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Prokaryotic promoter reporting system based on lacZ gene and pUC replicon, and construction method and application thereof

A reporter system and construction method technology, applied in the field of prokaryotic promoter reporter system and its construction, can solve the problems of inconvenient promoter cloning, reduce the detection sensitivity of weak promoters, interfere with measurement results, etc., so as to reduce design and experimental operation costs, Improve stability and transformation efficiency, the effect of large adaptability

Active Publication Date: 2020-06-05
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, all kinds of promoter reporting systems currently on the market are mainly aimed at eukaryotic cells, and no commercialized prokaryotic promoter reporting systems have been seen. Most of the current prokaryotic promoter reporting systems based on lacZ are low-copy shuttle vectors with relatively small volumes. Large, inconvenient for the construction of recombinant plasmids during promoter cloning, and theoretically will reduce the detection sensitivity for weak promoters
In addition, there are generally few cloning sites in the promoter detection region immediately upstream of the lacZ gene in these vectors, and it is not convenient for the cloning of promoters containing the same restriction site in the sequence; finally, the host bacteria used in some reporter systems are not lacZ deletion mutations strain, potential background activity can also interfere with assay results
The existence of these problems will restrict the practical application of the promoter reporting system, which is not conducive to commercial promotion, and needs to be further improved or perfected

Method used

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  • Prokaryotic promoter reporting system based on lacZ gene and pUC replicon, and construction method and application thereof
  • Prokaryotic promoter reporting system based on lacZ gene and pUC replicon, and construction method and application thereof
  • Prokaryotic promoter reporting system based on lacZ gene and pUC replicon, and construction method and application thereof

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Experimental program
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Embodiment 1

[0040] Example 1 Construction of the prokaryotic promoter reporter system pFGH06 based on lacZ gene and pUC replicon 1. Primer design

[0041] To facilitate the design of subsequent experiments, the Cla I, Sac I, and Nde I restriction sites in the lacZ gene should be eliminated. To this end, site-directed mutagenesis primers were designed based on the principle of synonymous codons and according to the known lacZ gene, where lacZ-M1-F / lacZ-M1-R, lacZ-M2-F / lacZ-M2-R, lacZ-M3-F / lacZ-M3-R is reverse complementary, and can achieve C→T, G→A, T→C site-directed mutations on the third base of the Cla I, Sac I, and Nde I recognition sequences, respectively. lacZ-F1 and lacZ-R are amplification primers for both ends of the lacZ gene, and primers lacZ-F2 and lacZ-F3 are based on the lacZ-F1 primer and sequentially introduce the required sequence and restriction site (see Table 1). When designing the multiple cloning site sequence for replacement in the plasmid immediately adjacent to t...

Embodiment 2

[0057] Application test of embodiment 2 pFGH06

[0058] 1. Determination of background activity

[0059]First, referring to the method described by Sambrook et al. (Sambrook, J. and D.W.Russell, Molecular Cloning: A Laboratory Manual. 3rd ed. 2002, New York: Cold Spring Harbor Laboratory Press) and slightly modifying the preparation of Escherichia coli MC4100 competent cells, The specific operation is: use an inoculation loop to dip a small amount of bacterial solution from the frozen Escherichia coli MC4100 glycerin preservation solution and streak it on the LB plate, place it in a biochemical incubator at 37°C for 16-24 hours, and then pick a single colony with a sterile toothpick , inoculated in a test tube containing 5 mL of LB liquid medium, and cultured overnight at 37°C with a constant temperature shaker. The next day, take 100 μL of bacterial culture solution and culture it in 10 mL of LB liquid medium for 3-4 hours with shaking. When the OD value reaches 0.3-0.4, tak...

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Abstract

The invention discloses a prokaryotic promoter reporting system based on a lacZ gene and a pUC replicon. The reporting system comprises the pUC replicon derived from pFLX107, a Cat gene, an rrnbT1T2,a lambda t0 transcription terminator, and the lacZ gene derived from a plasmid pRCL, wherein ClaI, SacI and NdeI sites in the lacZ gene are subjected to silent mutation. The invention further discloses a construction method and application of the prokaryotic promoter reporting system. The size of a plasmid pFGH06 constructed by the invention is 4949 bp; the background activity of the plasmid pFGH06 under a culture condition of 28 DEG C is only 12.2 + / - 0.6, which is remarkably lower than the background activity (21.3 + / - 1.7) of the low-copy reference plasmid pRCL; the plasmid pFGH06 is applied to cloning and activity determination of an inducible promoter araBAD and a constitutive promoter rpsM; and when the plasmid pFGH06 is applied to promoter screening in a simulated mode, 100% recognition of a target promoter can be achieved through blue-white selection.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a prokaryotic promoter reporter system based on lacZ gene and pUC replicon, its construction method and application. Background technique [0002] A prokaryotic promoter is a specific DNA sequence located upstream of a gene that can be recognized, combined and initiated by RNA polymerase. Its main function is to control the intensity and mode of gene expression, and it is the center of gene transcription level regulation. Accurate identification and identification is the premise and basis for the study and analysis of gene function and its regulatory mechanism, and it is also of great significance for the construction of related genetic engineering vectors and the transformation of mutant strains for specific experimental purposes. [0003] With more and more bacterial and viral genome sequences being determined and released, and the development of bioinformatics, the id...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/65C12N1/21C12R1/19
CPCC12N15/65C12N15/70
Inventor 付立霞徐敬潇杨辉黄志斌龚建森韩先干
Owner YANGZHOU UNIV
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