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Recombinant strain as well as preparation method and application thereof

A technology of recombinant strains and recombinases, applied in the field of genetic engineering, can solve the problems of insufficient substrate concentration, bacterial strain death, unstable yield, etc., and achieve the effects of increasing yield, improving stability and good biological activity.

Active Publication Date: 2021-11-05
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Synthesizing indigo with Escherichia coli, yeast, Bacillus subtilis, etc. is an important research direction in this field, but it still needs to rely on the plasmid system, and the yield is unstable
Not only that, in order to increase the yield of indigo fermentation and improve the fermentation conditions, fermenters are mostly used for batch fermentation at present, but batch fermentation has nutrients and insufficient concentration of substrates, which leads to the death of bacteria, which eventually leads to unstable and unsustainable indigo production.

Method used

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  • Recombinant strain as well as preparation method and application thereof
  • Recombinant strain as well as preparation method and application thereof
  • Recombinant strain as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] The construction of embodiment 1 recombinant vector

[0108] 1.1 Primer design

[0109] Some primers of this application are shown in Table 2.

[0110] Table 2 Primer list

[0111]

[0112]

[0113] 1.2 PCR amplification of homology arm, indigo synthesis-related genes styA and styB

[0114]Using E.coli DH5αtrpR as the integration site to amplify the left and right arms, the size of the left arm is 358bp (sequence shown in SEQ ID NO.3), and the size of the right arm is 357bp (sequence shown in SEQ ID NO.4); Use E.coli DH5αtrpB as the integration site to amplify the left and right arms, the size of the left arm is 353bp (sequence shown in SEQ ID NO.5), and the size of the right arm is 351bp (sequence shown in SEQ ID NO.6) ; Using E.coli DH5αhisD as the integration site to amplify the left arm and the right arm, the size of the left arm is 346bp (sequence shown in SEQ ID NO.7), and the size of the right arm is 369bp (sequence shown in SEQ ID NO.8 ).

[0115] The...

Embodiment 3

[0142] The construction of embodiment 3 recombinant strains

[0143] 3.1 Induced expression of Red gene

[0144] Transform the pKD46 plasmid into E.coli DH5α competent medium, resume culture at 30°C for 1 h, and apply ampicillin (Amp + ) plates were incubated overnight at 30°C. Single clones were picked and cultured overnight in LB test tubes containing Amp.

[0145] Inoculate into LB medium with Amp at a ratio of 1:100, add L-arabinose to a final concentration of 1 mM, culture at 30°C, OD 600nm If it is not more than 0.6, prepare electroporation competent.

[0146] 3.2 Electroporation targeting fragments

[0147] Mix the targeting DNA fragments obtained in 2.2 with the electroporation competent cells (100 μL competent cells + about 200 ng targeting DNA fragments, transfer to a pre-cooled electroporation cup, and take three electroporation competent plates prepared in step 3.1 Electric shock transformation of monoclonal. After electric shock, add LB medium, place at 30°C ...

Embodiment 4

[0159] Example 4 Target gene expression analysis

[0160] 4.1 Total RNA Extraction of Engineering Bacteria

[0161] The total RNA in the bacteria was extracted using the spin-column type ultra-pure total RNA rapid extraction kit from Beijing Biotech Co., Ltd. The specific operation steps can be found in the kit instruction manual.

[0162] 4.2 Reverse transcription of total RNA into cDNA

[0163] The ReverTra Ace qPCR RT Master Mix reverse transcription kit from TOYOBO (Shanghai) Co., Ltd. was selected for cDNA synthesis, and the specific operation method is shown in the instruction manual.

[0164] 4.3 RT-PCR analysis

[0165] According to the sequence information of styA and styB genes, primers were designed and specificity was evaluated by Beacon Design software. The primer sequences are shown in Table 7.

[0166] Table 7 RT-PCR primers

[0167] Gene Primer name number in the sequence listing sequence (5'-3') styA F-A SEQ ID NO.20 GGCGAGCTGAT...

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Abstract

The invention discloses a recombinant strain as well as a preparation method and application thereof. The recombinant strain is formed by integrating a fragment I on a genome of a host; and the fragment I contains a styrene monooxygenase gene styA and a styrene monooxygenase gene styB. The styrene monooxygenase gene styA and the styrene monooxygenase gene styB are integrated on the genome of the recombinant strain, so that the recombinant strain has good stability and relatively high indigo pigment yield.

Description

technical field [0001] The application relates to a recombinant bacterial strain and its preparation method and application, belonging to the technical field of genetic engineering. Background technique [0002] At present, indigo production includes natural extraction, chemical synthesis, and microbial synthesis. Using E. coli, yeast, and Bacillus subtilis to synthesize indigo is an important research direction in this field. However, it still needs to rely on the plasmid system and the yield is unstable. Not only that, in order to increase the yield of indigo fermentation and improve the fermentation conditions, fermenters are mostly used for batch fermentation at present, but there are nutrients and substrate concentration in batch fermentation, which leads to the death of bacteria, which eventually leads to unstable and unsustainable indigo production. Contents of the invention [0003] According to one aspect of the present application, a recombinant strain is provid...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/70C12N15/90C12P17/16C12R1/19
CPCC12N9/0071C12N15/70C12N15/90C12P17/165C12Y114/14011
Inventor 程雷王成涛袁栋栋潘紫荆王玥
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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