WO2020164195A1 - 一种基于双荧光报告基因系统鉴定生物元件的方法及基于该方法构建的生物元件库 - Google Patents

一种基于双荧光报告基因系统鉴定生物元件的方法及基于该方法构建的生物元件库 Download PDF

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WO2020164195A1
WO2020164195A1 PCT/CN2019/086173 CN2019086173W WO2020164195A1 WO 2020164195 A1 WO2020164195 A1 WO 2020164195A1 CN 2019086173 W CN2019086173 W CN 2019086173W WO 2020164195 A1 WO2020164195 A1 WO 2020164195A1
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reporter gene
fluorescent reporter
gene system
dual
seq
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PCT/CN2019/086173
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French (fr)
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杨世辉
杨永富
沈威
李闰霞
黄钜
王禺
易犁
马立新
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湖北大学
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Priority claimed from CN201910114932.1A external-priority patent/CN109852631A/zh
Priority claimed from CN201910114652.0A external-priority patent/CN109777862A/zh
Priority claimed from CN201910114635.7A external-priority patent/CN109913487B/zh
Application filed by 湖北大学 filed Critical 湖北大学
Priority to US16/769,558 priority Critical patent/US11248258B2/en
Publication of WO2020164195A1 publication Critical patent/WO2020164195A1/zh

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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for identifying biological elements based on a dual fluorescent reporter gene system and a biological element library constructed based on the method.
  • Z. mobilis is a gram-negative, facultative anaerobic bacteria, with a peripheral flagellum, the size is 1.4-2.0*4.0-5.0 ⁇ m; the suitable growth temperature is 30°C, which can be tolerated
  • the pH range is between 3.5-9, which can efficiently utilize glucose, fructose and sucrose to produce ethanol through the Enter-Doudoroff pathway (ED pathway).
  • Z.mobilis has many advantages such as high ethanol yield and tolerance, high osmotic pressure tolerance, low biomass, and no need to add oxygen during fermentation, making it one of the main bioethanol production strains. In order to increase the scope of application of this strain, research on system biology for Z. mobilis has been successively carried out. At the same time, with the development of metabolic engineering and synthetic biology, the demand for the biological components available in Z. mobilis has also increased.
  • Promoter is an important element for regulating gene expression, and is a site where RNA polymerase recognizes and specifically binds. Promoter strength is generally used to describe the frequency at which RNA polymerase initiates transcription at the promoter. The selection of a certain strength promoter during biosynthesis can assist the smooth progress of biosynthesis.
  • the traditional method of screening promoters is to randomly shear the genome, and then use the expression of a single reporter gene downstream to identify whether the sequence obtained is a promoter sequence and how strong its activation is.
  • Most of the traditional detection systems are single reporter gene systems, but the expression of the reporter gene in a single reporter gene system will be affected by multiple factors from inside and outside the strain, which will affect the identification of the promoter by the system.
  • the speed of obtaining the promoter to be tested by the previous method is slow, and the strength identification cannot be performed quickly and with high throughput, which is not conducive to the efficient quantitative study of the promoter.
  • the existing system biology data is used to develop and mine biological elements that can be used in the metabolic engineering of Z. mobilis, especially the specific strength of the promoter and ribosome binding site, and the sRNA-UTR effect is equivalent, and
  • the establishment of an efficient, fast, and high-throughput method for quantitative analysis of promoters and other biological elements in vivo is of great significance for expanding the element library of Z. mobilis promoters and other biological elements; at the same time, the method can also be applied In other microbial systems, it provides diverse biological functions and regulatory elements for metabolic engineering and synthetic biology
  • the present invention provides a method for identifying biological elements based on a dual fluorescent reporter gene system and a biological element library constructed based on the method.
  • a method for identifying biological elements based on a dual fluorescent reporter gene system including the following steps:
  • step S6 Transform the biological element to be tested and the skeleton of the dual fluorescent reporter gene system obtained in step S5 into E. coli DH5 ⁇ through the improved Gibson assembly method, verify the positive clone on the plate by PCR, and extract the plasmid after overnight culture;
  • step S7 Transform the plasmid extracted in step S6 into ZM4 competent cells, activate the culture to the logarithmic phase, and use flow cytometry to detect and verify.
  • the fluorescent protein is all activated by the promoter PlacUV5, and the fluorescent protein is EGFP, mCherry, RFP, CFP, and any one of opEGFP, opmCherry and opCFP optimized by codons.
  • promoter number 1 is Ptet
  • promoter number 2 is PlacUV5
  • fluorescent reporter gene number 1 and fluorescent reporter gene number 2 are EGFP and opmCherry, respectively.
  • forward and reverse primers in step S3 are primer 1 and primer 2
  • sequences of primer 1 and primer 2 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • forward and reverse primers in step S5 are primers Prtt-F and Prtt-R respectively, and the sequences of primers Prtt-F and Prtt-R are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively;
  • the forward and reverse primers in step S5 are primers Prtt-F and PgapTSS-R, respectively, and the sequences of primers Prtt-F and PgapTSS-R are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the biological element to be tested in step S6 is an endogenous promoter of different strengths, or a promoter containing synthetic RBS sequences of different strengths, or a terminator of different strengths, or a sRNA-UTR action pair.
  • the biological element to be tested is endogenous promoters of different strengths or promoters containing synthetic RBS sequences of different strengths
  • the method for obtaining the promoters containing RBS sequences of different strengths includes the following steps:
  • Sa Predict the sequences of ribosome binding sites of different strengths
  • the biological element to be tested is terminator of different strength
  • the method of obtaining the terminator of different strength includes the following steps:
  • S01 Screening gene sets with large differences in expression of adjacent genes in the same transcription direction, and sorting them according to expression differences;
  • S02 Use bioinformatics methods to predict the terminator sequence between adjacent genes, and use the expression difference of adjacent genes to represent the terminator strength;
  • S03 Design primers for the target terminator sequence, perform PCR amplification, and obtain terminator fragments.
  • the biological element to be tested is an sRNA-UTR interaction pair
  • the method for obtaining the UTR fragment includes the following steps:
  • S0a Analyze the target sequence using bioinformatics methods, and after determining the transcription start site, reserve the target 5’UTR sequence from the transcription start site to 99-bp after the start codon ATG;
  • S0b Using Z.mobilis genome as a template, design forward and reverse primers for PCR amplification to obtain target UTR sequence fragments;
  • PCR amplification is performed to obtain the dual fluorescent reporter gene system skeleton.
  • step S7 the plasmid is transformed into ZM4 competent cells by electrotransformation, and the specific transformation conditions are as follows:
  • step S7 a flow cytometer is used to verify the intensity detection, and the specific steps are as follows:
  • the biological elements to be tested in step S6 are promoters of different strengths or promoters containing RBS sequences of different strengths.
  • the method for obtaining the promoters containing RBS sequences of different strengths includes the following steps:
  • the second objective of the present invention is to provide a biological element library constructed based on the method for identifying biological elements by a dual fluorescent reporter gene system.
  • the present invention uses Zymomonas mobilis as a model strain, and establishes a method for predicting and screening promoter sequences of different strengths using system biology data.
  • the predictive screening method is efficient and fast, and can be used as a guiding basis for experiments.
  • the present invention can integrate existing system biology data to quickly and efficiently screen promoters of specific strength; this method predicts the strength of RBS and promoters to be screened, and has good correlation with experimental data (respectively R 2 > 0.9, R 2 >0.7), indicating that the method of screening promoters based on system biology data proposed by the present invention can be used for predictive screening of promoters and other biological elements with different requirements.
  • this method can quickly and high-throughput screen and quantify promoters of different strengths and other biological elements, and the quantitative analysis of the promoters is completed in the cell body, which is affected by the inside and outside of the cell. The impact of environmental changes is small, and the quantification is accurate. It can quickly expand the biological element library of Zymomonas mobilis and be used for metabolic engineering of different needs.
  • the present invention uses the existing system biology data and bioinformatics methods to predict and screen biological elements of different intensities, and conducts intensity identification and verification through the dual fluorescence reporter gene system developed by our laboratory, which is used for quantitative analysis of promoters and other biological elements.
  • the strength of the components find biological components that can be used in metabolic engineering and synthetic biology. Combine the two methods to establish a strategy that can quickly select biological components to be used for rational design and modification of microorganisms in the era of synthetic biology.
  • the dual fluorescence reporter gene system developed by this method can be used to quantitatively analyze other biological elements, and can also be used in other species except Zymomonas mobilis.
  • the advantages of the present invention are shown in the following table.
  • the traditional method of identifying the sRNA-UTR interaction relationship is very cumbersome. Moreover, the detection is generally performed in vitro, which cannot avoid the trouble of obtaining experimental materials in vitro and conducting related experiments in vitro, which makes it impossible to quickly determine whether there is an interaction between sRNA and the target UTR, which is not convenient for batch operation and cannot be used for batch quantification. Analyze the strength of the interaction between the target sRNA and its target 5'UTR.
  • the invention is convenient and quick, and compared with the traditional method for identifying the sRNA-UTR interaction relationship, the operation is very simple.
  • the present invention is based on the dual fluorescence reporter gene system of flow cytometry, which can be detected in vivo, eliminating the trouble of obtaining experimental materials in vitro and conducting related experiments in vitro, can quickly determine whether there is an interaction relationship between sRNA and target UTR, and is convenient Batch operation.
  • the dual fluorescent reporter gene system used in the method of the present invention can be used to quantitatively analyze the strength of the interaction, and can also be transplanted to other species except Zymomonas mobilis.
  • the present invention provides a 5'UTR for identifying sRNA and its target mRNA. The interaction method is established to quickly and efficiently determine the interaction relationship between the target sRNA and its target 5'UTR, and quantitative analysis can be performed.
  • the present invention screened and identified 37 promoters and 4 ribosomal binding sites (RBS) strengths, 4 pairs of sRNA-UTR action pairs and The strength of the 6 terminators greatly expands the element library of Zymomonas mobilis. It can fully exploit and utilize the system biology of Zymomonas mobilis and different microbial systems and expand its biological element library for metabolic engineering and synthetic biology practice. The method is simple and easy to operate, has a wide range of applicable biological elements, and can be extended to other species.
  • RBS ribosomal binding sites
  • A is a single fluorescent reporter gene system diagram
  • B is a screened fluorescent protein expression result diagram
  • Figure 2 is a diagram of the dual fluorescent reporter gene system
  • Figure 3 is a schematic diagram of the verification results of the dual fluorescent reporter gene system at different levels
  • A is the flow cytometer mode;
  • B is a schematic diagram of data correlation;
  • Figure 5 is a schematic diagram of Wien analysis and screening of promoters of different strengths
  • A is a schematic diagram of the verification results of different intensities of RBS;
  • B is a schematic diagram of the correlation between different intensities of RBS and tetracycline concentration;
  • FIG. 7 is a flow chart of the method for identifying sRNA-UTR interaction provided by the present invention.
  • TSS expresses the transcription start site
  • ATG represents the start codon
  • WT represents the wild-type strain
  • AsRNA represents the target sRNA knockout strain
  • OE_sRNA represents the target sRNA overexpression strain
  • Figure 8 is a schematic diagram of the experimental results of the interaction between sRNA and its target UTR provided by the present invention.
  • the present invention discloses a method for identifying biological elements based on a dual fluorescent reporter gene system and a biological element library constructed based on the method, as shown in the following examples.
  • Embodiment 1 A method for identifying biological elements based on a dual fluorescent reporter gene system
  • S1 Use the pEZ15Asp plasmid as the backbone to construct a single fluorescent reporter gene system, as shown in Figure 1A, to screen fluorescent proteins.
  • the fluorescent protein is EGFP, mCherry, RFP, CFP, and opEGFP, opmCherry and opCFP which are optimized by codons.
  • the fluorescent protein is all activated by the promoter PlacUV5.
  • S2 Send each fluorescent protein gene sequence to a third-party company for gene sequence synthesis; use pEZ15Asp as a template, and use primer 1 and primer 2 for PCR amplification to obtain the pEZ15Asp backbone; use Gibson assembly to combine the PlacUV5-fluorescent reporter gene with The pEZ15Asp backbone is connected, and the single fluorescent reporter gene system is obtained by transforming E.
  • Primer 1 5'-GCGCTAGCGGAGTGTATACTGGCTTACTATGTT-3', SEQ ID NO: 1;
  • Primer 2 5'-ACGGTGAGCTGGTGACCTGCCTTATC-3', SEQ ID NO: 2.
  • fluorescent reporter gene No. 1 and fluorescent reporter gene No. 2 According to the fluorescence expression intensity of different fluorescent protein genes in Zymomonas mobilis, select suitable fluorescent reporter genes and named them fluorescent reporter gene No. 1 and fluorescent reporter gene No. 2, respectively.
  • Promoter No. 1 uses Ptet and Promoter No. 2 No. chooses PlacUV5.
  • FIG. 1B The expression results are shown in FIG. 1B.
  • the fluorescent proteins EGFP and opmCherry have high fluorescence intensity expression in Z. mobilis, therefore, they were used as fluorescent reporter gene No. 1 and fluorescent reporter gene No. 2 to participate in subsequent experiments.
  • primer 1 and primer 2 are designed for PCR amplification to obtain the pEZ15Asp backbone, and the PCR product is recovered and purified according to the PCR recovery kit.
  • PCR program settings 98°C pre-denaturation for 3min, 98°C denaturation for 10s, 55°C annealing for 10s, 72°C extension for 35s, 29 cycles.
  • step S4 Using the improved Gibson assembly method, connect Ptet-fluorescent reporter gene No. 1 or Ptet-EGFP, and PlacUV5-fluorescence reporter gene No. 2, or PlacUV5-opmCherry, to the pEZ15Asp skeleton obtained in step S3, and connect them to the A terminator is added to the reporter gene to obtain an inducible dual fluorescence reporter gene system, as shown in Figure 2.
  • the terminator added in the middle of the fluorescent reporter gene is BBa_B0014, and the sequence is:
  • the terminator used in the 3'of the two fluorescent protein genes is the rrnB T1 terminator, and the sequence is:
  • antibiotics can be selected according to actual needs. After 12-24 hours, select 5-10 single colonies with clear edges for colony PCR to select recombinants, and then select the 2 clearest bands and send them to the sequencing company for sequencing verification. After obtaining the sequencing sequence, compare it with the original sequence to confirm Its sequence correctness.
  • the sequencing After the sequencing is correct, use the conventional plasmid extraction kit (Plasmid Miniprep Kit, Tsingke) to extract the plasmid. After the plasmid is obtained, the plasmid is electroporated into wild-type Z. mobilis, and the final strain is determined by colony PCR and sequencing. The sequencing is completed by Tsingke After obtaining the sequencing sequence, compare it with the original sequence to confirm its correctness.
  • the conventional plasmid extraction kit Plasmid Miniprep Kit, Tsingke
  • Rich media (RM) medium formula: RMG5: 50g/L glucose, 10g/L yeast extract, and 2g/L KH 2 PO 4 , spectinomycin resistance is 200 ⁇ g/mL. Culture conditions: 30°C, 100 rpm.
  • FCM flow cytometry
  • the single clone verified by PCR positive clones and loaded into the dual fluorescence reporter gene system is activated and cultured in RM medium with 200 ⁇ g/mL spectinomycin; other types can also be selected according to actual needs in other embodiments And the concentration of antibiotics.
  • sample 200 ⁇ L After incubating to the logarithmic phase, sample 200 ⁇ L, centrifuge at 12000 rpm for 1 min, remove the supernatant, and wash twice with 1 ⁇ PBS before resuspending.
  • the excitation wavelength of EGFP is 488nm
  • the detector is FITC
  • the excitation wavelength of opmCherry is 561nm
  • the detector is PC5.5. Take the average of three replicate samples and take the logarithm of log2 for subsequent data analysis.
  • qPCR fluorescent quantitative PCR
  • the transcription levels of EGFP and opmCherry in the dual fluorescence reporter system are performed under the same thermal cycling conditions.
  • the TRIzol (Invitrogen, USA) method was used to extract the total RNA of the log phase samples, and the NanoDrop 8000 was used to detect the quality of the extracted RNA. Then use the commercial kit iScript TM gDNA Clear cDNA Synthesis Kits (Bio-Rad, USA) instructions to remove genomic DNA and reverse transcription into cDNA. iTaq TM Universal The Green Supermix (Bio-Rad, USA) kit was performed on the CFX96 Real-Time System (Bio-Rad, USA) instrument.
  • the primers used in the experiment are high-purity salt-free primers whose annealing temperature is the same at 60°C.
  • the primer sequence is as follows:
  • EGFP or mCherry primary antibody (Proteintech, China) at a ratio of 1:5000. After 1 hour in the greenhouse, wash with 1x PBST every 5 minutes and wash 3 times. Then apply EGFP or mCherry's secondary antibody (Peroxidase-conjugated goat anti-Mouse IgG) at a ratio of 1:5000. After 1 hour in the greenhouse, wash with 1x PBST every 5 minutes and wash 3 times.
  • the ImmobilonTM Western Chemiluminescent HRP Substrate was prepared at a ratio of 1:1, and the AI600 Imaging System (GE, USA) was used for imaging.
  • PCR program settings pre-denaturation at 98°C for 3min, denaturation at 98°C for 10s, annealing at 55°C for 10s, extension at 72°C for 50s, 29 cycles.
  • Prtt-R 5'-ACTAGTAGCGGCCGCTG-3' SEQ ID NO: 4.
  • step S6 Transform the biological element to be tested and the skeleton of the dual fluorescent reporter gene system obtained in step S5 into E. coli DH5 ⁇ by the Gibson assembly method.
  • antibiotics can be selected according to actual needs. After 12-24 hours, select 5-10 single colonies with clear edges for colony PCR to select recombinants, and then select the 2 clearest bands and send them to the sequencing company for sequencing verification. After obtaining the sequencing sequence, compare it with the original sequence to confirm Its sequence correctness.
  • Colony PCR program settings pre-denaturation at 98°C for 3 minutes, denaturation at 98°C for 10 seconds, annealing at 55°C for 10 seconds, extension at 72°C for 30 seconds, 25 cycles.
  • the sequencing After the sequencing is correct, use the conventional plasmid extraction kit (Plasmid Miniprep Kit, Tsingke) to extract the plasmid. After the plasmid is obtained, the plasmid is electroporated into wild-type Z. mobilis, and the final strain is determined by colony PCR and sequencing. The sequencing is completed by Tsingke After obtaining the sequencing sequence, compare it with the original sequence to confirm its correctness.
  • the conventional plasmid extraction kit Plasmid Miniprep Kit, Tsingke
  • step S7 Transform the recombinant plasmid extracted in step S6 into ZM4 competent cells, activate the culture to the logarithmic phase, and use flow cytometry to detect and verify.
  • Example 2 Method for obtaining endogenous promoters of different strengths and identification of endogenous promoters of different strengths
  • Screening of endogenous promoters Screen out genes with strong downstream expression according to different omics data, and conduct Venn analysis, as shown in Figure 5.
  • the common genes of each omics data are screened out.
  • 19 strong promoters, 9 medium-strength promoters, and 10 weak promoters were screened, as shown in the table below.
  • step S6 an endogenous Pgap promoter with a certain strength is used as the biological element to be tested for the experiment. In other embodiments, other strengths can also be used Promoters or promoters containing different strength RBS sequences or other biological elements for detection.
  • the primers P0177-F and P0177-R were used for PCR amplification to obtain the promoter fragments.
  • the lowercase letters at the 5'end of the primers are the homology arms of the dual fluorescent reporter gene system.
  • the PCR product was recovered and purified according to the PCR recovery kit.
  • PCR program settings 98°C pre-denaturation for 3 minutes, 98°C denaturation for 10 seconds, 55°C annealing for 10 seconds, 72°C extension for 10 seconds, 29 cycles.
  • step S7 Transform the recombinant plasmid extracted in step S6 into ZM4 competent cells, activate the culture to the logarithmic phase, and use flow cytometry to detect and verify.
  • the recombinant plasmid was electrotransformed into ZM4 competent cells, the specific method is as follows: take ZM4 competent cells on ice, after the competent cells have melted, take 50 ⁇ L into the electrorotor cup, and add 1 ⁇ g plasmid to the electrorotor cup .
  • the electric transfer conditions are 1600V, 25 ⁇ F, 200 ⁇ .
  • Colony PCR program settings pre-denaturation at 98°C for 3 minutes, denaturation at 98°C for 10 seconds, annealing at 55°C for 10 seconds, extension at 72°C for 30 seconds, 25 cycles.
  • Flow cytometry detection activate 38 correct monoclonals in RM medium with 200 ⁇ g/mL spectinomycin (in other embodiments, other types and concentrations of antibiotics can be selected according to actual needs). Incubate 3 samples in parallel. After culturing to the logarithmic phase, sample 200 ⁇ L, centrifuge at 12000 rpm for 1 min, remove the supernatant, wash twice with 1 ⁇ PBS and resuspend, and perform flow cytometry detection with the set program. To prevent small probability and accidental events, the present invention sets the cell collection event to 20,000.
  • FIG. 4 a schematic diagram of the flow cytometer mode and data correlation provided by the embodiment of the present invention.
  • the results show that loading the biological element promoter in the dual fluorescent reporter gene system can be used for rapid quantitative analysis by flow cytometry.
  • the ratio of EGFP/opmCherry indicates the relative strength of the tested promoter in the system, and the experimental results are consistent with the group.
  • the correlation between the predicted intensities of the scientific data is relatively high, indicating that the dual fluorescent reporter gene system of the present invention can be used for the identification of promoter strength.
  • the Z.mobilis 16S rRNA sequence is as follows, see SEQ ID NO: 10 for details.
  • Table 2 shows the different strength RBS sequences of Zymomonas mobilis provided in the embodiments of the present invention.
  • RBS-10 is taken as an example, using a dual fluorescent reporter gene system containing a Ptet promoter as a template, and primers pEZ-tetR-F, RBS-10-R Carry out PCR amplification, wherein the lowercase letter at the 5'end of the primer is the homology arm of the double fluorescent reporter gene system, and a promoter with a specific strength RBS sequence is obtained.
  • PCR program settings 98°C pre-denaturation 3min, 98°C denaturation 10s, 55°C annealing 10s, 72°C extension 20s, 29 cycles.
  • the strength of the obtained promoter is verified based on the dual fluorescent reporter gene system, as follows.
  • the obtained promoter containing the specific strength RBS sequence and the double fluorescent reporter gene system skeleton obtained in Example 1 were transformed into E. coli DH5 ⁇ by the improved Gibson assembly method, and the positive clones on the plate were verified by PCR. After overnight culture The plasmid is extracted to obtain the recombinant plasmid, and the plasmid extraction is performed in accordance with the standard procedure of the plasmid extraction kit.
  • the extracted recombinant plasmid is electrotransformed to ZM4 competent cells, the specific steps are: take ZM4 competent cells on ice, take 50 ⁇ L into the electroporation cup after the competent cells are thawed, and add 1 ⁇ g plasmid to the electroporation cup.
  • the electric transfer conditions are 1600V, 25 ⁇ F, 200 ⁇ .
  • Intensity verification The single clone verified by PCR positive clones and correctly loaded into the dual fluorescence reporter gene system is activated in RM medium with 200 ⁇ g/mL spectinomycin (in other embodiments, other types and concentrations can be selected according to actual needs) After activation, each sample is cultured in three parallels, and tetracycline at a concentration of 0, 0.2, 0.4, 0.6, 0.8, or 1.0 ⁇ g/mL is used for induction culture. After the culture reaches the logarithmic phase, 200 ⁇ L is sampled. Centrifuge at 12000rpm for 1min, remove the supernatant, wash twice with 1 ⁇ PBS and resuspend, and perform flow cytometry detection with a set program. To prevent small probability and accidental events, the present invention sets the cell collection event to 20. 000.
  • Embodiment 4 Method for obtaining terminator of different strength
  • S01 Screen gene sets with large differences in expression of adjacent genes in the same transcription direction, and sort them according to the differences in expression.
  • S02 Use bioinformatics methods to predict the terminator sequence between adjacent genes, and use the expression difference of adjacent genes to represent the terminator strength.
  • S03 Design primers for the target terminator sequence, perform PCR amplification, and obtain terminator fragments. Take terminator T1929f as an example, use primers T1929f-F/T1929f-R, and use ZM4 genome as a template for PCR amplification to obtain terminator T1929f fragments.
  • a modified Gibson method was used to insert the terminator sequence into the transcription start site of the promoter before EGFP.
  • the promoter used here is Pgap.
  • step S7 After obtaining the recombinant material by the method of step S6 in Example 1, the method of step S7 is used for electrotransformation.
  • step S7 use the method of step S7 for strength verification.
  • the terminator only affects transcription, not the translation process. In this case, the stronger the termination intensity, the smaller the value of EGFP/opmCherry, and the experimental results are consistent with the predicted intensity. Table 3 shows the relevant information and verification results of the terminator identified by the method of the present invention.
  • Table 3 is related information of different strength terminator sequences of Zymomonas mobilis provided in the embodiment of the present invention
  • S0a Use bioinformatics methods to analyze the target sequence, and after determining the transcription initiation site, reserve the target 5'UTR sequence from the transcription initiation site to 99-bp after the initiation codon ATG.
  • S0b Using the Z.mobilis genome as a template, design forward and reverse primers for PCR amplification to obtain target UTR sequence fragments.
  • PCR amplification of UTR1754-F/R was carried out with primers to obtain UTR1754 sequence.
  • the PCR product was recovered and purified according to the PCR recovery kit. (The small letters in the primer sequence are the homology arms, and the capital letters are the primer sequence.)
  • PCR program settings 98°C pre-denaturation for 3 minutes, 98°C denaturation for 10 seconds, 55°C annealing for 10 seconds, 72°C extension for 10 seconds, 29 cycles.
  • S0c Take the dual fluorescent reporter gene system containing the Pgap promoter as a template, and use the primers Prtt-F and PgapTSS-R to perform PCR amplification to obtain the dual fluorescent reporter gene system skeleton.
  • the PCR product was recovered and purified according to the PCR recovery kit.
  • Example 1 of the present invention When verifying the strength of the Pgap promoter in Example 1 of the present invention, a dual fluorescent reporter gene system containing the Pgap promoter has been obtained, which is used as a template in this operation step of this example.
  • SEQ ID NO: 3 Prtt-F 5’-ATGGTGAGCAAGGGCGAG-3’;
  • SEQ ID NO: 65 PgapTSS-R 5'-AAACATCAATACCATAACGAAGACC-3'.
  • S0d Obtain the recombinant plasmid: After obtaining the UTR sequence and the skeleton of the dual fluorescent reporter gene system, transform E. coli DH5 ⁇ by the improved Gibson assembly method, verify the positive clone on the plate by PCR, and extract the plasmid after overnight culture. (Plasmid extraction follows the standard procedure of plasmid extraction kit).
  • S0e Obtain the strain of the dual fluorescent reporter gene system plasmid containing the specific UTR sequence: use the extracted plasmid to electrotransform the Z.mobilis wild-type strain, Zms4 knockout and overexpression strain. Take the corresponding competent cells on ice. After the competent cells are thawed, add 50 ⁇ L to the electrorotor cup, and add 1 ⁇ g plasmid to the electrorotor cup. The electric transfer conditions are 1600V, 25 ⁇ F, 200 ⁇ . After electroporation is completed, it is recovered in RM liquid medium at 30°C. The culture recovered for 6-12 hours was centrifuged at 6000 rpm for 1 min, and the supernatant was removed.
  • SEQ ID NO: 8 Pdual-F CCGCTCACAATTCCACACATTATAC
  • SEQ ID NO: 9 Pdual-R ACCAGGATGGGCACCAC.
  • S0f Flow cytometry detection: activate the validated single clone in RM medium with 300 ⁇ g/mL kanamycin. After activation, each sample will be cultured in three parallels. After culturing to the logarithmic phase, 200 ⁇ g will be sampled. Centrifuge at 12000 rpm for 1 min, remove the supernatant, wash twice with 1 ⁇ PBS and resuspend, and perform flow cytometry detection with the set program. To prevent small probability and accidental events, set the cell collection event to 20,000.
  • FIG. 8 a schematic diagram of the interaction between Zms4 and its target UTR provided by an embodiment of the present invention.
  • the results show that the detection of the dual-fluorescence reporter gene system is consistent with the bioinformatics prediction results.
  • Zms4 is predicted to have a stabilizing effect on the target UTR (Zms4-UTR1754)
  • the value of EGFP/opmCherry in the Zms4 knockout strain is significantly lower than that in the WT strain.
  • Zms4-UTR1993 the value of EGFP/opmCherry in the Zms4 knockout strain is significantly higher than that in the WT strain.
  • the experimental results obtained by using the system of the present invention are consistent with those obtained by the traditional experimental method, but the present invention is convenient, fast, safe and efficient, has a short experimental period (saves at least one week time compared with the traditional method), and can be operated in batches.
  • nucleotide sequence of Zms4 is SEQ ID NO: 66
  • Zms6 nucleotide sequence is SEQ ID NO: 67
  • UTR1754 nucleotide sequence Is SEQ ID NO: 68
  • UTR1993 nucleotide sequence is SEQ ID NO: 69
  • UTR0170 nucleotide sequence is SEQ ID NO: 70
  • UTR1934 nucleotide sequence is SEQ ID NO: 71
  • UTR0149 nucleotide sequence is SEQ ID NO: 72.
  • step Sa of Example 2 of the present invention is as follows:

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Abstract

本发明提供了一种基于双荧光报告基因系统鉴定生物元件的方法及基于该方法构建的生物元件库。该方法包括利用pEZ15Asp质粒为骨架，构建单荧光报告基因系统用于筛选荧光蛋白并确定荧光报告基因；获取pEZ15Asp骨架；组装荧光基因；获得双荧光报告基因系统骨架；构建重组质粒，最终转化至感受态细胞中进行荧光强度的定量分析。

Description

一种基于双荧光报告基因系统鉴定生物元件的方法及基于该方法构建的生物元件库 技术领域
本发明属于生物技术领域，特别涉及一种基于双荧光报告基因系统鉴定生物元件的方法及基于该方法构建的生物元件库。
背景技术
运动发酵单胞菌(Zymomonas mobilis，Z.mobilis)为革兰氏阴性、兼性厌氧细菌，周生鞭毛，大小1.4-2.0*4.0-5.0μm；适宜的生长温度为30℃，能耐受的pH的范围在3.5-9之间，其能通过Enter-Doudoroff途径(ED途径)高效利用葡萄糖、果糖和蔗糖产生乙醇。Z.mobilis具有高乙醇产率及耐受性，高渗透压耐受性，生物量少，发酵时不需要加氧等诸多优点而成为生物乙醇的主要生产菌株之一。为提高对该菌种的适用范围，针对Z.mobilis的系统生物学研究相继展开，同时，随着代谢工程及合成生物学的发展，对Z.mobilis中可用的生物元件的需求也日益上升。
但是，目前对于Z.mobilis的系统生物学数据的挖掘依然不够，特别是针对基因组范围内的启动子、核糖体结合序列等生物元件挖掘开发的研究较少，使得已鉴定的可用的Z.mobilis的生物元件少之又少，从而导致对Z.mobilis进行的精准的代谢工程改造及合成生物学时代的发展受到了极大的限制。
启动子是调控基因表达的重要元件，是RNA聚合酶识别并特异性结合的位点。启动子强度一般用来描述RNA聚合酶在启动子处起始转录的频率，生物合成时选用具有一定强度的启动子能够辅助生物合成的顺利进行。
传统的筛选启动子的方法是对基因组随机剪切，之后通过下游的单一报告基因的表达情况来鉴定得到的序列是否为启动子序列以及其启动强度如何。传统的检测系统大多为单一报告基因系统，但是单一报告基因系统中报告基因的表达会受到来自菌株内部和外部的多种因素的影响，从而会影响该系统对启动子的鉴定。此外，通过以往的方法获取待测启动子的速度慢，且无法快速高通量的进行强度鉴定，不利于启动子的高效定量研究。随着各项技术的发展，虽然又有一些新的方法被开发，比如电泳迁移率分析、原子力显微镜等体外检测技术，但是都因为准确度不高而导致使用受到限制。
综上，利用已有的系统生物学数据开发挖掘可供运动发酵单胞菌代谢工程使用的生物元件，尤其是特定强度的启动子和核糖体结合位点，及sRNA-UTR作用对等，并建立一种高效、快速、高通量的启动子及其它生物元件体内定量的分析方法，对扩大运动发酵单胞菌的启动子及其它生物元件的元件库具有重大意义；同时该方法也可以应用于其它微生物体系，为代谢工程和合成生物学提供多样的生物功能及调控元件
发明内容
针对现有技术存在的问题，本发明提供了一种基于双荧光报告基因系统鉴定生物元件的方法及基于该 方法构建的生物元件库。
本发明的上述技术目的是通过以下技术方案得以实现的：
一种基于双荧光报告基因系统鉴定生物元件的方法，包括以下步骤：
S1：以pEZ15Asp质粒为骨架，构建单荧光报告基因系统，筛选荧光蛋白；
S2：根据不同荧光蛋白基因在运动发酵单胞菌中的表达情况，筛选合适的荧光报告基因，分别命名为荧光报告基因1号和荧光报告基因2号；
S3：以pEZ15Asp为模板，分别设计正、反向引物，进行PCR扩增，得到pEZ15Asp骨架；
S4：利用改良后的Gibson装配方法，将启动子1号-荧光报告基因1号和启动子2号-荧光报告基因2号与pEZ15Asp骨架连接，并在两荧光报告基因中间加入终止子，得到双荧光报告基因系统；
S5：以双荧光报告基因系统为模板，分别设计正、反向引物进行PCR扩增获得双荧光报告基因系统骨架；
S6：将待测生物元件与步骤S5中获得的双荧光报告基因系统骨架通过改良后的Gibson装配方法转化至大肠杆菌DH5α，PCR验证平板上的阳性克隆，过夜培养后提取质粒；
S7：将步骤S6中提取的质粒转化至ZM4感受态细胞中，活化培养至对数期后，使用流式细胞仪检测验证。
进一步地，步骤S1中所述荧光蛋白均由启动子PlacUV5启动，所述荧光蛋白为EGFP,mCherry,RFP，CFP，和分别经密码子优化后的opEGFP,opmCherry和opCFP中的任一种。
进一步地，步骤S2中启动子1号为Ptet，启动子2号为PlacUV5；荧光报告基因1号和荧光报告基因2号分别为EGFP和opmCherry。
进一步地，步骤S3中所述正、反向引物分别为引物1和引物2，引物1和引物2的序列分别见SEQ ID NO:1和SEQ ID NO:2。
进一步地，步骤S5中所述正、反向引物分别为引物Prtt-F和Prtt-R，引物Prtt-F和Prtt-R序列分别见SEQ ID NO:3和SEQ ID NO:4；
或者，步骤S5中所述正、反向引物分别为引物Prtt-F和PgapTSS-R，引物Prtt-F和PgapTSS-R序列分别见SEQ ID NO:3和SEQ ID NO:4。
进一步地，步骤S6中所述待测生物元件为内源不同强度的启动子，或含不同强度合成RBS序列的启动子，或不同强度的终止子，或sRNA-UTR作用对。
进一步地，所述待测生物元件为内源不同强度的启动子或含不同强度合成RBS序列的启动子，获取所述含不同强度RBS序列的启动子的方法包括以下步骤：
Sa：对不同强度核糖体结合位点序列进行预测；
Sb：以双荧光报告基因系统为模板，引物pEZ-tetR-F、RBS-R进行PCR扩增，其中引物5’端的小写字母为与双荧光报告基因系统的同源臂，获得含特定强度RBS序列的启动子。
进一步地，所述待测生物元件为不同强度的终止子，获取所述不同强度的终止子的方法包括以下步骤：
S01：筛选同一转录方向的相邻基因表达差异大的基因集，并根据表达差异进行排序；
S02：使用生物信息学方法预测相邻基因间的终止子序列，并用相邻基因的表达差异代表终止子强度；
S03：针对目标终止子序列设计引物，进行PCR扩增，获取终止子片段。
进一步地，所述待测生物元件为sRNA-UTR作用对，获取所述UTR片段的方法包括以下步骤：
S0a：利用生物信息学方法对靶序列进行分析，确定转录起始位点后，保留从转录起始位点开始至起始密码子ATG后99-bp，作为靶5’UTR序列；
S0b：以Z.mobilis基因组为模板，设计正、反向引物进行PCR扩增，获得靶UTR序列片段；
以含有Pgap启动子的双荧光报告基因系统为模板，进行PCR扩增得到双荧光报告基因系统骨架。
进一步地，步骤S7中质粒采用电转化的方法转化至ZM4感受态细胞中，具体转化条件如下：
(1)取ZM4感受态细胞于冰上，待感受态细胞融化后取50μL加入电转杯中，并在电转杯中加入1μg质粒；电转条件为1600V，25μF，200Ω；
(2)电转完毕后于RM液体培养基中于30℃复苏；
(3)复苏6-12小时的培养物于6000rpm，1min离心，除去上清；
(4)加入200μL新鲜的RM培养基，取100μL涂布于含相应抗生素的抗性平板，30℃培养2天；
(5)再用引物Pdual-F,Pdual-R进行PCR阳性克隆验证；
Pdual-F CCGCTCACAATTCCACACATTATAC，见SEQ ID NO:8；
Pdual-R ACCAGGATGGGCACCAC，见SEQ ID NO:9。
进一步地，步骤S7中使用流式细胞仪对强度检测验证，具体步骤如下：
(1)将经PCR阳性克隆验证正确的装入双荧光报告基因系统的单克隆于含相应抗生素的RM培养基中进行活化、培养；
(2)培养至对数期后，取样200μL，12000rpm离心1min，去上清，并用1×PBS洗两次后重悬；
(3)用流式细胞仪检测，细胞收集事件设置为20,000。
进一步地，步骤S6中所述待测生物元件为不同强度的启动子或含不同强度RBS序列的启动子。
进一步地，获取所述含不同强度RBS序列的启动子的方法包括以下步骤：
Sa：根据不同的组学数据筛选出下游表达强的基因，并进行韦恩分析，筛选出各组学数据共有基因；
Sb：对不同强度核糖体结合位点序列进行预测；
Sc：以双荧光报告基因系统为模板，引物pEZ-tetR-F、RBS-R进行PCR扩增，其中引物5’端的小写字母为与双荧光报告基因系统的同源臂，获得含特定强度RBS序列的启动子。
本发明的第二目的是提供一种基于双荧光报告基因系统鉴定生物元件的方法构建的生物元件库。
与现有技术相比，本发明的有益效果是：
本发明以运动发酵单胞菌为模式菌株，建立了利用系统生物学数据预测筛选不同强度的启动子序列的方法，该预测筛选方法高效快捷，可作为实验的指导基础。本发明可以整合已有系统生物学数据，快速高效的筛选特定强度的启动子；本方法预测筛选的RBS强度和启动子强度，与实验数据之间有较好的相关性(分别为R 2>0.9,R 2>0.7)，说明本发明提出的基于系统生物学数据筛选启动子的方法，可以用于不同需求启动子及其他生物元件的预测筛选。该方法相较于现有的启动子筛选鉴定方法，可以快速高通量的筛选并定量不同强度的启动子以及其他的生物元件，并且启动子的定量分析在细胞体内完成，受细胞内、外部环境变化的影响小，定量准确，可以快速扩充运动发酵单胞菌的生物元件库，用于不同需求的代谢工程改造。
本发明利用已有的系统生物学数据及生物信息学方法预测筛选不同强度的生物元件，并通过本实验室开发的双荧光报告基因系统进行强度鉴定与验证，用于定量分析启动子及其他生物元件的强度，找到可以用于代谢工程和合成生物学的生物元件。结合两种方法，建立一种可以快速选择待用生物元件的策略，以供在合成生物学时代理性设计和改造微生物使用。
另外，本方法开发的双荧光报告基因系统可用于定量分析其他的生物元件，也可以利用到除运动发酵单胞菌以外的其他物种中使用。本发明所拥有的优势如下表所示。
本发明与现有技术的优劣势比较
Figure PCTCN2019086173-appb-000001
Figure PCTCN2019086173-appb-000002
传统的鉴定sRNA-UTR相互作用关系的方法，操作十分繁琐。而且一般在在体外进行检测，不能免去体外获取实验材料及体外进行相关实验的麻烦，造成不能快速确定sRNA与靶UTR之间是否存在相互作用关系，不便于批量操作，并且不能用于批量定量分析目的sRNA与其靶5’UTR之间的相互作用的强度。本发明方便快捷，相比于传统的鉴定sRNA-UTR相互作用关系的方法，操作十分简单。本发明基于流式细胞术的双荧光报告基因系统，在体内进行检测，免去了体外获取实验材料及体外进行相关实验的麻烦，能快速确定sRNA与靶UTR之间是否存在相互作用关系，便于批量操作。本发明的方法利用的双荧光报告基因系统可用于定量分析相互作用的强度，也可以移植到除运动发酵单胞菌以外的其他物种中使用本发明提供一种鉴定sRNA与其靶mRNA的5’UTR相互作用的方法，建立可以快速高效的确定目的sRNA与其靶5’UTR之间的相互作用关系，并可以进行定量分析。
本发明利用公共数据库和生物学数据、双荧光报告基因系统，筛选并鉴定了37个启动子和4个核糖体结合位点(ribosomal binding site,RBS)的强度，4对sRNA-UTR作用对以及6个终止子的强度，极大扩展了运动发酵单胞菌的元件库。可以达到对运动发酵单胞菌及不同微生物体系系统生物学的充分挖掘利用及扩大其生物元件库，用于代谢工程及合成生物学实践。本方法简单易于操作，适用生物元件范围广，且可推广到其它物种。
附图说明
图1中A是单荧光报告基因系统图；B是筛选的荧光蛋白表达结果图；
图2是双荧光报告基因系统图；
图3是双荧光报告基因系统经不同层面验证结果示意图；
图4中A是流式细胞仪模式；B是数据相关性示意图；
图5是韦恩分析筛选不同强度启动子的示意图；
图6中A是不同强度RBS强度验证结果示意图；B是不同强度RBS与四环素浓度之间的相关性示意图；
图7是本发明提供的鉴定sRNA-UTR相互作用的方法流程图，图中：TSS表达转录起始位点，ATG表示起始密码子，WT表示野生型菌株，ΔsRNA表示目的sRNA敲除菌株，OE_sRNA表示目的sRNA过表达菌株；
图8是本发明提供的sRNA与其靶UTR之间的相互作用实验结果示意图。
具体实施方式
下面将结合具体实施例，对本发明的技术方案进行清楚、完整地描述。所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明的实施例，本领域普通技术人员在没有做出创造性劳 动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明披露了一种基于双荧光报告基因系统鉴定生物元件的方法及基于该方法构建的生物元件库，具体如下各实施例所示。
实施例1 一种基于双荧光报告基因系统鉴定生物元件的方法
S1：以pEZ15Asp质粒为骨架，构建单荧光报告基因系统，如图1A所示，用来筛选荧光蛋白。荧光蛋白为EGFP,mCherry,RFP，CFP，和分别经密码子优化后的opEGFP,opmCherry和opCFP中的任一种，荧光蛋白均由启动子PlacUV5启动。
S2：将各荧光蛋白基因序列送至第三方公司进行基因序列合成；以pEZ15Asp为模板，分别以引物1和引物2进行PCR扩增，得到pEZ15Asp骨架；利用Gibson装配，将PlacUV5-荧光报告基因与pEZ15Asp骨架连接，通过转化大肠杆菌DH5α，得到单荧光报告基因系统；对得到的重组菌株进行PCR验证平板上的阳性克隆，过夜培养后提取质粒，并测序验证连接及序列正确性；将提取的质粒转化至ZM4感受态细胞中，活化培养至对数期后，使用流式细胞仪检测表达强度。
引物1：5’-GCGCTAGCGGAGTGTATACTGGCTTACTATGTT-3’，SEQ ID NO：1；
引物2：5’-ACGGTGAGCTGGTGACCTGCCTTATC-3’，SEQ ID NO：2。
根据不同荧光蛋白基因在运动发酵单胞菌中的荧光表达强度情况，筛选合适的荧光报告基因，分别命名为荧光报告基因1号和荧光报告基因2号，启动子1号选用Ptet，启动子2号选用PlacUV5。表达结果如图1B所示，本实施例中荧光蛋白EGFP和opmCherry在运动发酵单胞菌中的荧光强度表达较高，因此，分别作为荧光报告基因1号和荧光报告基因2号参与后续实验。
S3：以pEZ15Asp为模板，设计引物1和引物2进行PCR扩增，得到pEZ15Asp骨架，按照PCR回收试剂盒对PCR产物回收纯化。
PCR体系配置：
Figure PCTCN2019086173-appb-000003
PCR程序设置：98℃预变性3min，98℃变性10s，55℃退火10s，72℃延伸35s，29个循环。
S4：利用改良后的Gibson装配方法，将Ptet-荧光报告基因1号即Ptet-EGFP，和PlacUV5-荧光报告基因2号即PlacUV5-opmCherry，与步骤S3中得到的pEZ15Asp骨架连接，并在两荧光报告基因中间加入 终止子，得到可诱导的双荧光报告基因系统，如图2所示。
本实施例中，在荧光报告基因中间加入的终止子为BBa_B0014，序列为：
5’-CCTTTTTTCTCCTGCCACATGAAGCACTTCACTGACACCCTCATCAGTGCCAACATAGTAAGCCAGTATACACTCCGCTAGCGCAAATAATAAAAAAGCCGGATTAATAATCTGGCTTTTTATATTCTCTCTCTAGTATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACCTGCAGCGGCCGCTACTAGT-3’，见SEQ ID NO：73。
两个荧光蛋白基因3’所用的终止子均为rrnB T1终止子，序列为：
5’-CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTA-3’，见SEQ ID NO：74。
改良后的Gibson装配的实验方案：连接过程使用5μL的体系，目的片段与载体的摩尔比为3：1，0.5μL的T5外切酶，0.5μL的buffer 4,体积不足时用去离子水补充。体系混匀后在冰上反应5分钟，接着加入100μL的DH5α化学感受态细胞，冰上孵育30分钟后，于42℃热激45秒，接着冰上静置2-3分钟，使重组载体更多的进入感受态细胞。再于37℃，250rpm恢复1小时后，涂布到加有200μg/mL壮观霉素的LB固体培养基于37℃培养箱培养，其他实施例中也可根据实际需要选用其他种类及浓度的抗生素。12-24小时后挑选5-10个边缘清晰的单菌落进行菌落PCR以选出重组子，再选2个条带最清晰的送测序公司测序验证，得到测序序列后与原始序列进行比对确定其序列正确性。
测序正确后使用常规质粒提取试剂盒(Plasmid Miniprep Kit，Tsingke)提取质粒，得到质粒后，将质粒电转到野生型运动发酵单胞菌中，通过菌落PCR及测序确定最终菌株，测序由Tsingke公司完成，得到测序序列后与原始序列进行比对确定其序列正确性。
系统验证：得到含有可诱导的双荧光报告基因系统的菌株后，利用浓度为0,0.2,0.4,0.6,0.8,或1.0μg/mL的浓度的四环素进行诱导培养，取样时间均为对数期，并利用流式细胞术(FCM)、荧光定量PCR(qPCR)、Western Blot在不同层面验证双荧光报告基因系统。结果如图3所示，图中：(A,B)qPCR，(C,D)FCM，(E)WB,(F)qPCR与FCM之间的相关性。
Rich media(RM)培养基配方：RMG5:50g/L葡萄糖,10g/L酵母提取物,and 2g/L KH 2PO 4，壮观霉素抗性为200μg/mL。培养条件：30℃，100rpm。
流式细胞术(FCM)具体步骤如下：
(1)将经PCR阳性克隆验证正确的装入双荧光报告基因系统的单克隆于200μg/mL壮观霉素的RM培养基中进行活化、培养；其他实施例中也可根据实际需要选用其他种类及浓度的抗生素。
(2)培养至对数期后，取样200μL，12000rpm离心1min，去上清，并用1×PBS洗两次后重悬。
(3)用流式细胞仪检测，事件开始记录为1,000个细胞，直到检测到20,000细胞停止。
(4)以无荧光以及含有单荧光的EGFP，opmCherry的菌株作为对照，进行画门，补偿为软件自动补偿。
(5)EGFP的激发波长为488nm，检测器为FITC，opmCherry的激发波长为561nm检测器为PC5.5。取三个重复样品的平均值并取log2的对数进行后续数据分析。
荧光定量PCR(qPCR)具体步骤如下：
双荧光报告系统中的EGFP和opmCherry的转录水平在相同的热循环条件下进行。使用TRIzol(Invitrogen,USA)法提取对数期样品的总RNA，并用NanoDrop 8000检测提取的RNA的质量。接着使用商品试剂盒iScript TM gDNA Clear cDNA Synthesis Kits(Bio-Rad,USA)的操作说明去除基因组DNA以及反转录为cDNA。qPCR荧光定量反应使用iTaq TM Universal
Figure PCTCN2019086173-appb-000004
Green Supermix(Bio-Rad,USA)试剂盒在CFX96 Real-Time System(Bio-Rad,USA)仪器上进行。实验过程中使用的引物为退火温度同为60℃的高纯度无盐引物，引物序列如下：
Figure PCTCN2019086173-appb-000005
通过熔解曲线分析PCR产物的特异性后，使用以下程序进行检测：95℃ 5min变性；(95℃ 15s,60℃10s,and 72℃ 30s)40个扩增循环；并定量检测单荧光。使用基于内参校准曲线的绝对定量方法进行qPCR数据分析。
Western Blot的具体实验方案如下：
细胞裂解和总蛋白提取使用蛋白提取试剂盒(Zomanbio,China)。使用Bradford方法将蛋白上样量定量到200ng。聚丙烯酰胺凝胶电泳(SDS–PAGE)使用5％的浓缩胶和12％分离胶。使用预染蛋白Marker(10-170kDa,Thermo,Lithuania)作Marker分辨蛋白条带大小。电泳完后，使用
Figure PCTCN2019086173-appb-000006
Semi-Dry Electrophoretic Transfer Cell(Bio-Rad,USA)系统将目的蛋白(EGFP或opmCherry)转印到甲醇浸泡的PVDF膜上，条件为25V，20分钟。转膜后，用5％的脱脂奶粉室温封闭1小时。封闭完后以1：5000的比例敷EGFP或mCherry的一抗(Proteintech,China)，温室敷育1小时后，用1x PBST每5分钟洗一次，洗3次。接着同样以1：5000的比例敷EGFP或mCherry的二抗(Peroxidase-conjugated goat anti-Mouse IgG)，温室敷育1小时后，用1x PBST每5分钟洗一次，洗3次。以1：1的比例配制照胶显色液(ImmobilonTM Western Chemiluminescent HRP Substrate)，使用AI600Imaging System(GE,USA)进行成像。
结果如图3所示，qPCR，WB和流式细胞术被用来检测在不同四环素浓度下两种荧光蛋白在转录和翻译水平的表达(图3)。为了消除胞内和胞外环境的影响，EGFP/opmCherry的比值被用来表示待测启动子及其他生物元件的强度。实验结果表明，在不同的水平，opmCherry的表达都相对稳定，而EGFP的表达随四环素浓度的提高而升高，并且EGFP/opmCherry的比值与四环素浓度成线性正相关。此外，流式细胞术与qPCR和WB之间也成线性正相关，这暗示了使用高通量的流式细胞术来定量鉴定潜在遗传元件的可行性。
S5：以双荧光报告基因系统为模板，引物Prtt-F,Prtt-R进行PCR扩增获得双荧光报告基因系统骨架。
PCR体系配置：
Figure PCTCN2019086173-appb-000007
PCR程序设置：98℃预变性3min，98℃变性10s，55℃退火10s，72℃延伸50s，29个循环。
Prtt-F 5’-ATGGTGAGCAAGGGCGAG-3’，SEQ ID NO：3；
Prtt-R 5’-ACTAGTAGCGGCCGCTG-3’，SEQ ID NO：4。
S6：将待测生物元件与步骤S5中获得的双荧光报告基因系统骨架通过Gibson装配的方法转化至大肠杆菌DH5α。
改良后的Gibson装配的实验方案：连接过程使用5μL的体系，目的片段与载体的摩尔比为3：1，0.5μL的T5外切酶，0.5μL的buffer 4,体积不足时用去离子水补充。体系混匀后在冰上反应5分钟，接着加入100μL的DH5α化学感受态细胞，冰上孵育30分钟后，于42℃热激45秒，接着冰上静置2-3分钟，使重组载体更多的进入感受态细胞。再于37℃，250rpm恢复1小时后，涂布到加有200μg/mL壮观霉素的LB固体培养基于37℃培养箱培养，其他实施例中也可根据实际需要选用其他种类及浓度的抗生素。12-24小时后挑选5-10个边缘清晰的单菌落进行菌落PCR以选出重组子，再选2个条带最清晰的送测序公司测序验证，得到测序序列后与原始序列进行比对确定其序列正确性。
菌落PCR体系配置：
Figure PCTCN2019086173-appb-000008
Figure PCTCN2019086173-appb-000009
菌落PCR程序设置：98℃预变性3min，98℃变性10s，55℃退火10s，72℃延伸30s，25个循环。
Pseq-F 5’-GCCATTGACGCTACCTT-3’
Pseq-R 5’-TGGTGGCATCGCCCTCG-3’
测序正确后使用常规质粒提取试剂盒(Plasmid Miniprep Kit，Tsingke)提取质粒，得到质粒后，将质粒电转到野生型运动发酵单胞菌中，通过菌落PCR及测序确定最终菌株，测序由Tsingke公司完成，得到测序序列后与原始序列进行比对确定其序列正确性。
S7：将步骤S6中提取的重组质粒转化至ZM4感受态细胞中，活化培养至对数期后，使用流式细胞仪检测验证。
实施例2 获取不同强度内源启动子的方法及对不同强度内源启动子进行鉴定
内源启动子的筛选：根据不同的组学数据筛选出下游表达强的基因，并进行韦恩(Venn)分析，如图5所示。筛选出各组学数据共有基因。在不同的组学数据中，根据每个基因在所有条件下的平均值进行排序，定义排在90％以上的为强启动子，排在40-60％的为中等强度启动子，排在10％以下的为弱启动子。筛选到19个强启动子，9个中等强度启动子，10个弱启动子，具体如下表所示。
表1 本发明实施例提供的运动发酵单胞菌的不同强度内源启动子基因及其对应的系统生物学数据与FACS验证结果
Figure PCTCN2019086173-appb-000010
Figure PCTCN2019086173-appb-000011
内源启动子的鉴定：按照实施例1中步骤S1-S6进行操作，其中步骤S6中以具有一定强度的内源Pgap启动子作为待测生物元件进行实验，其它实施例中也可以选用其它强度的启动子或含不同强度RBS序列的启动子或其他生物元件进行检测。
Pgap启动子序列:
Figure PCTCN2019086173-appb-000012
Pgap启动子的获取：以Zymomonas mobilis ZM4为模板，引物P0177-F,P0177-R进行PCR扩增获得启动子片段，引物5’端的小写字母为与双荧光报告基因系统的同源臂。按照PCR回收试剂盒对PCR产物回收纯化。
PCR体系配置：
Figure PCTCN2019086173-appb-000013
PCR程序设置：98℃预变性3min，98℃变性10s，55℃退火10s，72℃延伸10s，29个循环。
P0177-F，5’-gcggccgctactagtGTTCGATCAACAACCCGAATC-3’，SEQ ID NO：6；
P0177-R，5’-gcccttgctcaccatGTTTATTCTCCTAACTTATTAAGTAGC-3’，SEQ ID NO：7。
S7：将步骤S6中提取的重组质粒转化至ZM4感受态细胞中，活化培养至对数期后，使用流式细胞仪检测验证。
本实施例中将重组质粒电转化至ZM4感受态细胞中，具体方法如下：取ZM4感受态细胞于冰上，待感受态细胞融化后取50μL加入电转杯中，并在电转杯中加入1μg质粒。电转条件为1600V，25μF，200Ω。电转完毕后于RM液体培养基中于30℃复苏。复苏6-12小时的培养物于6000rpm，1min离心，除去上清。加入200μL新鲜的RM培养基，取100μL涂布于200μg/mL壮观霉素抗性平板(其他实施例中也可根据实际需要选用其他种类及浓度的抗生素)，30℃培养2天。再用引物Pdual-F,Pdual-R进行PCR阳性克隆验证。
菌落PCR体系配置：
Figure PCTCN2019086173-appb-000014
菌落PCR程序设置：98℃预变性3min，98℃变性10s，55℃退火10s，72℃延伸30s，25个循环。
Pdual-F CCGCTCACAATTCCACACATTATAC，SEQ ID NO：8；
Pdual-R ACCAGGATGGGCACCAC，SEQ ID NO：9。
流式细胞术检测：将38个验证正确的单克隆于200μg/mL壮观霉素的RM培养基中进行活化(其他实施例中也可根据实际需要选用其他种类及浓度的抗生素)，活化后每个样培养3个平行，培养至对数期后，取样200μL，12000rpm离心1min，去上清，并用1×PBS洗两次后重悬，用设定好的程序进行流式 细胞仪检测，为防止小概率及偶然事件，本发明将细胞收集事件设置为20，000。
结果分析：根据流式细胞仪得到的数据，每个样品取所有事件的EGFP和opmCherry的平均荧光值进行计算，并用EGFP/opmCherry的比值进行标准化处理，以排除来自细胞内部及外部的干扰，结果如图4。
如图4所示，本发明实施例提供的流式细胞仪模式和数据相关性示意图。
(A)不同强度启动子的流式细胞仪模式图；(B)实验数据与组学数据的相关性。
结果表明，在双荧光报告基因系统中装载生物元件启动子可以用流式细胞仪进行快速定量分析，EGFP/opmCherry的比值说明所测试的启动子在该系统中的相对强度，并且实验结果与组学数据预测的强度之间的相关性较高，说明本发明中的双荧光报告基因系统可以用于启动子强度的鉴定。
实施例3 获取不同强度合成RBS序列的启动子的方法
Sa：对不同强度核糖体结合位点序列进行预测：根据Z.mobilis的16s rRNA使用RBS Calculator V2.0(https://salislab.net/software/)进行预测。
Z.mobilis 16S rRNA序列如下，具体可见SEQ ID NO：10。
Figure PCTCN2019086173-appb-000015
Figure PCTCN2019086173-appb-000016
表2是本发明实施例提供的运动发酵单胞菌的不同强度RBS序列。
Figure PCTCN2019086173-appb-000017
Sb：含特定强度RBS序列的启动子的获取：本实施例中以RBS-10为例，以含Ptet启动子的双荧光报告基因系统为模板，引物pEZ-tetR-F、RBS-10-R进行PCR扩增，其中引物5’端的小写字母为与双荧光报告基因系统的同源臂，获得含特定强度RBS序列的启动子。
PCR体系配置：
Figure PCTCN2019086173-appb-000018
PCR程序设置：98℃预变性3min，98℃变性10s，55℃退火10s，72℃延伸20s，29个循环。
pEZ-tetR-F，
5’-gcggccgctactagtTTAAGACCCACTTTCACATTTAAGTTGTTTTTC-3’，见SEQ ID NO：15。
RBS-10-R，
5’-gcccttgctcaccatGTGCTTACTTTCTCTAGATTATGGAGATCaTTTGAATaCTTTTCTCTATCACTGATAGGGAGTGG-3’，见SEQ ID NO：16。
在获得含特定强度RBS序列的启动子后，基于双荧光报告基因系统对获得的启动子的强度进行验证，具体如下。
将获得的含特定强度RBS序列的启动子与实施例1中获得的双荧光报告基因系统骨架，通过改良后的Gibson装配的方法转化至大肠杆菌DH5α，PCR验证平板上的阳性克隆，过夜培养后提取质粒，得到重组质粒，质粒提取按照质粒提取试剂盒标准步骤操作。
将提取的重组质粒对ZM4感受态细胞进行电转化，具体步骤为：取ZM4感受态细胞于冰上，待感受态细胞融化后取50μL加入电转杯中，并在电转杯中加入1μg质粒。电转条件为1600V，25μF，200Ω。电转完毕后于RM液体培养基中于30℃复苏。复苏6-12小时的培养物于6000rpm，1min离心，除去上清。加入200μL新鲜的RM培养基，取100μL涂布于200μg/mL壮观霉素抗性平板(其他实施例中也可根据实际需要选用其他种类及浓度的抗生素)，30℃培养2天。再用引物Pdual-F,Pdual-R进行PCR阳性克隆验证。引物Pdual-F,Pdual-R序列分别见SEQ ID NO：8和SEQ ID NO：9。
强度验证：将经PCR阳性克隆验证正确的装入双荧光报告基因系统的单克隆于200μg/mL壮观霉素的RM培养基中进行活化(其他实施例中也可根据实际需要选用其他种类及浓度的抗生素)，活化后每个样培养3个平行，利用浓度为0,0.2,0.4,0.6,0.8,或1.0μg/mL的浓度的四环素进行诱导培养，培养至对数期后，取样200μL，12000rpm离心1min，去上清，并用1×PBS洗两次后重悬，用设定好的程序进行流式细胞仪检测，为防止小概率及偶然事件，本发明将细胞收集事件设置为20，000。
结果分析：
根据流式细胞仪得到的数据，每个样品取所有事件的EGFP和opmCherry的平均荧光值进行计算，并用EGFP/opmCherry的比值进行标准化处理，以排除来自细胞内部及外部的干扰，结果如图6所示。图6反应了本发明实施例提供的不同强度RBS强度验证(A)及与四环素浓度(B)之间的相关性。实验结果说明本发明中的基于组学数据预测启动子强度的方法可以用于筛选不同强度的启动子。
实施例4 获取不同强度终止子的方法
S01：筛选同一转录方向的相邻基因表达差异大的基因集，并根据表达差异进行排序。
S02：使用生物信息学方法预测相邻基因间的终止子序列，并用相邻基因的表达差异代表终止子强度。
S03：针对目标终止子序列设计引物，进行PCR扩增，获取终止子片段。以终止子T1929f为例，以引物T1929f-F/T1929f-R，以ZM4基因组为模板进行PCR扩增，获得终止子T1929f片段。
Figure PCTCN2019086173-appb-000019
使用改良后的Gibson方法将终止子序列插入在EGFP前的启动子转录起始位点后，这里使用的启动子为Pgap。
通过实施例1中的步骤S6的方法获得重组质料后使用步骤S7的方法进行电转化。
再使用步骤S7的方法进行强度验证。
终止子只影响转录，不影响翻译过程。在本方明中终止强度越强，EGFP/opmCherry的值越小，实验结果与预测强度相符。表3为利用本发明的方法鉴定的终止子的相关信息及验证结果。
表3是本发明实施例提供的运动发酵单胞菌的不同强度终止子序列相关信息
Figure PCTCN2019086173-appb-000020
实施例5 对作用对sRNA-UTR相互作用关系进行鉴定
S0a：利用生物信息学方法对靶序列进行分析，确定转录起始位点后，保留从转录起始位点开始至起始密码子ATG后99-bp，作为靶5’UTR序列。
以验证sRNA Zms4与基因ZMO1754 UTR(Zms4-UTR1754)的相互作用进行详细说明，实验原理如图7所示。
取基因ZMO1754基因与前一个基因之间的基因间区序列，用BPROM(http://www.softberry.com/berry.phtml？topic＝bprom&group＝programs&subgroup＝gfindb)进行启动子分析，得到转录起始位点(TSS)后，保留从TSS到ZMO1754基因起始密码子ATG后的99-bp序列为UTR1754序列，SEQ ID NO：62。
UTR1754
Figure PCTCN2019086173-appb-000021
S0b：以Z.mobilis基因组为模板，设计正、反向引物进行PCR扩增，获得靶UTR序列片段。
以Z.mobilis基因组为模板，用引物对UTR1754-F/R进行PCR扩增，以获得UTR1754序列。按照PCR回收试剂盒对PCR产物回收纯化。(引物序列中小字母为同源臂，大写字母为引物序列。)
PCR体系配置：
Figure PCTCN2019086173-appb-000022
PCR程序设置：98℃预变性3min，98℃变性10s，55℃退火10s，72℃延伸10s，29个循环。
SEQ ID NO：63：
UTR1754-F
5’-atggtattgatgtttGATCATTTCACAAAAAATGAGAAAAAATTAAGGATGAG-3’
SEQ ID NO：64：
UTR1754-R 5’-gcccttgctcaccatCGCCGCCCGATCAACG-3’
S0c：以含有Pgap启动子的双荧光报告基因系统为模板，用引物Prtt-F、PgapTSS-R进行PCR扩增得到双荧光报告基因系统骨架。按照PCR回收试剂盒对PCR产物回收纯化。
在本发明实施例1中对Pgap启动子的强度进行验证时已获得含有Pgap启动子的双荧光报告基因系统，将其作为本实施例该操作步骤中的模板使用。
SEQ ID NO：3：Prtt-F 5’-ATGGTGAGCAAGGGCGAG-3’；
SEQ ID NO：65：PgapTSS-R 5’-AAACATCAATACCATAACGAAGACC-3’。
S0d：获得重组质粒：获得UTR序列和双荧光报告基因系统骨架后，通过改良后的Gibson装配的方法转化大肠杆菌DH5α，PCR验证平板上的阳性克隆，过夜培养后提取其中的质粒。(质粒提取按照质粒提取试剂盒标准步骤)。
S0e：获取含有特定UTR序列的双荧光报告基因系统质粒的菌株：用提取的质粒对Z.mobilis野生型菌株，Zms4敲除和过表达菌株进行电转化。取相应的感受态细胞于冰上，待感受态细胞融化后取50μL加入电转杯中，并在电转杯中加入1μg质粒。电转条件为1600V，25μF，200Ω。电转完毕后于RM液体培养基中于30℃复苏。复苏6-12小时的培养物于6000rpm，1min离心，除去上清。加入200μL新鲜的RM培养基，取100μL涂布于300μg/mL卡那霉素抗性平板，30℃培养2天。再用引物Pdual-F,Pdual-R进行PCR阳性克隆验证。
SEQ ID NO：8：Pdual-F CCGCTCACAATTCCACACATTATAC
SEQ ID NO：9：Pdual-R ACCAGGATGGGCACCAC。
S0f：流式细胞术检测：将验证正确的单克隆于300μg/mL卡那霉素的RM培养基中进行活化，活化 后每个样培养3个平行，培养至对数期后，取样200μg，12000rpm离心1min，去上清，并用1×PBS洗两次后重悬，用设定好的程序进行流式细胞仪检测，为防止小概率及偶然事件，将细胞收集事件设置为20,000。
结果分析：根据流式细胞仪得到的数据，每个样品取所有事件的EGFP和opmCherry的平均荧光值进行计算，并用EGFP/opmCherry的比值进行标准化处理，以排除来自细胞内部及外部的干扰，结果如图8。
同理进行了Zms4-UTR1993相互作用关系的鉴定。
如图8所示，本发明实施例提供的Zms4与其靶UTR之间的相互作用示意图。结果表明，双荧光报告基因系统的检测与生物信息学预测结果相符合。当预测Zms4对靶UTR有稳定作用时(Zms4-UTR1754)，在Zms4敲除菌株中EGFP/opmCherry的值相较WT菌株中显著性降低。当预测Zms4对靶UTR有降解作用时(Zms4-UTR1993)，在Zms4敲除菌株中EGFP/opmCherry的值相较WT菌株中显著性升高。
利用本发明系统得到的实验结果，与传统实验方法得到的结果一致，但本发明方便快捷，安全高效，实验周期短(比传统方法至少节约一个星期的时间)，可批量操作。
下表是利用本发明的技术方案鉴定的作用对的相关信息，其中，Zms4核苷酸序列为SEQ ID NO：66，Zms6:核苷酸序列为SEQ ID NO：67，UTR1754:核苷酸序列为SEQ ID NO：68，UTR1993核苷酸序列为SEQ ID NO：69，UTR0170核苷酸序列为SEQ ID NO：70，UTR1934核苷酸序列为SEQ ID NO：71，UTR0149:核苷酸序列为SEQ ID NO：72。
表4 本发明实施例提供的运动发酵单胞菌中sRNA-UTR作用对相互作用关系鉴定的结果
Figure PCTCN2019086173-appb-000023
本发明实施例2的步骤Sa中筛选的启动子的序列如下：
SEQ ID NO：17
P0177
Figure PCTCN2019086173-appb-000024
Figure PCTCN2019086173-appb-000025
SEQ ID NO：18
P1360
Figure PCTCN2019086173-appb-000026
SEQ ID NO：19
P0516
Figure PCTCN2019086173-appb-000027
SEQ ID NO：20
P1608
Figure PCTCN2019086173-appb-000028
SEQ ID NO：21
P0997
Figure PCTCN2019086173-appb-000029
SEQ ID NO：22
P0367
Figure PCTCN2019086173-appb-000030
SEQ ID NO：23
P1719
Figure PCTCN2019086173-appb-000031
SEQ ID NO：24
P1609
Figure PCTCN2019086173-appb-000032
SEQ ID NO：25
P0689
Figure PCTCN2019086173-appb-000033
SEQ ID NO：26
P1721
Figure PCTCN2019086173-appb-000034
SEQ ID NO：27
P0514
Figure PCTCN2019086173-appb-000035
Figure PCTCN2019086173-appb-000036
SEQ ID NO：28
P1596
Figure PCTCN2019086173-appb-000037
SEQ ID NO：29
P1141
Figure PCTCN2019086173-appb-000038
SEQ ID NO：30
P0241
Figure PCTCN2019086173-appb-000039
SEQ ID NO：31
P0244
Figure PCTCN2019086173-appb-000040
SEQ ID NO：32
Po1721
Figure PCTCN2019086173-appb-000041
SEQ ID NO：33
P0493
Figure PCTCN2019086173-appb-000042
SEQ ID NO：34
P1779
Figure PCTCN2019086173-appb-000043
SEQ ID NO：35
P1351
Figure PCTCN2019086173-appb-000044
SEQ ID NO：36
P0056
Figure PCTCN2019086173-appb-000045
SEQ ID NO：37
P0559
Figure PCTCN2019086173-appb-000046
SEQ ID NO：38
P1385
Figure PCTCN2019086173-appb-000047
SEQ ID NO：39
P0127
Figure PCTCN2019086173-appb-000048
SEQ ID NO：40
P1100
Figure PCTCN2019086173-appb-000049
SEQ ID NO：41
P1392
Figure PCTCN2019086173-appb-000050
SEQ ID NO：42
P0326
Figure PCTCN2019086173-appb-000051
SEQ ID NO：43
P0570
Figure PCTCN2019086173-appb-000052
SEQ ID NO：44
P1231
Figure PCTCN2019086173-appb-000053
SEQ ID NO：45
P1980
Figure PCTCN2019086173-appb-000054
SEQ ID NO：46
P1484
Figure PCTCN2019086173-appb-000055
SEQ ID NO：47
P0145
Figure PCTCN2019086173-appb-000056
SEQ ID NO：48
P0101
Figure PCTCN2019086173-appb-000057
SEQ ID NO：49
P1194
Figure PCTCN2019086173-appb-000058
SEQ ID NO：50
1644
Figure PCTCN2019086173-appb-000059
SEQ ID NO：51
P1582
Figure PCTCN2019086173-appb-000060
SEQ ID NO：52
P0005
Figure PCTCN2019086173-appb-000061
SEQ ID NO：53
P0300 GGCATGTTCGCGGCCGCCGGTTCCGATAAGCAGGACGTTC

Claims (12)

  1. 一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于，包括以下步骤：
    S1：以pEZ15Asp质粒为骨架，构建单荧光报告基因系统，筛选荧光蛋白；
    S2：根据不同荧光蛋白基因在运动发酵单胞菌中的表达情况，筛选合适的荧光报告基因，分别命名为荧光报告基因1号和荧光报告基因2号；
    S3：以pEZ15Asp为模板，分别设计正、反向引物，进行PCR扩增，得到pEZ15Asp骨架；
    S4：利用改良后的Gibson装配方法，将启动子1号-荧光报告基因1号和启动子2号-荧光报告基因2号与pEZ15Asp骨架连接，并在两荧光报告基因中间加入终止子，得到双荧光报告基因系统；
    S5：以双荧光报告基因系统为模板，分别设计正、反向引物进行PCR扩增获得双荧光报告基因系统骨架；
    S6：将待测生物元件与步骤S5中获得的双荧光报告基因系统骨架通过改良后的Gibson装配方法转化至大肠杆菌DH5α，PCR验证平板上的阳性克隆，过夜培养后提取质粒；
    S7：将步骤S6中提取的质粒转化至ZM4感受态细胞中，活化培养至对数期后，使用流式细胞仪检测验证。
  2. 根据权利要求1所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于：步骤S1中所述荧光蛋白均由启动子PlacUV5启动，所述荧光蛋白为EGFP,mCherry,RFP，CFP，和分别经密码子优化后的opEGFP,opmCherry和opCFP中的任一种。
  3. 根据权利要求2所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于：步骤S2中启动子1号为Ptet，启动子2号为PlacUV5；荧光报告基因1号和荧光报告基因2号分别为EGFP和opmCherry。
  4. 根据权利要求1所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于：步骤S3中所述正、反向引物分别为引物1和引物2，引物1和引物2的序列分别见SEQ ID NO:1和SEQ ID NO:2。
  5. 根据权利要求1所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于：步骤S5中所述正、反向引物分别为引物Prtt-F和Prtt-R，引物Prtt-F和Prtt-R序列分别见SEQ ID NO:3和SEQ ID NO:4；
    或者，步骤S5中所述正、反向引物分别为引物Prtt-F和PgapTSS-R，引物Prtt-F和PgapTSS-R序列分别见SEQ ID NO:3和SEQ ID NO:4。
  6. 根据权利要求1-5任一所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于：步骤S6中所述待测生物元件为内源不同强度的启动子，或含不同强度合成RBS序列的启动子，或不同强度的终止子，或sRNA-UTR作用对。
  7. 根据权利要求6所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于，所述待测生物元件为内源不同强度的启动子或含不同强度合成RBS序列的启动子，获取所述含不同强度RBS序列的启 动子的方法包括以下步骤：
    Sa：对不同强度核糖体结合位点序列进行预测；
    Sb：以双荧光报告基因系统为模板，引物对pEZ-tetR-F、RBS-R进行PCR扩增，其中引物5’端的小写字母为与双荧光报告基因系统的同源臂，获得含特定强度RBS序列的启动子。
  8. 根据权利要求6所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于，所述待测生物元件为不同强度的终止子，获取所述不同强度的终止子的方法包括以下步骤：
    S01：筛选同一转录方向的相邻基因表达差异大的基因集，并根据表达差异进行排序；
    S02：使用生物信息学方法预测相邻基因间的终止子序列，并用相邻基因的表达差异代表终止子强度；
    S03：针对目标终止子序列设计引物，进行PCR扩增，获取终止子片段。
  9. 根据权利要求6所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于，所述待测生物元件为sRNA-UTR作用对，获取所述UTR片段的方法包括以下步骤：
    S0a：利用生物信息学方法对靶序列进行分析，确定转录起始位点后，保留从转录起始位点开始至起始密码子ATG后99-bp，作为靶5’UTR序列；
    S0b：以Z.mobilis基因组为模板，设计正、反向引物进行PCR扩增，获得靶UTR序列片段；
    以含有Pgap启动子的双荧光报告基因系统为模板，进行PCR扩增得到双荧光报告基因系统骨架。
  10. 根据权利要求7-9任一所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于：步骤S7中质粒采用电转化的方法转化至ZM4感受态细胞中，具体转化条件如下：
    (1)取ZM4感受态细胞于冰上，待感受态细胞融化后取50μL加入电转杯中，并在电转杯中加入1μg质粒；电转条件为1600V，25μF，200Ω；
    (2)电转完毕后于RM液体培养基中于30℃复苏；
    (3)复苏6-12小时的培养物于6000rpm，1min离心，除去上清；
    (4)加入200μL新鲜的RM培养基，取100μL涂布于含相应抗生素的抗性平板，30℃培养2天；
    (5)再用引物Pdual-F,Pdual-R进行PCR阳性克隆验证；
    Pdual-F CCGCTCACAATTCCACACATTATAC，见SEQ ID NO:8；
    Pdual-R ACCAGGATGGGCACCAC，见SEQ ID NO:9。
  11. 根据权利要求10所述的一种基于双荧光报告基因系统鉴定生物元件的方法，其特征在于：步骤S7中使用流式细胞仪对强度检测验证，具体步骤如下：
    (1)将经PCR阳性克隆验证正确的装入双荧光报告基因系统的单克隆于含抗生素的RM培养基中进行活化、培养；
    (2)培养至对数期后，取样200μL，12000rpm离心1min，去上清，并用1×PBS洗两次后重悬；
    (3)用流式细胞仪检测，细胞收集事件设置为20，000。
  12. 一种利用权利要求1或权利要求11所述的基于双荧光报告基因系统鉴定生物元件的方法构建的生物元件库。
PCT/CN2019/086173 2019-02-14 2019-05-09 一种基于双荧光报告基因系统鉴定生物元件的方法及基于该方法构建的生物元件库 WO2020164195A1 (zh)

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