aiiA_EL222_35EB10, and pTD103aiiA_EL222_35EB10EB with sfGFP elements through recombination reaction. PCR reaction primers and amplified fragments are shown in Table 7 and Table 8.

Table 7: PCR reaction primers

Table 8: PCR amplified fragments

(3) Separation of target DNA fragments and recovery of target DNA fragments by agarose gel electrophoresis

The steps are the same as above.

(4) Recombination reaction

Add samples according to the reaction system in Table 9, 50 ℃ water bath, 30min.

Table 9: Recombination reaction system

(5) Conversion:

MG1655 competent cells were used for transformation, and the steps were the same as above.

(6) Selection of recombinant recipient bacteria and preserved strains.

(7) After PCR and agarose gel electrophoresis are positive, the plasmid is sent to a sequencing company for sequencing identification. The constructed plasmids are pTD103aiiA_EL222_10EB_sfGFP, pTD103aiiA_EL222_35EB10_sfGFP, and pTD103aiiA_EL222_35EB10EB_sfGFP.

1.4 Improvement of Population Synchronized Gene Oscillator

1.4.1 Acquisition of fluorescent protein

(1) Acquisition of mTagBFP fluorescent protein

①Add 10μl of sterile ddH2O to well 19I of iGEM 2015 kit plate 1, the solution turns red after the plasmid dry powder is dissolved. (Element source: iGEM 2015 kit plate 1 19I, plasmid backbone is pSB1C3, resistance is Cm.)

② Take 5 μl of plasmid and transform it into T1 competent cells, shake the bacteria for culture, and store the strains for future use.

③ Send the plasmid or bacterial solution to the sequencing company for sequencing and identification, and follow-up operations after confirming that the sequence is correct.

(2) Acquisition of mTagBFP2

① Design and synthesize primers for PCR reaction to change the mTagBFP sequence to mTagBFP2 (the first and second amino acids of mTagBFP are replaced with six amino acids of MVSKGE, and the amino acid I at position 174 is replaced with A). The template plasmid for PCR was pSB1C3-mTagBFP. PCR primers and amplified fragments are shown in Table 10-11.

Table 10: PCR reaction primers

Table 11: PCR amplified fragments

②Separation of target DNA fragments by agarose gel electrophoresis

③ Recovery of target DNA fragments

④ Recombination reaction

Add sample according to the reaction system in Table 12, 50°C water bath, 30min.

Table 12: Recombination reaction system

⑤Conversion

Use T1 competent cells for transformation, and the steps are the same as above.

⑥ Selection of recombinant recipient bacteria and preserved strains

⑦ After PCR and agarose gel electrophoresis are positive, the plasmid is sent to a sequencing company for sequencing identification.

1.4.2 Performance comparison of fluorescent proteins

In order to compare the protein brightness of mTagBFP fluorescent protein and mTagBFP2 fluorescent protein, and choose a more suitable fluorescent protein, in this experiment, the promoter before the fluorescent protein gene was replaced with pBAD, so that the protein expression could be controlled by the concentration of arabinose. Meanwhile, sfGFP fluorescent protein was used as a control in this experiment.

(1) Construction of fluorescent protein performance detection plasmid

First, the ligated fragments were obtained by PCR amplification, and then the target DNA fragments were separated by agarose gel electrophoresis. After recovery, the Gibson Assembly reaction was performed, and then operations such as transformation and plating were performed. The PCR primers and amplified fragments are shown in Table 13-14.

Table 13: PCR reaction primers

Table 14: PCR amplified fragments

(2) Performance comparison of fluorescent proteins

① Add the three performance-testing bacteria to the LB liquid medium containing 1% arabinose solution, and cultivate to log phase.

②Escherichia coli population fluorescence microscopic observation was carried out to compare the brightness of the three fluorescent proteins.

2. Construction of Gene Oscillating Elements

2.1 Replacement of fluorescent proteins for population-synchronized gene oscillatory elements

The construction of the population synchronization gene oscillator plasmid (pTD103luxI_mTagBFP2) with mTagBFP2 as the reporter protein requires three steps. First, construct the pTD_luxR_mTagBFP2 plasmid, then insert the luxI fragment to construct the pTD 103luxI_mTagBFP2V plasmid, and finally add the rapid degradation tag after the CDS region of the mTagBFP2 element.

The experimental method is Gibson Assembly, and the experimental steps are roughly the same as above. After the plasmid was constructed, it was amplified with primers P1 and m2-PV and digested with EcoR I endonuclease (the length of the fragment after digestion was 1864 bp and 3675 bp), and then agarose gel electrophoresis was carried out for inspection. The PCR amplified fragments and reaction primers are shown in Table 15-16.

Table 15: PCR amplified fragments

Table 16: PCR reaction primers

3. Integration of blue light-controlled gene oscillators

The pTD103luxI_mTagBFP2 plasmid and the pTD103aiiA_EL222_35EB10(Cm) plasmid were co-transformed into the MG1655 strain to form a blue light-controlled gene oscillator.

(1) Plasmid donor bacteria culture and plasmid extraction

The strains containing pTD103luxI_mTagBFP2, pTD103aiiA_EL222_35EB10(Cm) plasmids were shaken overnight to extract plasmids.

(2) Conversion

The above two plasmids were co-transformed into MG1655 competent cells.

(3) Selection of target colonies and preservation of strains

The experimental steps are the same as above.

(4) Enzyme digestion and sequencing test

①Enzyme digestion test: Digest with BglII restriction endonuclease, it is a single restriction site in both pTD103luxI_mTagBFP2 and pTD103aiiA_EL222_35EB10 plasmids, and linear fragments of 5539bp and 4589bp are formed after restriction enzyme digestion.

②Sequencing test

The primer sequences of the sequencing reaction are shown in Table 17.

Table 17: Sequencing reaction primers

Example 2 Construction of red light-controlled gene oscillator

1. Construction of red light control elements

1.1 Construction of pTD103aiiA_ho1_pcyA_aiiA plasmid

The integration of light control elements and oscillator elements on different plasmids is mainly carried out in two steps.

1.1.1 Construction of pEO100c_aiiA plasmid

(1) Plasmid donor bacteria culture and plasmid extraction: The strains containing pEO100c plasmid and pTD103aiiA(Cm) plasmid were shaken overnight to extract plasmid.

(2) Design primers for PCR amplification to obtain insert fragments and vector fragments for subsequent GibsonAssembly reaction. The PCR amplified fragments and reaction primers are shown in Table 18-19.

Table 18: PCR amplified fragments

Table 19: PCR reaction primers

(3) Separation and recovery of target DNA fragments by agarose gel electrophoresis

The experimental steps are the same as above.

(4)Gibson Assembly

Add samples according to the reaction system in Table 20, and bath at 50°C for 30min.

Table 20: Gibson Assembly reaction system

(5) Conversion

The plasmid obtained by Gibson Assembly was transformed into T1 competent.

(6) Selection of recombinant recipient bacteria and preserved strains

(7) Enzyme digestion and sequencing identification

The pEO100c_aiiA plasmid was digested with PstI restriction endonuclease to obtain a linear fragment with a length of 5372 bp. After the positive detection by agarose gel electrophoresis, the plasmid was sent to a sequencing company for sequencing and identification.

1.1.2 Construction of target plasmid

The pEO100c_aiiA plasmid described above was integrated with the pTD103aiiA_ho1_pcyA plasmid.

(1) Insert PCR

The template DNA is pEO100c_aiiA, a PompC+aiiA fragment is formed after PCR, and the length is 2193 bp. The PCR reaction primers used are shown in Table 21.

Table 21: PCR reaction primers

(2) Enzyme digestion of vector fragments

The vector plasmid was pTD103aiiA_ho1_pcyA, and a linear fragment with a length of 5372 bp was obtained after SpeI restriction endonuclease digestion.

Table 22: Enzymatic digestion reaction system

In the PCR tube, according to the principle of increasing the system first and then adding the smaller system, add the reaction systems shown in Table 24, mix well, flick to remove air bubbles, and centrifuge at low speed. Incubate for 1 h at 37°C for digestion.

(3) Separation of target DNA fragments and recovery of target DNA fragments by agarose gel electrophoresis

After the insert fragment and the vector fragment were separated by agarose gel electrophoresis, the target DNA fragment was recovered.

(4) Recombination reaction

Add the reaction system shown in Table 25 to the PCR tube, mix well and centrifuge at low speed. 50℃, warm bath for 30min.

Table 23: Recombination reaction system

(5) Transformation and coating

Transform into T1 competent, the steps are the same as above.

(6) Selection of recombinant recipient bacteria and preserved strains

(7) Enzyme digestion test and delivery test

The target plasmid was digested with Pst1 endonuclease, and it was estimated that the plasmid was cut into two parts with lengths of 4625bp and 2918bp, respectively. After agarose gel electrophoresis was positive, the plasmid was sent to a sequencing company for sequencing and identification.

2 Construction of gene oscillatory elements

Construction of pTD103luxI_sfGFP_Cph8(Amp) plasmid:

Because the resistance of the pTD103luxI_sfGFP_Cph8(Kan) plasmid is the same as that of the host strain JT2, it is easy to cause the loss of the plasmid during the culture process, so it is necessary to replace the Kan gene with the Amp gene.

(1) Using pEO100c_aiiA as a template and AMP-fwd and AMP-rev as primers, the target fragment was amplified, and two restriction sites, BglII and SpeI, were added to both ends of the Amp gene fragment. The length of the target fragment is 1172bp, the PCR reaction system and reaction procedure are the same as above, and the reaction primers are shown in Table 24.

Table 24: PCR reaction primers

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

① Take 50 μl of the PCR product of the Amp gene fragment, add 250 μl of solution BB, mix well, add to the spin column, stand for 1 min, centrifuge at 10,000 × g for 1 min, and discard the effluent.

② Add 650 μl of solution WB, centrifuge at 10,000 × g for 1 min, and discard the effluent.

③ Centrifuge at 10,000 × g for 2 min to completely remove residual WB.

④Put the spin column in a clean centrifuge tube, and add 30 μl of solution EB to the center of the column. After standing at room temperature for 1 min, centrifuged at 10,000 × g for 1 min to elute the DNA to obtain the purified PCR product.

(3) Enzyme cleavage reaction

The PCR-purified product of Amp gene fragment and pTD103luxI_sfGFP_Cph8(Kan) plasmid were both digested with SpeI and BglII. Because the optimal buffers of the two enzymes are different, the products were first digested with BglII, and then the product was purified, and then digested with SpeI.

BglII digestion: In the PCR tube, according to the principle of increasing the system first and then reducing the system, add the reaction systems shown in Table 25, mix well, flick to remove air bubbles, and centrifuge at low speed. Incubate for 15 min at 37°C for digestion.

Table 25: Enzymatic digestion reaction system

②Enzyme digestion product purification (the steps are the same as PCR product purification)

SpeI digestion: Add the reaction system shown in Table 26 to the PCR tube, and incubate at 37°C for 1 hour for digestion.

Table 26: Enzymatic digestion reaction system

(4) Separation of target DNA fragments by agarose gel electrophoresis and recovery of target DNA fragments

The steps are the same as above. The length of the Amp gene fragment after digestion is 1155bp, and the length of the pTD103luxI_sfGFP_Cph8(Kan) vector fragment after digestion is 7129bp.

(5) Enzyme ligation reaction (DNA Ligation Kit Ver.2.1)

Add samples to a centrifuge tube according to the reaction system shown in Table 27, and react at 16 °C for 30 min.

Table 27: DNA ligation reaction system

(6) Transformation, coating plate

Transform into T1 competent, the steps are the same as above.

(7) Selection of target strains and preservation strains

(8) PCR, test delivery

PCR reactions were carried out with AMP-fwd, AMP-rev and CPH8-fwd, CPH8-rev shown in Table 26 as primers, respectively, to form Amp and CPH6 fragments, which are estimated to be 1197 and 2235 bp, respectively. Then, agarose gel electrophoresis was carried out for detection. After the detection was positive, the plasmid was sent to a sequencing company for sequencing identification.

3 Integration of red light-controlled gene oscillators

(1) The pTD103luxI_sfGFP_Cph8(Amp) and pTD103aiiA_ho1_pcyA_aiiA plasmids were co-transformed into the JT2 E. coli strain, and the experimental steps were the same as above.

(2) Selection of target colonies and preservation of strains

(3) Enzyme digestion and agarose gel electrophoresis verification

In a PCR tube, add samples according to the system shown in Table 30. 37 ℃, warm bath for 1h.

After digestion with PstI, the plasmid pTD103LuxI_sfGFP_Cph8(Amp) has 3 restriction sites, and the plasmid pTD103 aiiA_ho1_pcyA_aiiA has 2 restriction sites. The two plasmids were digested with PstI to obtain 5 fragments with the lengths of 1867bp, 2379bp and 2920bp respectively. , 3948bp, 4609bp.

Table 28: Enzymatic digestion reaction system

(4) A positive result by agarose gel electrophoresis indicates that the target plasmid has been transferred into JT2.

Example 3 Fluorescence observation of Escherichia coli

1 Escherichia coli smear observation

1.1 Preparation of Escherichia coli smear

(1) Get the MG1655 E. coli containing pBAD_sfGFP, pBAD_mTagBFP, pBAD_mTagBFP2 plasmids, and culture them in LB broth containing arabinose solution and Cm antibiotics for 3-4h to the mid-log phase. (The above culture is the selection experiment of the reporter protein of the oscillatory system, and the population fluorescence brightness detection is carried out; the blue light control system effect comparison experiment, the MG1655 E. culture, and perform single-cell fluorescence brightness detection.)

(2) Take a 1.5mL sterilized centrifuge tube, add 1.5mL bacterial solution, centrifuge at 12000×g and remove the supernatant. Using the remaining little supernatant, the Escherichia coli pellet was vortexed on a high-speed vortexer to form a uniform thick bacterial liquid, which was used for the fluorescence brightness detection of the Escherichia coli population. (Escherichia coli single cell fluorescence brightness detection: take 0.9% normal saline and dilute the Escherichia coli culture solution by 1%.)

(3) Take a clean glass slide, suck 5 μl of the above bacterial solution, and after the slide glass is evenly coated, gently cover it with a cover glass, taking care not to have air bubbles. Finally, use absorbent paper to clean up excess liquid around the coverslip.

1.2 Fluorescence Microscopic Observation

(1) Place the prepared smear on the microscope stage, and press it with a tablet clamp, at this time, the sample is facing the light-transmitting hole.

(2) Start observation from a low magnification lens (10×), adjust the focus, rotate the coarse focus screw, and slowly raise the stage until the objective lens is close to the smear. At this time, look at the objective lens to prevent the objective lens from hitting the smear. Then look at the eyepiece with both eyes, turn the coarse focus screw in the opposite direction to lower the stage until the object image is observed, and then turn the fine focus screw to adjust slightly to make the object image seen more clearly.

(3) After finding the corresponding observation point, convert the low magnification objective lens to a high magnification objective lens (40×), and then turn the fine focus screw slightly to see a clear object image.

(4) Switch the microscope to the fluorescence observation mode, select the corresponding excitation light, and perform fluorescence observation.

1.3 Microfluidic chip observation

1.3.1 Culture and concentration of bacterial liquid

(1) Add 30 μl of the revived bacterial solution to 3 mL of LB medium containing the corresponding antibiotics, place it on a constant temperature shaker, and incubate at 37°C for 3-4 hours to reach the mid-log phase.

(2) Take a 1.5mL centrifuge tube, add 1.5mL bacterial solution, centrifuge at 2000r/min for 5min, remove the supernatant, leave only a little residual culture solution, and then vortex and mix the bacterial precipitate on a vortexer. Uniform thick bacterial liquid.

1.3.2 Sample loading

(1) Prepare three capillaries of appropriate length, place them and microfluidic chips and other materials in an ultra-clean bench, and sterilize them by ultraviolet light for 20min.

(2) Put the microfluidic chip in a vacuum hood and vacuumize it, which takes about 10 minutes.

(3) In the ultra-clean bench, after sucking part of the concentrated bacterial solution with a 10 μl pipette, insert the pipette tip into the liquid inlet of the chip, and start injecting the bacterial liquid from the liquid inlet.

(4) Select the syringe needle of the corresponding specification, and cover the capillary over the needle, so that the two are closely connected. Use a syringe to draw the culture solution containing the corresponding antibiotics, connect the syringe to the needle, press the piston handle to fill the entire capillary with the culture solution, and then insert the other end of the capillary into the liquid inlet of the chip. At the same time, insert the other two capillaries into the liquid outlet of the chip.

1.3.3 Photogenic observation

Fix the microfluidic chip on the stage, and fix the syringe on the syringe pump, and start injection and observation. Pay attention to pressing the plunger handle before observation, so that the culture medium can quickly pass through to flush the chip channel, so as to avoid a large number of bacteria growing in the channel and cause blockage.

Example 4 Virtual oscillator modulates light-controlled gene oscillator to achieve stable oscillation

The present invention writes a population-synchronized genetic oscillator simulation program (i.e., a virtual oscillator) based on the mathematical model of the population-synchronized genetic oscillator (as shown in Figure 1B). The oscillation period and amplitude of the virtual oscillator are 50 min and 201, respectively, and the oscillation period and amplitude are relatively stable. The oscillation pattern is shown in Figure 1C. The virtual oscillator can control the illumination light source of the microscope according to the oscillation observation of its gene oscillator, thereby affecting the expression of the aiiA gene. When the virtual oscillation value is less than 100, the light intensity emitted by the microscope changes linearly; when the oscillation value exceeds 100, the microscope is illuminated at 100% of the maximum intensity (as shown in Figure 1A). This regulatory mechanism makes the light intensity applied by us exhibit a stable oscillatory change, so that the expression of the aiiA element in the gene oscillator exhibits a stable pattern, and finally makes the oscillatory behavior of the entire gene oscillator more robust.

In the computer, the virtual oscillator is an independent program that can write the amplitude value of the virtual oscillation into a file for other programs to read. During the observation and photographing process, a program was written in the microscope software to read the virtual oscillation value, so that the microscope light source emits illumination light of corresponding intensity, and fluorescence photographing was performed at intervals of 15 min.

Through the regulation of the above virtual oscillator, the oscillation results of the light-controlled gene oscillator are as follows:

1. Blue light-controlled robust oscillation system

Continuous microscopic observation was performed on the blue light-controlled gene oscillator in the microfluidic chip, and the fluorescence brightness changes of mTagBFP2 fluorescent protein were recorded. As shown in Figure 2A, when no regulation is applied, the oscillation phenomenon of the blue light-controlled gene oscillator is not stable enough, and the period and amplitude of the oscillation vary greatly (σ period = 0.195, σ amplitude = 0.048); as shown in Figure 2B, The robustness of the oscillator was greatly increased after imposition of the virtual oscillation regulation, and the period and amplitude gradually became more stable (σ period = 0.083, σ amplitude = 0.038). When the blue light regulation was just carried out, the amplitude of each oscillation changed greatly, which may be due to the hysteresis of regulation, which led to a large fluctuation in the synthesis amount of the reporter protein. In summary, in the blue light-controlled stable oscillation system, the virtual oscillator is effective in regulating the blue light-controlled gene oscillator. (Note: Standard deviation σ=sqrt(((x1-x)2+(x2-x)2+...(xn-x)2)/n), where x1, x2.... ..xn is each data value, x is the average value of the data, and n is the amount of data.)

2. Red light-controlled robust oscillation system

Continuous microscopic observation was performed on the same chamber of the microfluidic chip. As shown in Figure 3A, when no regulation was applied, the oscillation phenomenon of the red light-controlled gene oscillator was relatively unstable, and the period and amplitude of each oscillation changed significantly. large (σ period = 0.291, σ amplitude = 0.068); as shown in Figure 3B, after applying virtual oscillator regulation, the oscillator's robustness is greatly increased, and the period and amplitude become stable (σ period = 0.079, σ amplitude = 0.013 ). This result indicates that the regulation of virtual oscillators enhances the robustness of red light-controlled gene oscillators. (Note: Standard deviation σ=sqrt(((x ₁ -x) ² +(x ₂ -x) ² +...(x _n -x) ² )/n), where x ₁ , x ₂ ......x _n is each data value, x is the average value of the data, and n is the amount of data.)

The above-described embodiments only represent several embodiments of the present invention, and the descriptions thereof are more specific and detailed, but should not therefore be construed as limiting the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several replacements, modifications and improvements can be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

SEQUENCE LISTING

<110> Huazhong Agricultural University

<120> A Light-Controlled Robust Gene Oscillation System

<130> 5.6

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 4589

<212> DNA

<213> Synthetic

<400> 1

gcctaatatt tttgaaatat cccaagagct ttttccttcg catgcccacg ctaaacattc 60

ttttttctctt ttggttaaat cgttgtttga ttttattattt 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 ttttttattta 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

gattttttgtc 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 ttattttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt 3240

ctggttatag gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca 3300

ttgggatata tcaacggtgg tatatccagt gattttttttc 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> Synthetic

<400> 2

ctcgagttaa ttttttaaagt 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 ttttttctgaa 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 ggttttttttg 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> Synthetic

<400> 3

ctcgagttaa ttttttaaagt 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 ttttttctgaa 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 ttttttcttta 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> Synthetic

<400> 4

ctcgagttaa ttttttaaagt 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 ttttttctgaa 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 ttattttgca 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 atttttattatt tacagcattg ttttcaaata 2340

tattccaatt aattggtgaa tgattggagt tagaataatc tactatagga tcatatttta 2400

ttaaattagc gtcatcataa tattgcctcc atttttttagg 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 ggtggtttttt 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 ccgttatgg 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 light-controlled robust gene oscillation system, characterized in that it comprises a light-controlled gene oscillator and a regulation light source; the light-controlled gene oscillator is formed by integrating a light-controlled element and a gene oscillation element in a cell; the The control light source includes a calculation control component and a light source, the calculation control component can send a control signal in real time based on the oscillation observation value of the light-controlled gene oscillator, and adjust the illumination intensity and illumination time of the light source; the light-controlled gene oscillator According to the adjusted light source irradiation intensity and irradiation time, its oscillation behavior is adjusted; wherein, the light control element is a plasmid containing a gene fragment encoding AiiA protein; the gene oscillation element is a plasmid containing a gene fragment encoding a fluorescent reporter protein, and the The oscillation observation value is the fluorescence detection value emitted by the fluorescent reporter protein detected by the calculation control component. 2 . The light-controlled robust gene oscillation system according to claim 1 , wherein the light-controlled element is an optogenetic gene expression control module. 3 . 3 . The light-controlled robust gene oscillation system of claim 1 , wherein the light source is a light source of a specific wavelength capable of causing an optogenetic molecular response. 4 . 4. The light-controlled robust gene oscillation system according to claim 1, characterized in that, the calculation control component comprises a computer program with digital logic operation and logic processing functions, an intelligent mobile device, an embedded hardware system, a digital circuit, Any one or a combination of digital optical path, analog operation circuit or analog operation optical path. 5. The light-controlled robust gene oscillation system according to claim 1, wherein the light-controlled gene oscillator includes but is not limited to a blue light-controlled gene oscillator and a red-light light-controlled gene oscillator, and the blue light The control gene oscillator is regulated by blue light, and the red light light control gene oscillator is regulated by red light, wherein the fluorescent reporter protein of the blue light control gene oscillator is mTagBFP2, and the fluorescent reporter protein of the red light light control gene oscillator is sfGFP. 6 . The light-controlled robust gene oscillation system according to claim 5 , wherein the light-controlled element of the blue light-controlled gene oscillator is a plasmid as shown in SEQ NO: 1. 7 . 7 . The light-controlled robust gene oscillation system according to claim 5 , wherein the gene oscillation element of the blue light-controlled gene oscillator is a plasmid as shown in SEQ NO: 2. 8 . 8 . The light-controlled robust gene oscillation system according to claim 5 , wherein the light control element of the red light light-controlled gene oscillator is a plasmid as shown in SEQ NO: 3. 9 . 9 . The light-controlled robust gene oscillation system according to claim 5 , wherein the gene oscillation element of the red light light-controlled gene oscillator is a plasmid as shown in SEQ NO: 4. 10 .

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