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RRNA mosaic promoter and expression vector containing same

A technology of chimeric promoters and expression vectors, applied in the field of molecular biology, can solve problems such as difficult regulation and achieve high-efficiency expression

Inactive Publication Date: 2011-03-30
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the rRNA promoter will continue to be active during the pre-induction phase, so it is difficult to regulate it

Method used

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  • RRNA mosaic promoter and expression vector containing same
  • RRNA mosaic promoter and expression vector containing same
  • RRNA mosaic promoter and expression vector containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Promoter Fragment P 16S-2 Amplification and construction of intermediate plasmid p16S-2

[0044] according to E. coli rRNA rrnB P1 promoter sequence and lac operon lacO Sequence design PCR primers, specifically:

[0045] Upstream primer S1: SEQ ID NO. 3 (containing Sph I restriction site), downstream primer S2: SEQ ID NO. 4. P 16S-2 Promoter fragment. The PCR conditions are: 10 μL 5×PrimeSTAR Buffer (Mg 2+ plus), 50 μM dNTP Mixture, 0.5 μM S1, 0.5 μM S2, 1.25 U PrimeSTAR HS DNA Polymerase (TAKARA), and adjust the reaction volume to 50 μL with sterile water. The PCR amplification reaction program was: 98 °C, 10 s, 55 °C, 15 s, 72 °C, 30 s, 29 cycles; 72 °C, 10 min.

[0046] PCR products were purified by 2% agarose gel electrophoresis, and Taq The enzyme can add deoxyadenosine to the end of the amplified fragment, and PCR is performed again to fill in the sticky end of each fragment and add a dA. The PCR conditions are: 2.5 μL 10× Taq Buf...

Embodiment 2

[0051] Embodiment 2: Construction of expression vector pRNP2

[0052] The p16S-2 and pET29a plasmids were double-digested with NdeI and SphI to obtain the promoter fragment P16S-2 and the double-digested fragment of pET29a with the T7 promoter removed. After purification and recovery, they were mixed with enzymes at a ratio of 2:1. The enzyme-linked product was transformed into E.coli DH10B competent cells, and LB plates containing 50 μg·mL-1 kanamycin were spread. The transformant was identified by PCR and sequencing to obtain the expression vector pRNP2.

Embodiment 3

[0053] Example 3: Construction of recombinant plasmid pRNP2-cel5G and expression strain containing β-1,4 endoglucanase gene (cel5G)

[0054] pRNP2 and the recombinant plasmid pET29a- cel5 G (Cui Zhongli et al., Chinese patent application number: 201010018298.0) used respectively xho I and Nde I perform double digestion. The enzyme digestion system is:

[0055] Nde I enzyme (10 U·μL -1 )

1.0 μL

xho I enzyme (10 U·μL -1 )

1.0 μL

10× universal buffer

5.0 μL

pET29a- cel5 G

20.0 μL

double distilled water

24.0 μL

total capacity

50.0 μL

[0056] After reacting in a water bath at 37 °C for more than 3 h, the digested products were recovered by 0.75% agarose gel electrophoresis, and the method was the same as above.

[0057] The digested products were separated and purified by agarose gel electrophoresis, and then recovered with a DNA gel recovery kit. Then the double digested cel5 T...

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Abstract

The invention belongs to the field of molecular biology, and discloses an rRNA mosaic promoter and an expression vector containing the same. The mosaic promoter forms an annular DNA structure by inserting two lacO1 promoters of a lac operon into the upstream and downstream of an escherichia coli rRNArrnB operon promoter P1 respectively or inserting araI1 and araO2 promoters of an arabinose operon into the upstream and downstream of an escherichia coli rRNA operon promoter P1 respectively to realize control of the promoter P1. The induced expression vector is obtained by substituting the rRNArrnBP1 mosaic promoter for a promoter T7 of a vector pET29a. The mosaic promoter and the expression vectors pRNP2 and pRBB can conveniently realize efficient expression of exogenous genes in multiple escherichia coli strains, wherein the pRBB also can be used for directed evolution of genes.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to an rRNA chimeric promoter and an expression vector containing the chimeric promoter, in particular to an rRNA containing a DNA ring regulatory element rrnB P1 chimeric promoter, and an expression vector containing the chimeric promoter. Background technique [0002] In E. coli, the number of ribosomes is controlled at the transcriptional level by seven copies of the rRNA operon on the chromosome. When the generation time of E. coli was 20 minutes, the number of ribosomes in each cell was about 70,000, and when the growth rate was slower, there were about 20,000 ribosomes in each cell. Such a large number of ribosomes in the cell illustrates the superpower of the rRNA operator promoter. [0003] E. coli The promoter P1 in the rRNA operator is composed of a core promoter including the -10 region and the -35 region, an AT base-rich UP element located upstream of the -35 region, ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/70C12N15/56C12N9/42
Inventor 崔中利曹慧赵晓丽刘娟
Owner NANJING AGRICULTURAL UNIVERSITY
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