Recombinant cell screening system and building method thereof

A technology of recombinant cells and construction methods, applied in the field of recombinant cell screening, can solve the problems of uneven addition of substrates and inability to enter cells normally

Inactive Publication Date: 2015-12-09
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention provides a recombinant cell screening system and a preparation method thereof. When the system is used, no precursor substrate is added, which avoids the problem of uneven substrate addition or the inability to enter cells normally. The blue pigment produced by the system Intuitive results can be obtained by visual observation under visible light, which is convenient to use and reduces the cost of screening

Method used

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  • Recombinant cell screening system and building method thereof
  • Recombinant cell screening system and building method thereof
  • Recombinant cell screening system and building method thereof

Examples

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Comparison scheme
Effect test

Embodiment 1

[0031] Example 1, optimization of idgS codon and total gene synthesis

[0032] The idgS gene (see SEQ ID NO: 6 for the specific sequence) is a non-ribosomal peptide synthetase coding gene cloned from Streptomyces lavendulae CGMCC No. 4.1386 in our laboratory. In order to enable the gene to be better expressed in Escherichia coli, according to the codon usage preference of Escherichia coli, the idgS gene sequence (named as idgS Ec , see SEQ ID NO: 1 for the specific sequence. At the same time in idgS- E.coli Upstream, a strong promoter (T5promoter, see SEQ ID NO: 2 for the specific sequence) and a strong ribosome binding site (RBS S , see SEQ ID NO: 3 for the specific sequence). idgS containing a strong promoter and strong ribosome binding site Ec named T5p-Rs-idgS Ec , and commissioned a DNA synthesis company (Beijing Qingke Xinye Biotechnology Co., Ltd.) to carry out the whole gene synthesis, and the full sequence is shown in SEQ ID NO: 4.

Embodiment 2

[0033] Embodiment 2, construction engineering strain E.coliidgS01

[0034] Such as figure 2 As shown, the Escherichia coli integrative plasmid pOSIP-KO was used as a vector (St-Pierre, F., Cui, L., Priest, D.G., Endy, D., Dodd, I.B., and Shearwin, K.E. (2013) One-step cloning and chromosomalintegrationofDNA. ACSsyntheticbiology2,537-541.), the DNA fragment synthesized in Example 1 (T5p-Rs-idgS- Ec ) was integrated into the genome of E.coliJM109, and the Escherichia coli engineering strain E.coliidgS01 was constructed.

[0035] The detailed construction process of E.coliidgS01 is as follows:

[0036] 2.1 Synthetic primer T5F-SpeI: 5'-CGACTAGTAAGAATCATAAAAAATTTATTTGCT-3' and primer idgsmR-bamHI: 5'-GGGGATCCTTATTCACCCAG-3'.

[0037]2.2 Using the synthesized DNA fragment (T5p-Rs-idgS-Ec) as a template, PCR amplification was performed using T5F-SpeI and idgsmR-bamHI primers. The 50 μl amplification system contained the following components: 5XPhusionHFbuffer 10 μl, 2.5mMdNTPs 4...

Embodiment 3

[0050] Embodiment 3, the construction of engineering bacterial strain E.coliidgS02

[0051] In the engineering strain E.coliidgs01, there are two genetic elements integrated in a single copy, one is the pOSIP-KO vector and the other is the idgS- Ec expression element. The FLP recombinase recognition site FRT is included on both sides of the main part of the pOSIP-KO vector, so the main part of the vector pOSIP-KO can be eliminated by inducing the expression of FLP recombinase, and there is no antibiotic resistance selectable marker and only containing idgS- Ec The bacterial strain expressing element, we name it as E.coliidgS02 (such as figure 2 shown). The specific construction process is as follows:

[0052] The FLP recombinase expression plasmid pCP20 (Datsenko, K.A., and Wanner, B.L. (2000) One-stepinactivationofchromosomalgenesinEscherichiacoliK-12usingPCRproducts.ProceedingoftheNationalAcademyofScienceoftheUnitedStatesofAmerica97, 6640-6645.) was transformed into com...

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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 [0001] The invention relates to the technical field of recombinant cell screening, in particular to a recombinant cell screening system and a construction method thereof. Background technique [0002] Reporter gene-based screening system is an important tool in molecular biology research, and plays an important role in the research of gene expression, protein interaction and drug screening. The currently applied screening systems are mainly based on the coding genes of the following proteins or enzymes: β-galactosidase (β-Lactamase), β-glucuronidase (β-glucuronidase), luciferase (luciferase) and various fluorescent proteins (fluorescent protein) and so on. [0003] However, these screening systems have certain deficiencies. They either need to add additional precursor substrates, or require special hardware detection equipment. For example, β-galactosidase, β-glucosidase, and luciferase need to provide X-gal, X-gluc, and luciferin respectively as substrates...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N1/15C12N5/10C12N15/63C12N15/54C12N15/61
Inventor 陈义华谢周杰
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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