CN105132349A - Recombinant cell screening system and building method thereof - Google Patents
Recombinant cell screening system and building method thereof Download PDFInfo
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Abstract
The invention relates to a recombinant cell screening system and a building method thereof. The recombinant cell screening system comprises the receptor cells of constitutive expression non-ribosomal peptide synthetase encoding genes and plasmids containing phosphopantetheine transferase encoding genes. The recombinant cell screening system has the advantages that exogenous substrate needs not to be added, special detecting equipment is not needed, and a screening process can be completed fast, simply, economically and efficiently. The application example, namely clone screening of gene segments, of the system is specifically elaborated in the embodiment of the invention, and the application example proves that the system can be effectively used for screening recombinant cells.
Description
Technical field
The present invention relates to reconstitution cell triage techniques field, specifically, relate to a kind of reconstitution cell screening system and construction process thereof.
Background technology
Screening system based on reporter gene is the important tool in molecular biology research, does mutually and play an important role in the research such as drug screening at genetic expression, protein.The screening system applied at present is mainly based on the encoding gene of following albumen or enzyme: beta-galactosidase enzymes (β-Lactamase), beta-glucosidase (β-glucuronidase), luciferase (luciferase) and various fluorescin (fluorescentprotein) etc.
But there is certain weak point in these screening systems, they or need additionally to add precursor substrate, or need special hardware detection.Such as beta-galactosidase enzymes, beta-glucosidase, luciferase needs to provide X-gal respectively, the substrate that X-gluc and luciferin produces as signal, often because substrate interpolation is uneven or substrate can not normally enter cell interior thus greatly have impact on screening effect in actual mechanical process, the price of these precursor substrate is general higher in addition.Screening system based on luciferase needs photon detection apparatus, and the screening system based on fluorescin needs the excitation light source of specific wavelength to produce equipment.In a word, the demand of precursor substrate and hardware device not only adds the complicacy of screening system and the false positive of screening, and adds the Financial cost of screening.
Summary of the invention
The invention provides a kind of reconstitution cell screening system and preparation method thereof, without the need to adding precursor substrate when using this system, avoid substrate add uneven or can not normally enter cell interior produce problem, the blue pigment that this system produces detects by an unaided eye under visible light and just can obtain result intuitively, easy to use and reduce screening cost.
One aspect of the present invention, provides a kind of reconstitution cell screening system, comprising: can the recipient cell of constitutive expression NRPS114 encoding gene and the plasmid containing phosphopantetheine transferring enzyme encoding gene.
In above-described reconstitution cell screening system, after the 5th codon of described phosphopantetheine transferring enzyme encoding gene, insert multiple clone site.
In above-described reconstitution cell screening system, the sequence of described NRPS114 encoding gene is as shown in SEQIDNO:1.
In above-described reconstitution cell screening system, the promoter sequence of described NRPS114 encoding gene is as shown in SEQIDNO:2.
In above-described reconstitution cell screening system, the ribosome bind site sequence of described NRPS114 encoding gene is as shown in SEQIDNO:3.
In above-described reconstitution cell screening system, described recipient cell is prokaryotic cell prokaryocyte, fungal cell or mammalian cell; Particularly, described recipient cell is intestinal bacteria; More specifically, described intestinal bacteria are E.coliJM109.
Another aspect of the present invention, provides the construction process of the reconstitution cell screening system described in more than one, comprises the steps:
(1) DNA sequence dna containing NRPS114 encoding gene and promotor and its ribosome bind site is incorporated in recipient cell genome;
(2) plasmid containing phosphopantetheine transferring enzyme encoding gene is built.
Above-described construction process, the sequence of described NRPS114 encoding gene is as shown in SEQIDNO:1, the promoter sequence of described NRPS114 encoding gene is as shown in SEQIDNO:2, and the ribosome bind site sequence of described NRPS114 encoding gene is as shown in SEQIDNO:3.
Above-described construction process, described recipient cell is prokaryotic cell prokaryocyte, fungal cell or mammalian cell; Particularly, described recipient cell is intestinal bacteria; More specifically, described intestinal bacteria are E.coliJM109.
Above-described construction process, wherein phosphopantetheine transferring enzyme encoding gene described in step (2) substitutes the lacZ α gene in plasmid pUC19, and inserts multiple clone site by after the 5th codon of described phosphopantetheine transferring enzyme encoding gene; Particularly, described multiple clone site is from pUC19; More specifically, described phosphopantetheine transferring enzyme encoding gene derives from subtilis.
Above-described construction process, the described DNA sequence dna containing NRPS114 encoding gene and promotor thereof and its ribosome bind site by intestinal bacteria integrative plasmid pOSIP-KO be vector integration on the genome of E.coliJM109, then remove the integrated element in pOSIP-KO source and resistance gene element only retains NRPS114 Expression element.
The structure of present system is the phosphopantetheine transferring enzyme encoding gene sfp in NRPS114 encoding gene idgS based on Streptomyces and genus bacillus source.Active IdgS (Holo-idgS) can generate blue compound-indigo element (indigoidine) by catalysis L-glutaminate (L-Gln), and the direct expression product (Apo-IdgS) of idgS does not have activity, just need can become activity form through posttranslational modification process (this process is by the catalysis of phosphopantetheine transferring enzyme), Sfp albumen can complete the activation of IdgS.L-glutaminate (L-Gln) is present in all cells, add without the need to external source, be in cell as long as therefore the IdgS of activity is pure, thus this cell with endogenous L-glutaminate for substrate synthesizing blue compound-indigo element (indigoidine), will present macroscopic blueness.
The present invention inserts multiple clone site district with frame after the 5th codon of sfp gene in addition, does not affect the function of sfp, and provides convenience with the use that the sfp in multiple clone site district is this system.
Compared with traditional screening system, system of the present invention without the need to adding xenobiotic substrates, also without the need to special test set, can quick, easy, economical, complete screening process efficiently.The present invention is also specifically described the colony screening of this systematic difference example-gene fragment in an embodiment, demonstrates the screening that system of the present invention can be effective to reconstitution cell.
Accompanying drawing explanation
Fig. 1 is that reconstitution cell screening system of the present invention builds principle schematic.
The engineering strain idgS01 that Fig. 2 provides for the embodiment of the present invention and idgS02 building process schematic diagram.
The result electrophorogram of the E.coliidgs01 that Fig. 3 provides for the embodiment of the present invention; Wherein, the transformant of swimming lane (1,2,3) and corresponding three random chooses of swimming lane (4,5,6) difference.
Fig. 4 plasmid pSFPHE and multiple clone site schematic diagram.
The bacterial strain E.coliidgS02 that Fig. 5 contains plasmid pSFPHE presents blue phenotype.
Fig. 6 is GFP gene clone screening blue hickie phenotype under visible light in the embodiment of the present invention.
Fig. 7 is the phenotype of GFP gene clone screening under ultraviolet lamp in the embodiment of the present invention, and the successful insertion of result display white bacterium colony and GFP gene exists good corresponding relation.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in more details, the advantage of the solution of the present invention and its all respects can be understood better.But embodiment described below and embodiment are only the objects illustrated, instead of limitation of the present invention.The present invention is only for intestinal bacteria, but this system also has great application potential in fungal cell and mammalian cell.
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Optimization and the full genome of embodiment 1, idgS codon synthesize
IdgS gene (concrete sequence is shown in SEQIDNO:6) is the NRPS114 encoding gene that this laboratory is cloned into from StreptomyceslavendulaeCGMCCNo.4.1386.In order to enable this gene better be expressed in intestinal bacteria, according to colibacillary codon usage bias, redesign idgS gene order (called after idgS
ec, concrete sequence is shown in SEQIDNO:1).Simultaneously at idgS-
e.coliupstream, devises a strong promoter (T5promoter, concrete sequence is shown in SEQIDNO:2) and a strong ribosome bind site (RBS in the lump
s, concrete sequence is shown in SEQIDNO:3).Include the idgS of strong promoter and strong ribosome bind site
eccalled after T5p-Rs-idgS
ec, and trust carries out full genome synthesis by DNA Synesis Company (Beijing Qing Kexin industry Bioisystech Co., Ltd), complete sequence is shown in SEQIDNO:4.
Embodiment 2, structure engineering strain E.coliidgS01
As shown in Figure 2, utilize intestinal bacteria integrative plasmid pOSIP-KO as carrier (St-Pierre, F., Cui, L., Priest, D.G., Endy, D., Dodd, I.B., andShearwin, K.E. (2013) One-stepcloningandchromosomalintegrationofDNA.ACSsynthet icbiology2,537-541.), by the DNA fragmentation (T5p-Rs-idgS-of synthesis in embodiment 1
ec) be incorporated on the genome of E.coliJM109, construct colibacillus engineering strain E.coliidgS01.
The detailed building process of E.coliidgS01 is as follows:
2.1 synthetic primer T5F-SpeI:5 '-CGACTAGTAAGAATCATAAAAAATTTATTTGCT-3 ' and primer idgsmR-bamHI:5 '-GGGGATCCTTATTCACCCAG-3 '.
2.2 with the DNA fragmentation (T5p-Rs-idgS-Ec) of synthesis for template, adopt T5F-SpeI, idgsmR-bamHI primer carries out pcr amplification, containing, for example lower component in 50 μ l amplification systems: 5XPhusionHFbuffer10 μ l, 2.5mMdNTPs4 μ l, primer 1 (10 μMs of T5F-SpeI) 2 μ l, primer 2 (10 μMs of idgsmR-bamHI) 2 μ l, template (100ng/ μ lT5p-Rs-idgS-Ec) 1 μ l, PhusionDNApolymerase (2U/ul) 0.5 μ l, deionized water 30.5 μ l.Pcr amplification condition is as follows: 98 DEG C 30 seconds; 98 DEG C 10 seconds, 57 DEG C 10 seconds, 72 DEG C 1 point 20 seconds, totally 30 circulations; 72 DEG C 5 minutes.
After 2.3PCR amplified production carries out purifying, employing restriction enzyme SpeI (purchased from TakaraBioCompany), BamHI (purchased from TakaraBioCompany) carry out double digestion, the 50 μ l enzyme systems of cutting comprise following component: 10XKbuffer5 μ l, pcr amplification product (500ng/ μ l) 5 μ l, SpeI (10U/ μ l) 1 μ l, BamHI (10U/ μ l) 1 μ l, deionized water: 38 μ l.Enzyme tangent condition is: 37 DEG C, 3 hours.
2.4 similarly, and plasmid vector pOSIP-KO adopts SpeI, BamHI to carry out double digestion process, and enzyme tangent condition is the same.
2.5 carry out ligation by after above-mentioned digestion products purifying.20 μ l ligation systems are as follows: 10XT4DNA ligase enzyme buffer2 μ l, T5p-Rs-idgS-Ec digestion products (500ng/ μ l) 4 μ l, pOSIP-KO digestion products (500ng/ μ l) 4 μ l, T4DNA ligase enzyme 2 μ l, deionized water: 8 μ l.Ligation condition is 16 DEG C, 2 hours.
2.6 will connect product conversion E.coliJM109 (purchased from TakaraBioCompany) competent cell, converted product is the enterprising row filter (SambrookJ.etal.Molecularcloning, Laboratorymanuals.2001) of LB solid medium containing kantlex (50 μ g/ml).The transformant obtained is named as E.coliidgs01.
The checking of 2.7E.coliidgs01.Plasmid vector pOSIP-KO has a major integration site (attB1) and a secondary integration site (attB2) in intestinal bacteria, in order to determine the integration site of carrier pOSIP-KO in engineering strain E.coliidgs01, synthesize following primer:
P1：5’-CTCATTCGAAACCACCCACCG-3’，
P2：5’-ACTTAACGGCTGACATGG-3’，
P3：5’-ACGAGTATCGAGATGGCA-3’，
P4：5’-GATCATCATGTTTATTGCGTGG-3’，
SP1：5’-TCCGGAATGCCTGCATTG-3’，
SP4：5’-CCCTGGAGCCAAAATATCC-3’。
The wherein sequence of both sides, attB1 site on the corresponding genome of P1, P4, SP1 and SP4 corresponding both sides, attB2 site sequence.Sequence (Fig. 2) on the corresponding carrier pOSIP-KO of P2 and P3.Using random choose three engineering strains (E.coliidgs01) as template, two groups of mix primer (P1, P2, P3, P4) and (SP1, P2, P3, SP4) is adopted to carry out pcr amplification respectively.PCR amplification system (50 μ l) is as follows: 10XTaqbuffer5 μ l, 2.5mMdNTPs4 μ l, primer (10 μMs) (1,2,3,4) often kind of primer 2 μ l, template (E.coliidgs01) 1 μ l, Taq DNA polymerase (2U/ul) 1 μ l, deionized water 31.0 μ l.PCR reaction conditions is: 96 DEG C 30 seconds; 96 DEG C 10 seconds, 50 DEG C 15 seconds, 72 DEG C 30 seconds, totally 30 circulations; 72 DEG C 5 minutes.PCR primer amplified production is through agarose gel electrophoresis.Result as shown in Figure 3, combination of primers (P1, P2, P3, P4) band (Fig. 3, swimming lane 1,2,3) of two prediction sizes is amplified, combination of primers (SP1, P2, P3, SP4) amplify the band (Fig. 3, swimming lane 4,5,6) that is predicted size.Show that, in engineering strain E.coliidgs01, carrier pOSIP-KO is integrated in attB1.
The structure of embodiment 3, engineering strain E.coliidgS02
In engineering strain E.coliidgs01, single copy is integrated with two genetic elements, and one is pOSIP-KO carrier, and another is idgS-
ecexpression element.Comprise FLP recombinase recognition site FRT in the main part both sides of pOSIP-KO carrier, therefore by abduction delivering FLP recombinase, the main part of carrier pOSIP-KO can be eliminated, obtain and there is no antibiotic resistance selected marker and only contain idgS-
ecthe bacterial strain of Expression element, we are called after E.coliidgS02 (as shown in Figure 2).Concrete building process is as follows:
By FLP recombinase expression plasmid pCP20 (Datsenko, K.A., andWanner, B.L. (2000) One-stepinactivationofchromosomalgenesinEscherichiacoliK-12usingPCRproducts.ProceedingsoftheNationalAcademyofScie ncesoftheUnitedStatesofAmerica97, 6640-6645.) transform enter in E.coliidgs01 competent cell, transformant is cultivated at 30 DEG C, and at the enterprising row filter of LB substratum containing 100 μ g/ml, the transformant called after obtained: E.coliidgs01/pCP20.E.coliidgs01/pCP20 is in the LB substratum not having resistance in inoculation, cultivates 6 hours for 37 DEG C, and gradient dilution also coats the LB flat board not having resistance.37 DEG C of overnight incubation, the mono-clonal of acquisition is through resistance assay, and the bacterial strain that kalamycin resistance and amicillin resistance disappear simultaneously is named as E.coliidgs02.Further by sequence verification, bacterial strain E.coliidgs02 is only containing idgS-
ecexpression element, integrated element and the resistance gene element in pOSIP-KO carrier source are all succeeded elimination.
The structure of embodiment 4, plasmid pSFPHE
The sfp gene (concrete sequence is shown in SEQIDNO:5) of being originated by subtilis substitutes the lacZ α gene in plasmid pUC19, obtains plasmid pSFP.Further, the multiple clone site district (MCS) coming from pUC19 is inserted the 5th codon of sfp gene in pSFP with frame after, plasmid pSFP-HE is obtained.Specifically, building process is as follows:
The structure of 4.1pSFP, adopts one-step cloning test kit (ClonExpressIIOneStepCloningKitC112, VazymeBiotechCo., Ltd.) to complete.First with plasmid pCIMt002 (Li, P., etal. (2015) Anefficientblue-whitescreeningbasedgeneinactivationsyste mforStreptomyces.Appliedmicrobiologyandbiotechnology99, 1923-1933. purchased from Nanjing Vazyme Biotechnology Co., Ltd.) be template, employing primer sfpF-puc18r (5 '-CCGGCTCGTATGTTGTGTGGACTCTGTCAGATCTCACTCTGC-3 ') and sfpR-puc18f (5 '-GTGTCGGGGCTGGCTTAACTATAAAAGCTCTTCGTACGAGACCA-3 ') carry out pcr amplification, obtain the DNA fragmentation comprising sfp gene.PCR system consists of: 5XPhusionHFbuffer10 μ l, 2.5mMdNTPs4 μ l, primer sfpF-puc18r (10 μMs) 2 μ l, primer sfpR-puc18f (10 μMs) 2 μ l, template (100ng/ μ lpCIMt002) 1 μ l, PhusionDNA polysaccharase (2U/ul) 0.5 μ l, deionized water 30.5 μ l.PCR response procedures be 98 DEG C 30 seconds; 98 DEG C 10 seconds, 63 DEG C 10 seconds, 72 DEG C 20 seconds, totally 30 circulations; 72 DEG C 5 minutes.Then, with pUC19 (purchased from ThermoFisherScientificInc.) for template, employing primer puc18F-sfp (5 '-TGGTCTCGTACGAAGAGCTTTTATAGTTAAGCCAGCCCCGACAC-3 ') and primer puc18R-sfp (5 '-GCAGAGTGAGATCTGACAGAGTCCACACAACATACGAGCCGG-3 ') carry out pcr amplification, obtain the DNA fragmentation that size is about 2.7kb.PCR system consists of: 5XPhusionHFbuffer10 μ l, 2.5mMdNTPs4 μ l, primer puc18F-sfp (10 μMs) 2 μ l, primer puc18R-sfp (10 μMs) 2 μ l, template (100ng/ μ lpCIMt002) 1 μ l, PhusionDNA polysaccharase (2U/ul): 0.5 μ l, deionized water 30.5 μ l.PCR response procedures is: 98 DEG C 30 seconds; 98 DEG C 10 seconds, 67 DEG C 10 seconds, 72 DEG C 1 minute, totally 30 circulations; 72 DEG C 5 minutes.Above-mentioned two amplified production ends include homologous sequence, therefore adopt one-step cloning test kit (ClonExpressIIOneStepCloningKitC112, purchased from VazymeBiotechCo., Ltd.) to obtain plasmid pSFP.
The structure of 4.2pSFPHE
Take pSFP as template, pucsfpF-EcorI (5 '-CTCGAATTCCTGTAAATCTTCATGTGCGTCCTC-3 ') and pucsfpR-HindIII (5 '-TGCAAGCTTGATTTATATGGACCGCCCGCTTTC-3 ') be primer, adopt PhusionDNA polysaccharase to carry out pcr amplification.Amplification condition is: 98 DEG C 30 seconds; 98 DEG C 10 seconds, 69 DEG C 10 seconds, 72 DEG C 1 point 05 second, totally 30 circulations; 72 DEG C 5 minutes.Obtain the PCR primer PSFP of about 3.1kb size.Synthesize two DNA single chain MCSF (5 '-AGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCG-3 ') and MCSR (5 '-AATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA-3 ') after renaturation, obtain the DNA fragmentation MCS comprising multiple clone site district.MCS end is respectively containing the sticky end with EcoRI and HindIII complementary pairing.Therefore PSFP fragment can be connected with MCS after EcoRI with HindIII double digestion, connects product conversion E.coliJM109 competent cell and obtains plasmid pSFPHE (as Fig. 4).
Embodiment 5, pSFPHE and E.coliidgS02 form effective blue hickie screening system
By plasmid pSFPHE Transformed E .coliidgS02 competent cell, and be applied on the LB substratum containing penbritin (100 μ g/ml), cultivate 18 hours for 30 DEG C, transformant presents blue phenotype.This result shows to insert multiple clone site district with frame after sfp the 5th codon, still can give expression to functional sfp albumen.Plasmid pSFPHE and bacterial strain E.coliidgS02 constitutes effective blue hickie screening system (Fig. 5).
This system is used for the example of DNA fragmentation clone:
With sfGFP gene (P é delacqJD
1, etal. (2006) Engineeringandcharacterizationofasuperfoldergreenfluores centprotein.NatBiotechnol.Jan; 24 (1): 79-88.) be template, GFPF-EcoRI (5 '-CAGGAATTCAAGAGGAGAAATACTAGATGCG-3 ') and GFPR-HindIII (5 '-ATCAAGCTTTCATCATTTGTACAGTTCATCC-3 ') be primer, adopt PhusionDNA polysaccharase to carry out pcr amplification.Amplification condition is: 98 DEG C 30 seconds; 98 DEG C 10 seconds, 61 DEG C 10 seconds, 72 DEG C 20 seconds, totally 30 circulations; 72 DEG C 5 minutes.Pcr amplification product, after EcoRI and HindIII double digestion, under the effect of T4 ligase enzyme, is connected with the pSFPHE cutting process through same enzyme.Connect product conversion E.coliidgS02 competent cell, transformant is at the enterprising row filter of LB substratum containing penbritin (100 μ g/ml), cultivate 18 hours through 30 DEG C, result as shown in Figure 6, this system presents the white phenotype of good good indigo plant, nearly all hickie all presents green fluorescence (Fig. 7) under burst of ultraviolel, shows the correct insertion of GFP gene, is greater than 96% through statistics positive rate.
Last it is noted that obviously, above-described embodiment is only for example of the present invention is clearly described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of amplifying out or variation be still among protection scope of the present invention.
Claims (11)
1. a reconstitution cell screening system, is characterized in that, comprising: can the recipient cell of constitutive expression NRPS114 encoding gene and the plasmid containing phosphopantetheine transferring enzyme encoding gene.
2. reconstitution cell screening system as claimed in claim 1, is characterized in that, insert multiple clone site after the 5th codon of described phosphopantetheine transferring enzyme encoding gene.
3. reconstitution cell screening system as claimed in claim 1, it is characterized in that, the sequence of described NRPS114 encoding gene is as shown in SEQIDNO:1.
4. reconstitution cell screening system as claimed in claim 1, it is characterized in that, the promoter sequence of described NRPS114 encoding gene is as shown in SEQIDNO:2.
5. reconstitution cell screening system as claimed in claim 1, it is characterized in that, the ribosome bind site sequence of described NRPS114 encoding gene is as shown in SEQIDNO:3.
6. reconstitution cell screening system as claimed in claim 1, it is characterized in that, described recipient cell is prokaryotic cell prokaryocyte, fungal cell or mammalian cell; Particularly, described recipient cell is intestinal bacteria; More specifically, described intestinal bacteria are E.coliJM109.
7. the construction process of reconstitution cell screening system as described in claim 1, is characterized in that, comprise the steps:
(1) DNA sequence dna containing NRPS114 encoding gene and promotor and its ribosome bind site is incorporated in recipient cell genome;
(2) plasmid containing phosphopantetheine transferring enzyme encoding gene is built.
8. construction process as claimed in claim 7, it is characterized in that, the sequence of described NRPS114 encoding gene is as shown in SEQIDNO:1, the promoter sequence of described NRPS114 encoding gene is as shown in SEQIDNO:2, and the ribosome bind site sequence of described NRPS114 encoding gene is as shown in SEQIDNO:3.
9. construction process as claimed in claim 8, it is characterized in that, described recipient cell is prokaryotic cell prokaryocyte, fungal cell or mammalian cell; Particularly, described recipient cell is intestinal bacteria; More specifically, described intestinal bacteria are E.coliJM109.
10. construction process as claimed in claim 9, it is characterized in that, phosphopantetheine transferring enzyme encoding gene described in step (2) substitutes the lacZ α gene in plasmid pUC19, and inserts multiple clone site by after the 5th codon of described phosphopantetheine transferring enzyme encoding gene; Particularly, described multiple clone site is from pUC19; More specifically, described phosphopantetheine transferring enzyme encoding gene derives from subtilis.
11. construction processs as claimed in claim 10, it is characterized in that, the described DNA sequence dna containing NRPS114 encoding gene and promotor thereof and its ribosome bind site by intestinal bacteria integrative plasmid pOSIP-KO be vector integration on the genome of E.coliJM109, then remove the integrated element in pOSIP-KO source and resistance gene element only retains NRPS114 Expression element.
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| CN112111506A (en) * | 2020-09-23 | 2020-12-22 | 江南大学 | Method for improving expression quantity of gamma-glutamine transpeptidase by RBS optimization |
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| EP1564294A1 (en) * | 1997-05-07 | 2005-08-17 | Genomics One Corporation | Improved cloning vector containing marker inactivation system |
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| EP1564294A1 (en) * | 1997-05-07 | 2005-08-17 | Genomics One Corporation | Improved cloning vector containing marker inactivation system |
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| HITOSHI TAKAHASHI,ET AL: "Cloning and Characterization of a Streptomyces Single Module Type Non-ribosomal Peptide Synthetase Catalyzing a Blue Pigment Synthesis", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
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| CN112111506A (en) * | 2020-09-23 | 2020-12-22 | 江南大学 | Method for improving expression quantity of gamma-glutamine transpeptidase by RBS optimization |
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Application publication date: 20151209 |