Escherichia coli resistant to sodium acetate, sodium chloride and isobutanol and its construction method
A technology of Escherichia coli and construction methods, applied in the directions of biochemical equipment and methods, microorganism-based methods, botanical equipment and methods, etc., can solve problems such as unreported, achieve cost savings, avoid complexity, and avoid blindness Effect
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Embodiment 1
[0029] Example 1 Construction of Escherichia coli Global Regulator Mutation Library
[0030] The construction of the global regulatory factor mutation library is divided into two parts: the design and synthesis of the global regulatory factor and the diversity library flora containing the global regulatory factor mutation.
[0031] Design and synthesis of global regulators
[0032] Based on CRISPR Traceable Genome Engineering System (CREATE), including 3 sets of plasmids:
[0033] Arabinose-induced Cas9 protein expression X2-cas9 plasmid,
[0034] Temperature-sensitive pSIM5 plasmid expressing λ-red recombination system induced by heat shock
[0035] and gRNA and donor DNA expression global regulator library plasmids (Garst et al., 2016).
[0036] Among them, the global regulatory factor library plasmid is a mixed plasmid, which is divided into 5 groups. The 5 groups of library plasmids are composed of saturation mutations at the corresponding sites of the corresponding glo...
Embodiment 2
[0043] Enrichment screening of different mutant library recombinant bacteria under the condition of sodium acetate stress in embodiment 2
[0044] Based on the Escherichia coli global regulatory factor mutant library flora constructed in Example 1, under the sodium acetate stress condition of 10g / L-30g / L, by gradually increasing the screening pressure, the forward library mutant flora was enriched. set. In this screening process, the LB medium was used for the activation medium, and the M9 basic salt medium was used for the screening medium.
[0045] Firstly, 1ml of the thawed bacterial solution of the 6 strains (G1, G2, G3, G4, G5, ntg) stored at -80°C were all placed in a 5ml LB test tube (15ml test tube) and cultured overnight at 37°C and 220rpm. Then all the 6ml bacterial solution was transferred to 50ml LB medium (250ml Erlenmeyer flask) and cultivated to OD 600 When it is 5-6, the initial cell concentration OD 600 =0.1 means that the initial OD was transferred to the ...
Embodiment 3
[0053] Embodiment 3. Construction of mutant strain CRP-G142I
[0054] The forward mutation of CRP potential tolerance obtained in Example 2 was used to carry out scarless editing of the genome of wild-type E. coli MG1655 by using I-Scel-based DNA double-strand break gene recombination technology, starting from wild-type E. coli MG1655, Introduce the target mutation into the genome to obtain the reconstituted strain. Then, the obtained mutated reconstituted strains were cultured under the tolerance condition of sodium acetate 30g / L to further verify the effect of the mutation of the Escherichia coli global regulatory factor obtained by screening. The specific operation is as follows:
[0055] Construction of mutant reconstituted strains:
[0056] (1). Preparation of recombinant fragments for crp gene editing
[0057] The preparation of the recombinant fragment is divided into 3 steps
[0058] In the first step, using wild-type Escherichia coli MG1655 genomic DNA as a templa...
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