Patent Grant
11248258
This is a U.S. National Stage under 35 U.S.C. 371 of the International Application PCT/CN2019/086173, filed May 9, 2019, which claims priority under 35 U.S.C. 119(a-d) to CN 201910114635.7, CN 201910114932.1 and CN 201910114652.0, all filed Feb. 14, 2019.
The present invention relates to a technical field of biology, and more particularly to a method for identifying biological parts based on a dual-fluorescent reporter gene system and a biological part library constructed thereon.
Zymomonas mobilis (Z. mobilis) is the gram-negative and facultative anaerobic bacteria, which is peritrichous and has a size of (1.4-2.0)*(4.0-5.0) μm. An appropriate growth temperature of Z. mobilis is 30° C., and a tolerant pH range is 3.5-9. Z. mobilis can high-efficiently utilize the glucose, fructose and saccharose to generate ethanol through the ED (Enter-Doudoroff) pathway. Because of the advantages of high ethanol yield and tolerance, high osmotic pressure tolerance, low biomass, and no need of adding oxygen when fermenting, Z. mobilis has become one of the main strains for producing the bio-ethanol. In order to improve the application range of Z. mobilis, the system biological researches on Z. mobilis are carried out successively. Meanwhile, with the development of metabolic engineering and synthetic biology, the demands on the available biological parts in Z. mobilis are increasing.
However, at present, mining of the system biological data of Z. mobilis is still not enough, and particularly there are fewer researches about mining and development of the biological parts such as the promoters and the ribosome binding sequences in the genome scale, causing that the identified available biological parts of Z. mobilis become fewer and fewer, which greatly limits the developments of the accurate metabolic engineering on Z. mobilis and the synthetic biology era.
The promoter is the important part for regulating the gene expression and is the site can be identified and specifically bound by the RNA polymerase. The intensity of the promoter is generally used for describing the initial transcription frequency of the RNA polymerase at the promoter. When conducting the biological synthesis, the promoter of certain intensity is selected for assisting the successful completion of the biological synthesis.
The conventional promoter screening method is to randomly cut the genome, and then identify whether the obtained sequence is the promoter sequence and how is the intensity thereof through the expression of the downstream single reporter system. The conventional detecting system is generally the single-reporter gene system. However, the expression of the reporter gene in the single-reporter gene system will be influenced by multiple factors from the internal and external of the strains, which influences the identification of the system to the promoter. Moreover, with the conventional method, the speed of obtaining the promoter to be detected is slow, and the intensity identification cannot be conducted with fast speed and high throughput, which is unbeneficial to the high-efficient quantitative research of the promoter. With the development of the various technologies, although some new methods are developed, such as the in vitro detecting techniques of electrophoretic mobility shift assay and atomic force microscope, the applications of these methods are limited due to the low accuracy.
In conclusion, it is urgent to develop and mine the biological parts available for metabolic engineering of Z. mobilis with the conventional system biological data, especially the promoters and the ribosome binding sites of certain intensities and sRNA-UTR interaction pairs, and to establish an in vivo quantitative analysis method for the promoters and other biological parts with high efficiency, fast speed and high throughput, which is significant for expanding the part library of promoters of Z. mobilis and other biological parts. Moreover, the method can also be applied in other microorganism systems, and provide various biological functions and regulation parts for metabolic engineering and synthetic biology.
Aiming at existing problems in prior art, the present invention provides a method for identifying biological parts based on a dual-fluorescent reporter gene system and a biological part library constructed thereon.
The above technical object of the present invention is realized through technical solutions as follows.
A method for identifying biological parts based on a dual-fluorescent reporter gene system comprises steps of:
(S1) with pEZ15Asp plasmids as a skeleton, constructing a single-fluorescent reporter gene system, and screening fluorescent proteins;
(S2) according to expressions of different fluorescent protein genes in Zymomonas mobilis (Z. mobilis hereinafter), screening suitable fluorescent reporter genes, respectively named as a first fluorescent reporter gene and a second fluorescent reporter gene; choosing suitable promoters for the first and second fluorescent reporter genes, respectively named as a first promoter and a second promoter;
(S3) with pEZ15Asp as a template, respectively designing a forward primer and a reverse primer, conducting PCR (Polymerase Chain Reaction) amplification, and obtaining a pEZ15Asp skeleton;
(S4) with utilizing a modified Gibson assembly method, connecting first promoter-first fluorescent reporter gene and second promoter-second fluorescent reporter gene to the pEZ15Asp skeleton, adding a terminator between the two fluorescent reporter genes, and obtaining the dual-fluorescent reporter gene system;
(S5) with the dual-fluorescent reporter gene system as a template, respectively designing a forward primer and a reverse primer, conducting PCR amplification, and obtaining a dual-fluorescent reporter gene system skeleton;
(S6) through the modified Gibson assembly method, transforming the biological parts to be detected and the dual-fluorescent reporter gene system skeleton obtained in the step of S5 into Escherichia coli DH5α; verifying positive clones on a plate by PCR; after culturing overnight, extracting plasmids; and
(S7) transforming the plasmids extracted in the step of S6 into ZM4 competent cells; activating and culturing to a logarithmic phase, then detecting and verifying with a flow cytometer.
Preferably, in the step of S1, the fluorescent proteins are all promoted by a promoter PlacUV5; the fluorescent proteins are one of EGFP, mCherry, RFP, CFP, and opEGFP, opmCherry and opCFP after being optimized by a codon.
Preferably, in the step of S2, the first promoter is Ptet; the second promoter is PlacUV5; the first fluorescent reporter gene and the second fluorescent reporter gene are respectively EGFP and opmCherry.
Preferably, in the step of S3, the forward primer and the reverse primer are respectively a first primer and a second primer; sequences of the first primer and the second primer respectively refer to SEQ ID NO: 1 and SEQ ID NO: 2.
Preferably, in the step of S5, the forward primer and the reverse primer are respectively a primer Prtt-F and a primer Prtt-R; sequences of the primer Prtt-F and the primer Prtt-R respectively refer to SEQ ID NO: 3 and SEQ ID NO: 4; or
in the step of S5, the forward primer and the reverse primer are respectively the primer Prtt-F and a primer PgapTSS-R; sequences of the primer Prtt-F and the primer PgapTSS-R respectively refer to SEQ ID NO: 3 and SEQ ID NO: 65.
Preferably, in the step of S6, the biological parts to be detected are endogenous promoters of different intensities, promoters containing synthetic RBS (Ribosome Binding Site) sequences of different intensities, terminators of different intensities, or sRNA-UTR (soluble Ribonucleic Acid-Untranslated Region) interaction pairs.
Further preferably, the biological parts to be detected are the endogenous promoters of different intensities or the promoters containing the synthetic RBS sequences of different intensities; the promoters containing the RBS sequences of different intensities are obtained through steps of:
(Sa) according to different omics data, screening out genes having strong downstream expressions; then conducting Venn analysis; and screening out shared genes of each omics data;
(Sb) predicting the RBS sequences of different intensities; and
(Sc) with the dual-fluorescent reporter gene system as a template, conducting PCR amplification with a primer pEZ-tetR-F and a primer RBS-R, wherein lowercases at a 5′ terminal of each primer are homologous arms of the dual-fluorescent reporter gene system; and obtaining the promoters containing the RBS sequences of specific intensities.
Further preferably, the biological parts to be detected are the terminators of different intensities; the terminators of different intensities are obtained through steps of:
(S01) screening a gene set whose contiguous genes in a same transcription direction have large expression differences, and ordering according to the expression differences;
(S02) with a bioinformatics method, predicting terminator sequences between the contiguous genes, and representing the intensities of the terminators by the expression differences between the contiguous genes; and
(S03) designing primers for a target terminator sequence, conducting PCR amplification, and obtaining terminator fragments.
Further preferably, the biological parts to be detected are the sRNA-UTR interaction pairs; UTR fragments are obtained through steps of:
(S0a) with a bioinformatics method, analyzing a target sequence; after determining a transcriptional start site, retaining the sequence from the transcriptional start site to 99-bp after an initiation codon ATG, as a target 5′ UTR sequence;
(S0b) with a Z. mobilis genome as a template, designing a forward primer and a reverse primer, conducting PCR amplification, and obtaining the target UTR sequence fragments;
with a dual-fluorescent reporter gene system containing a promoter Pgap as a template, conducting PCR amplification, and obtaining the dual-fluorescent reporter gene system skeleton.
Further preferably, in the step of S7, the plasmids are electronically transformed into the ZM4 competent cells, particularly comprising steps of:
(1) placing the ZM4 competent cells on ice; after the ZM4 competent cells melt, taking 50 μL of the competent cells and adding into an electro-transformation cup, and then adding 1 μg of the plasmids into the electro-transformation cup, wherein electro-transformation conditions are 1600 V, 25 μF and 200 Ω;
(2) after completing electro-transformation, resuscitating at 30° C. in RM (Rich Media);
(3) centrifuging a culture, which is obtained after 6-12 hours of resuscitation, with a rotational speed of 6000 rpm for 1 minute, so as to remove a supernatant;
(4) adding 200 μL of fresh RM; taking a sample of 100 μL and coating on a resistant plate containing corresponding antibiotics; and culturing at 30° C. for 2 days; and
(5) conducting PCR positive clone verification with a primer Pdual-F and a primer Pdual-R, wherein:
Further preferably, in the step of S7, the intensities are detected and verified with the flow cytometer, particularly comprising steps of:
(1) activating and culturing mono-clones, which are loaded into the dual-fluorescent reporter gene system and verified to be correct by PCR positive clone verification, in RM containing corresponding antibiotics;
(2) after culturing to the logarithmic phase, taking a sample of 200 μL; centrifuging with a rotational speed of 12000 rpm for 1 minute, so as to remove a supernatant; washing with 1×PBS (Phosphate Buffered Saline) for two times, and re-suspending;
(3) detecting with the flow cytometer, wherein a cell collection event is set to be 20,000.
A second object of the present invention is to provide a biological part library constructed on the method for identifying the biological parts based on the dual-fluorescent reporter gene system.
Compared with the prior art, the present invention has beneficial effects as follows.
With the Z. mobilis as the type strains, the present invention establishes the method for predicting and screening the promoter sequences of different intensities with utilizing the system biological data. The predicting and screening method is high-efficient and quick, and can serve as a guidance basis of the experiments. The present invention integrates the existing system biological data, and high-efficiently and quickly screens the promoters of specific intensities. The RBS intensities and the promoter intensities, predicted and screened by the present invention, have the relatively good correlation with the experimental data (respectively R2>0.9 and R2>0.7), illustrating that the method for screening the promoters based on the system biological data, provided by the present invention, can be applied in predicting and screening the promoters and other biological parts of different demands. Compared with the conventional promoter screening and identifying method, the method provided by the present invention can screen and quantify the promoters of different intensities and other biological parts with fast speed and high throughput, and the quantitative analysis of the promoters is completed inside the cells, which is less influenced by the internal and external environmental change of the cells and is accurate; moreover, the method provided by the present invention can quickly expand the biological part library of Z. mobilis, so as to be applied in metabolic engineering of different demands.
According to the present invention, with the conventional system biological data and the bioinformatics method, the biological parts of different intensities are predicted and screened, and the intensities are identified and verified through the dual-fluorescent reporter gene system developed by the laboratory of the inventors, for quantitatively analyzing the intensities of the promoters and other biological parts and finding the biological parts available for metabolic engineering and synthetic biology. Combined with the two methods, a strategy for quickly selecting the stand-by biological parts is established, which is applicable in the rational design of the synthetic biology era and the microorganism modification.
Moreover, the dual-fluorescent reporter gene system provided by the present invention can be applied in the quantitative analysis of other biological parts, and in the other species besides Z. mobilis. The advantages of the present invention are listed in the following table.
Comparison of advantages and disadvantages between present invention and prior art
The conventional method for identifying the interactive relationship of sRNA-UTR has the complex operation, and detecting is generally conducted in vitro, which cannot avoid the troubles of obtaining the experimental material in vitro and conducting the related experiments in vivo, causing that whether the interactive relationship exists between sRNA and the target UTR thereof cannot be quickly determined, so that it is unbeneficial to batch operation and cannot be applied in the batch quantitative analysis of the intensity of the interaction between the target sRNA and the target 5′ UTR thereof. Compared with the conventional method for identifying the interactive relationship of sRNA-UTR, the present invention is convenient and quick, and has the easy operation. With the dual-fluorescent reporter gene system based on the flow cytometry, the present invention conducts detecting in vivo, which avoids the troubles of obtaining the experimental material in vitro and conducting the related experiments in vivo, is able to quickly determine whether the interactive relationship exists between sRNA and the target UTR thereof, and is beneficial to batch operation. With utilizing the dual-fluorescent reporter gene system, the method provided by the present invention can be applied in quantitatively analyzing the intensity of the interaction and can be transplanted to other species besides Z. mobilis. The present invention provides a method for identifying the interaction between sRNA and the target mRNA 5′ UTR thereof, which is able to quickly and high-efficiently determine the interactive relationship between the target sRNA and the target 5′ UTR thereof and conducts the quantitative analysis.
With utilizing the common database, biological data and dual-fluorescent reporter gene system, the present invention screens and identifies the intensities of 37 promoters and 4 RBS (Ribosome Binding Site), and the intensities of 4 sRNA-UTR interaction pairs and 6 terminators, which greatly expand the part library of Z. mobilis. The present invention can realize the full mining and utilization of the system biology of Z. mobilis and different microorganism systems and expand the biological part library thereof, and can be applied in metabolic engineering and synthetic biology. The present invention is simple, has easy operation and wide range of applicable biological parts, and can be promoted to other species.
Technical solutions of the present invention will be clearly and completely described with following preferred embodiments. The described preferred embodiments are merely a part of the embodiments of the present invention, not all of the embodiments. Based on the preferred embodiments of the present invention, all of other embodiments made by one of ordinary skill in the art without creative efforts should be encompassed in the protection scope of the present invention.
The present invention provides a method for identifying biological parts based on a dual-fluorescent reporter gene system and a biological part library constructed thereon. Details are illustrated with the preferred embodiments as follows.
The method comprises steps of:
(S1) with pEZ15Asp plasmids as a skeleton, constructing a single-fluorescent reporter gene system as shown in
(S2) sending gene sequences of every fluorescent protein to a third-party company for gene sequence synthesis; with pEZ15Asp as a template, conducting PCR (Polymerase Chain Reaction) amplification respectively with a first primer and a second primer, and obtaining a pEZ15Asp skeleton; with utilizing a Gibson assembly method, connecting PlacUV5-fluorescent reporter gene to the pEZ5Asp skeleton; through transforming into Escherichia coli DH5α, obtaining the single-fluorescent reporter gene system; for obtained recombinant strains, verifying positive clones on a plate by PCR, extracting plasmids after culturing overnight, and sequencing for verifying connection and sequence correctness; transforming the extracted plasmids into ZM4 competent cells, activating and culturing to a logarithmic phase, and detecting expression intensities with a flow cytometer; wherein:
according to fluorescent expression intensities of different fluorescent protein genes in Zymomonas mobilis (Z. mobilis hereinafter), screening suitable fluorescent reporter genes, respectively named as a first fluorescent reporter gene and a second fluorescent reporter gene; wherein: Ptet is chosen as a first promoter, and PlacUV5 is chosen as a second promoter; it can be seen from expression results shown in
(S3) with pEZ15Asp as the template, designing the first primer and the second primer, conducting PCR amplification, and obtaining the pEZ15Asp skeleton; according to a PCR recovery kit, recovering and purifying a PCR product; wherein:
preparation of a PCR system is:
setting of a PCR program is: pre-denaturing at 98° C. for 3 minutes, denaturing at 98° C. for 10 seconds, annealing at 55° C. for 10 seconds, and extending at 72° C. for 35 seconds, totally 29 cycles;
(S4) with utilizing a modified Gibson assembly method, connecting Ptet-first fluorescent reporter gene, namely Ptet-EGFP, and PlacUV5-second fluorescent reporter gene, namely PlacUV5-opmCherry, to the pEZ15Asp skeleton obtained in the step of S3; adding a terminator between the two fluorescent reporter genes; and obtaining an inducible dual-fluorescent reporter gene system, as shown in
in the first preferred embodiment, the terminator added between the two fluorescent reporter genes is a terminator BBa_B0014, and a sequence thereof is:
terminators used by the two fluorescent protein genes 3′ are both a terminator rmB T1, and a sequence thereof is:
for the modified Gibson assembly method, a system of 5 μL is used in the connection process; a molar ratio of target fragments to vectors is 3:1, 0.5 μL of T5 excision enzyme and 0.5 μL of buffer 4 are added; if the volume is not enough, deionized water is supplemented; the method particularly comprises steps of: after uniformly mixing the system, reacting on ice for 5 minutes; then adding 100 μL of DH5α chemical competent cells; after incubating on ice for 30 minutes, heat-shocking at 42° C. for 45 seconds, and then standing on ice for 2-3 minutes, so that more recombinant vectors will enter the competent cells; after recovering at 37° C. with a rotational speed of 250 rpm for 1 hour, coating to LB (Lysogeny Broth) solid media containing 200 μg/mL of spectinomycin, and culturing in an incubator at 37° C., wherein in other embodiments, antibiotics of other types and concentrations can be selected according to actual requirements; after 12-24 hours, selecting 5-10 single colonies having clear edges for colony PCR, so as to pick out a recombinant; further selecting 2 single colonies having clearest bands, and sending to a sequencing company for sequencing and verifying; after obtaining the sequences, comparing with an original sequence, so as to determine a sequence correctness;
after the sequences are determined to be correct, extracting plasmids with a conventional plasmid miniprep kit, Tsingke; after obtaining the plasmids, electronically transforming the plasmids into the wild-type Z. mobilis; through colony PCR and sequencing, determining a final strain; wherein: sequencing is completed by the Tsingke Company; the obtained sequence is compared with an original sequence, so as to determine a sequence correctness;
the system verification process comprises steps of: after obtaining the strain containing the inducible dual-fluorescent reporter gene system, conducting inducing culture with tetracycline having a concentration of 0, 0.2 μg/mL, 0.4 μg/mL, 0.6 μg/mL, 0.8 μg/mL or 1.0 μg/mL, wherein a sampling time is the logarithmic phase; and verifying the dual-fluorescent reporter gene system in different aspects respectively with FCM (Flow Cytometry), qPCR (quantitative PCR), and WB (Western Blot); wherein: results thereof are shown in
a formula of the RM comprises components of RMG5: 50 g/L glucose, 10 g/L yeast extract, 2 g/L K H2PO4, and 200 μg/mL spectinomycin; and culture conditions are 30° C. and 100 rpm;
FCM particularly comprises steps of:
(1) activating and culturing mono-clones, which are loaded into the dual-fluorescent reporter gene system and verified to be correct by PCR positive clone verification, in the RM containing the spectinomycin of 200 μg/mL; wherein: in other embodiments, the antibiotics of other types and concentrations can be selected according to actual requirements;
(2) after culturing to the logarithmic phase, taking a sample of 200 μL; centrifuging with a rotational speed of 12000 rpm for 1 minute, so as to remove a supernatant; washing with 1×PBS (Phosphate Buffered Saline) for two times, and re-suspending;
(3) detecting with the flow cytometer, wherein an event start record is 1,000 cells, and detecting is ended until 20,000 cells;
(4) with non-fluorescent strains and strains containing the single fluorescent EGFP and opmCherry as controls, drawing a gate, wherein compensation is made automatically by the software; and
(5) for EGFP, adopting an excitation wavelength of 488 nm and a detector of FITC; for opmCherry, adopting an excitation wavelength of 561 nm and a detector of PC5.5; taking average values of three replicate samples and taking the logarithm of log 2, and conducting subsequent data analysis;
qPCR analyzes the transcriptional levels of EGFP and opmCherry in the dual-fluorescent reporter gene system at the same thermal cycling conditions, particularly comprises steps of: with a TRIzol (Invitrogen, USA) method, extracting the total RNA of the logarithmic-phase sample, and detecting the quality of the extracted RNA with NanoDrop 800; then, according to operation instructions of the commercial kit iScript™ gDNA Clear cDNA Synthesis Kits (Bio-Rad, USA), removing the genome DNA and inversely transcribing into cDNA; conducting the qPCR fluorescent quantitative reaction on the instrument CFX96 Real-Time System (Bio-Rad, USA) with the kit iTaq™ Universal SYBR® Green Supermix (Bio-Rad, USA); wherein: the primers used in the experiment are high-purity salt-free primers having an annealing temperature of 60° C.; sequences of the primers are listed as follows:
after analyzing the specificity of the PCR product through the melting curve, detecting according to a following program, particularly comprising steps of: denaturing at 95° C. for 5 minutes; completing 40 amplification cycles (95° C. 15 s, 60° C. 10 s, and 72° C. 30 s); quantitatively detecting the single fluorescent proteins; with the absolute quantitative method based on the internal-reference calibration curve, conducting the qPCR data analysis;
Western Blot particularly comprises steps of:
using the protein extraction kit (Zomanbio, China) for cell lysis and total protein extraction; with the Bradford method, quantifying the protein loading quantity to 200 ng; conducting SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) with 5% stacking gel and 12% separating gel; with the pre-stained protein Marker (10-170 kDa, Thermo, Lithuania), distinguishing the size of the protein bands; after completing electrophoresis, with the Trans-Blot® Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, USA) system, transferring the target protein (EGFP or opmCherry) to the PVDF (Polyvinylidene Fluoride) membrane immersed in methyl alcohol, wherein transferring conditions are 25 V and 20 minutes; after membrane transferring, sealing at a room temperature for 1 hour with 5% skim milk powders; after completing sealing, incubating the primary antibodies (Proteintech, China) of EGFP or opmCherry with a ratio of 1:5000; after incubating at the room temperature for 1 hour, washing with 1×PBST every 5 minutes, totally washing for three times; then, incubating the secondary antibodies (Peroxidase-conjugated goat anti-Mouse IgG) of EGFP or opmCherry also with a ratio of 1:5000; after incubating at the room temperature for 1 hour, washing with 1×PBST every 5 minutes, totally washing for three times; preparing the gel substrate (Immobilon Western Chemiluminescent HRP Substrate) with a ratio of 1:1; and imaging with AI600 Imaging System (GE, USA);
the results are shown in
(S5) with the dual-fluorescent reporter gene system as a template, conducting PCR amplification with a primer Prtt-F and a primer Prtt-R, and obtaining a dual-fluorescent reporter gene system skeleton; wherein:
preparation of a PCR system is:
setting of a PCR program is: pre-denaturing at 98° C. for 3 minutes, denaturing at 98° C. for 10 seconds, annealing at 55° C. for 10 seconds, and extending at 72° C. for 50 seconds, totally 29 cycles;
(S6) through the modified Gibson assembly method, transforming the biological parts to be detected and the dual-fluorescent reporter gene system skeleton obtained in the step of S5 into Escherichia coli DH5α; wherein:
for the modified Gibson assembly method, a system of 5 μL is used in the connection process; a molar ratio of target fragments to vectors is 3:1, 0.5 μL of T5 excision enzyme and 0.5 μL of buffer 4 are added; if the volume is not enough, deionized water is supplemented; the method particularly comprises steps of: after uniformly mixing the system, reacting on ice for 5 minutes; then adding 100 μL of DH5α chemical competent cells; after incubating on ice for 30 minutes, heat-shocking at 42° C. for 45 seconds, and then standing on ice for 2-3 minutes, so that more recombinant vectors will enter the competent cells; after recovering at 37° C. with a rotational speed of 250 rpm for 1 hour, coating to LB solid media containing 200 μg/mL of spectinomycin, and culturing in an incubator at 37° C., wherein in other embodiments, antibiotics of other types and concentrations can be selected according to actual requirements; after 12-24 hours, selecting 5-10 single colonies having clear edges for colony PCR, so as to pick out a recombinant; further selecting 2 single colonies having clearest bands, and sending to a sequencing company for sequencing and verifying; after obtaining the sequences, comparing with an original sequence, so as to determine a sequence correctness; wherein:
preparation of a colony PCR system is:
setting of a colony PCR program is: pre-denaturing at 98° C. for 3 minutes, denaturing at 98° C. for 10 seconds, annealing at 55° C. for 10 seconds, and extending at 72° C. for 30 seconds, totally 25 cycles;
after the sequences are determined to be correct, extracting plasmids with the conventional plasmid miniprep kit, Tsingke; after obtaining the plasmids, electronically transforming the plasmids into the wild-type Z. mobilis; through colony PCR and sequencing, determining a final strain; wherein: sequencing is completed by the Tsingke Company; the obtained sequence is compared with an original sequence, so as to determine a sequence correctness;
(S7) transforming the recombinant plasmids extracted in the step of S6 into ZM4 competent cells; activating and culturing to a logarithmic phase, then detecting and verifying with the flow cytometer.
The method for screening the endogenous promoters comprises steps of: according to different omics data, screening out genes having strong downstream expressions, and conducting Venn analysis, as shown in
The method for identifying the endogenous promoters comprises steps of operating according to steps of S1-S6 in the first preferred embodiment, wherein: in the step of S6, the endogenous promoters Pgap of certain intensity are experimented, as the biological parts to be detected; in other embodiments, the promoters of other intensities, the promoters containing the RBS (Ribosome Binding Site) sequences of different intensities, or other biological parts can be selected for being detected.
The promoter Pgap is obtained through steps of: with Z. mobilis ZM4 as a template, conducting PCR amplification with a primer P0177-F and a primer P0177-R, and obtaining promoter fragments, wherein lowercases at a 5′ terminal of each primer are homologous arms of the dual-fluorescent reporter gene system; according to a PCR recovery kit, recovering and purifying a PCR product; wherein:
preparation of a PCR system is:
setting of a PCR program is: pre-denaturing at 98° C. for 3 minutes, denaturing at 98° C. for 10 seconds, annealing at 55° C. for 10 seconds, and extending at 72° C. for 10 seconds, totally 29 cycles;
The method further comprises a step of: (S7) transforming the recombinant plasmids extracted in the step of S6 into ZM4 competent cells; activating and culturing to a logarithmic phase, then detecting and verifying with a flow cytometer.
According to the second preferred embodiment, the recombinant plasmids are electronically transformed into the ZM4 competent cells, particularly comprising steps of: placing the ZM4 competent cells on ice; after the ZM4 competent cells melt, taking 50 μL of the competent cells and adding into an electro-transformation cup, and then adding 1 μg of the plasmids into the electro-transformation cup, wherein electro-transformation conditions are 1600 V, 25 μF and 200Ω; after completing electro-transformation, resuscitating at 30° C. in RM; centrifuging a culture, which is obtained after 6-12 hours of resuscitation, with a rotational speed of 6000 rpm for 1 minute, so as to remove a supernatant; adding 200 μL of fresh RM; taking a sample of 100 μL and coating on a resistant plate containing spectinomycin of 200 μg/mL (in other embodiments, antibiotics of other types and concentrations can be selected according to actual requirements); culturing at 30° C. for 2 days; and, conducting PCR positive clone verification with a primer Pdual-F and a primer Pdual-R, wherein:
preparation of a colony PCR system is:
setting of a colony PCR program is: pre-denaturing at 98° C. for 3 minutes, denaturing at 98° C. for 10 seconds, annealing at 55° C. for 10 seconds, and extending at 72° C. for 30 seconds, totally 25 cycles;
The detecting process with FCM comprises steps of: activating 38 mono-clones, which are verified to be correct, in the RM containing the spectinomycin of 200 μg/mL (in other embodiments, antibiotics of other types and concentrations can be selected according to actual requirements); after activating, culturing three parallels for each sample; after culturing to the logarithmic phase, taking a sample of 200 μL; centrifuging with a rotational speed of 12000 rpm for 1 minute, so as to remove a supernatant; washing with 1×PBS for two times, and re-suspending; detecting with the flow cytometer according to a set program, wherein a cell collection event is set to be 20,000 in the second preferred embodiment, so as to avoid small-probability and accidental events.
Result analysis: according to data obtained by the flow cytometer, for each sample, calculating with average fluorescent values of EGFP and opmCherry of all the events; and standardizing with a ratio of EGFP/opmCherry, so as to eliminate interferences from the internal and external of the cells. The results thereof are shown in
The results indicate that: the biological part promoters loaded in the dual-fluorescent reporter gene system can be quickly and quantitatively analyzed with the flow cytometer; the ratio of EGFP/opmCherry represents the relative intensity of the tested promoters in the system; the correlation between the experimental results and the intensities predicted by the omics data is relatively high, illustrating that the dual-fluorescent reporter gene system can be applied in identifying the intensities of the promoters.
The method comprises steps of:
(Sa) according to different omics data, screening out genes having strong downstream expressions; then conducting Venn analysis; and screening out shared genes of each omics data;
(Sb) predicting the RBS sequences of different intensities with RBS Calculator V2.0 according to 16s rRNA of Z. mobilis; wherein:
Z. mobilis 16S rRNA is:
Table 2 RBS sequences of different intensities of Z. mobilis according to third preferred embodiment
(Sc) obtaining the promoters containing the RBS sequences of specific intensities, particularly comprising steps of: according to the third preferred embodiment, with RBS-10 as an example and the dual-fluorescent reporter gene system containing the promoter Ptet as a template, conducting PCR amplification with a primer pEZ-tetR-F and a primer RBS-10-R, wherein lowercases at a 5′ terminal of each primer are homologous arms of the dual-fluorescent reporter gene system; and obtaining the promoters containing the RBS sequences of specific intensities; wherein:
preparation of a PCR system is:
setting of a PCR program is: pre-denaturing at 98° C. for 3 minutes, denaturing at 98° C. for 10 seconds, annealing at 55° C. for 10 seconds, and extending at 72° C. for 20 seconds, totally 29 cycles;
After obtaining the promoters containing the RBS sequences of specific intensities, the intensities of the obtained promoters are verified based on the dual-fluorescent reporter gene system, particularly comprising steps of:
with the modified Gibson assembly method, transforming the obtained promoters containing the RBS sequences of specific intensities and the dual-fluorescent reporter gene system skeleton obtained in the first preferred embodiment into Escherichia coli DH5α; verifying positive clones on a plate by PCR; after culturing overnight, extracting plasmids, and obtaining recombinant plasmids; wherein: the plasmids are extracted according to standard steps of the plasmid extraction kit;
electronically transforming the extracted recombinant plasmids into the ZM4 competent cells, particularly comprising steps of: placing the ZM4 competent cells on ice; after the ZM4 competent cells melt, taking 50 μL of the competent cells and adding into an electro-transformation cup, and then adding 1 μg of the plasmids into the electro-transformation cup, wherein electro-transformation conditions are 1600 V, 25 μF and 200 S; after completing electro-transformation, resuscitating at 30° C. in RM; centrifuging a culture, which is obtained after 6-12 hours of resuscitation, with a rotational speed of 6000 rpm for 1 minute, so as to remove a supernatant; adding 200 μL of fresh RM; taking a sample of 100 μL and coating on a resistant plate containing spectinomycin of 200 μg/mL (in other embodiments, antibiotics of other types and concentrations can be selected according to actual requirements); culturing at 30° C. for 2 days; and, conducting PCR positive clone verification with a primer Pdual-F and a primer Pdual-R, wherein: sequences of the primer Pdual-F and the primer Pdual-R respectively refer to SEQ ID NO: 8 and SEQ ID NO: 9;
verifying the intensities, particularly comprising steps of: activating mono-clones, which are loaded into the dual-fluorescent reporter gene system and verified to be correct by PCR positive clone verification, in the RM containing the spectinomycin of 200 μg/mL (in other embodiments, antibiotics of other types and concentrations can be selected according to actual requirements); after activating, culturing three parallels for each sample; conducting inducing culture with tetracycline having a concentration of 0, 0.2 μg/mL, 0.4 μg/mL, 0.6 μg/mL, 0.8 μg/mL or 1.0 μg/mL; after culturing to the logarithmic phase, taking a sample of 200 μL; centrifuging with a rotational speed of 12000 rpm for 1 minute, so as to remove a supernatant; washing with 1×PBS for two times, and re-suspending; detecting with the flow cytometer according to a set program, wherein a cell collection event is set to be 20,000 in the third preferred embodiment, so as to avoid small-probability and accidental events.
Result analysis: according to data obtained by the flow cytometer, for each sample, calculating with average fluorescent values of EGFP and opmCherry of all the events; and standardizing with a ratio of EGFP/opmCherry, so as to eliminate interferences from the internal and external of the cells. The results thereof are shown in
The method comprises steps of:
(S01) screening a gene set whose contiguous genes in a same transcription direction have large expression differences, and ordering according to the expression differences;
(S02) with a bioinformatics method, predicting terminator sequences between the contiguous genes, and representing the intensities of the terminators by the expression differences between the contiguous genes; and
(S03) designing primers for a target terminator sequence, conducting PCR amplification, and obtaining terminator fragments; wherein: in the fourth preferred embodiment, with a terminator T1929f as an example, the PCR amplification is conducted with the primers T1929f-F and T1929f-R and the ZM4 genome as the template, and finally the terminator T1929f fragments are obtained.
With the modified Gibson assembly method, the terminator sequence is inserted after the transcriptional start site of the promoter and before EGFP, wherein the used promoter herein is the promoter Pgap.
Through the step of S6 in the first preferred embodiment, the recombinant plasmids are obtained; and then the plasmids are electronically transformed through the step of S7.
Through the step of S7, the intensities care verified.
The terminators only influence the transcription, not influence the translation process. According to the fourth preferred embodiment, the stronger the terminator intensity, the smaller the value of EGFP/opmCherry; the experimental results conform to the predicted intensities Table 3 shows the relevant information of the verified terminators and the verification results with the method provided by the fourth preferred embodiment.
mobilis according to fourth preferred embodiment
The method comprises steps of:
(S0a) with a bioinformatics method, analyzing a target sequence; after determining a transcriptional start site, retaining the sequence from the transcriptional start site to 99-bp after an initiation codon ATG, as a target 5′ UTR sequence; wherein:
verification of the interaction between sRNA Zms4 and the gene ZMO1754 UTR (Zms4-UTR1754) is described in detail as an example, and experimental principles thereof are shown in
taking the intermediate sequence between the gene ZMO1754 and the previous gene; conducting promoter analysis with a BPROM program; after obtaining the TSS (Transcriptional Start Site), retaining the sequence from the TSS to 99-bp after the initiation codon ATG of the gene ZMO1754 as the UTR1754 sequence, referring to SEQ ID NO: 62;
(S0b) with a Z. mobilis genome as a template, designing a forward primer and a reverse primer, conducting PCR amplification, and obtaining the target UTR sequence fragments; the step of S0b particularly comprising steps of:
with the Z. mobilis genome as the template, conducting PCR amplification with the primer UTR1754-F and the primer UTR1754-R, and obtaining the UTR1754 sequence; according to a PCR recovery kit, recovering and purifying a PCR product (the lowercases in the sequence of each primer are the homologous arms, and the uppercases are the primer sequences); wherein:
preparation of a PCR system is:
setting of a PCR program is: pre-denaturing at 98° C. for 3 minutes, denaturing at 98° C. for 10 seconds, annealing at 55° C. for 10 seconds, and extending at 72° C. for 10 seconds, totally 29 cycles;
(S0c) with the dual-fluorescent reporter gene system containing the promoter Pgap as the template, conducting PCR amplification with a primer Prtt-F and a primer PgapTSS-R, and obtaining the dual-fluorescent reporter gene system skeleton; according to the PCR recovery kit, recovering and purifying the PCR product; wherein:
in the first preferred embodiment, when verifying the intensity of the promoter Pgap, the dual-fluorescent reporter gene system containing the promoter Pgap has been obtained, and thus it is used as the template in the fifth preferred embodiment;
(S0d) obtaining recombinant plasmids, particularly comprising steps of: after obtaining the UTR sequence and the dual-fluorescent reporter gene system skeleton, with the modified Gibson assembly method, transforming into Escherichia coli DH5α; verifying positive clones on a plate by PCR; after culturing overnight, extracting plasmids (the plasmids are extracted according to standard steps of the plasmid extraction kit);
(S0e) obtaining the strains of the dual-fluorescent reporter gene system plasmids, which contain the specific UTR sequences, particularly comprising steps of: electronically transforming the Z. mobilis wild-type strains, Zms4 knock-out strains and over-expressed strains with the extracted plasmids; placing the corresponding competent cells on ice; after the competent cells melt, taking 50 μL of the competent cells and adding into an electro-transformation cup, and then adding 1 μg of the plasmids into the electro-transformation cup, wherein electro-transformation conditions are 1600 V, 25 μF and 200; after completing electro-transformation, resuscitating at 30° C. in RM; centrifuging a culture, which is obtained after 6-12 hours of resuscitation, with a rotational speed of 6000 rpm for 1 minute, so as to remove a supernatant; adding 200 μL of fresh RM; taking a sample of 100 μL and coating on a resistant plate containing kanamycin of 300 μg/mL; culturing at 30° C. for 2 days; and, conducting PCR positive clone verification with a primer Pdual-F and a primer Pdual-R wherein:
(S0f) detecting with FCM, particularly comprising steps of: activating mono-clones, which are verified to be correct, in the RM containing the kanamycin of 300 μg/mL; after activating, culturing three parallels for each sample; after culturing to the logarithmic phase, taking a sample of 200 μg; centrifuging with a rotational speed of 12000 rpm for 1 minute, so as to remove a supernatant; washing with 1×PBS for two times, and re-suspending; detecting with the flow cytometer according to a set program, wherein a cell collection event is set to be 20,000, so as to avoid small-probability and accidental events.
Result analysis: according to data obtained by the flow cytometer, for each sample, calculating with average fluorescent values of EGFP and opmCherry of all the events; and standardizing with a ratio of EGFP/opmCherry, so as to eliminate interferences from the internal and external of the cells. The results thereof are shown in
In a similar way, the interactive relationship of Zms4-UTR1993 is identified.
The experimental results obtained through the system provided by the present invention is consistent with the results obtained through the conventional experimental method. However, the present invention is quick, convenient, safe and high-efficient, has a short experimental period (save at least one week in comparison with the conventional method), and can conduct batch operation.
Table 4 shows relevant information of the identified interaction pairs with the technical solutions according to the fifth preferred embodiment, wherein: Zms4 nucleotide sequence refers to SEQ ID NO: 66, Zms6 nucleotide sequence refers to SEQ ID NO: 67; UTR1754 nucleotide sequence refers to SEQ ID NO: 68; UTR1993 nucleotide sequence refers to SEQ ID NO: 69; UTR0170 nucleotide sequence refers to SEQ ID NO: 70, UTR1934 nucleotide sequence refers to SEQ ID NO:71, and UTR149 nucleotide sequence refers to SEQ ID NO: 72.
The sequences of the promoters screened by the step of Sa in the third preferred embodiment are listed as follows.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910114635.7 | Feb 2019 | CN | national |
| 201910114652.0 | Feb 2019 | CN | national |
| 201910114932.1 | Feb 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/086173 | 5/9/2019 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2020/164195 | 8/20/2020 | WO | A |
| Number | Date | Country |
|---|---|---|
| 103382505 | Nov 2013 | CN |
| 105555947 | May 2016 | CN |
| 108018301 | May 2018 | CN |
| Entry |
|---|
| Yang et al. Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era. Published online Mar. 14, 2019. Biotechnology for Biofuels. vol. 12, No. 52, p. 1-13. (Year: 2019). |
| Geng et al., Application research progress of dual-luciferase reporter gene system, Science & Technology Information, 2012, No. 21, p. 1. |
| Number | Date | Country | |
|---|---|---|---|
| 20200385795 A1 | Dec 2020 | US |
