CN110616181A

CN110616181A - Engineering strain based on escherichia coli transformation

Info

Publication number: CN110616181A
Application number: CN201910929579.2A
Authority: CN
Inventors: 霍毅欣; 王靖格; 黄潮勇
Original assignee: Beijing University of Technology
Current assignee: Beijing University of Technology; Beijing Institute of Technology BIT
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2019-12-27

Abstract

MG1655 is the wild type colibacillus strain with obvious growth advantage, the invention has provided a engineering strain based on MG1655 and reforms transform, the strain has fast and high advantage of transformation potential at the same time, offer the new host for plasmid clone, offer the new underpan cell for metabolic engineering.

Description

Engineering strain based on escherichia coli transformation

Technical Field

The invention relates to an engineering strain modified based on escherichia coli MG1655, and belongs to the technical field of biological engineering.

Background

The engineering strain is a fungus cell strain obtained by modifying an original strain by using a genetic engineering method, and has the characteristics of multiple functions, high efficiency, strong adaptability and the like. Escherichia coli is used as a model organism of prokaryotes, and an engineering strain transformed from the Escherichia coli has wide application in synthetic biology, metabolic engineering and genetic engineering. For example: the engineering strain such as DH5 alpha can be used as a host cell for plasmid cloning, and the engineering strain such as BL21(DE3) can be used as a host cell for protein expression.

There are many engineered strains suitable for plasmid cloning currently on the market, such as DH5 alpha, TOP10, DH10B, BL21, Mach1-T1, XL10-gold, XL1-blue, etc. However, these engineered strains generally grow slowly and are less stress resistant than wild-type E.coli strains, and host cells cloned with these engineered strains as plasmids result in prolonged experimental cycles.

Wild-type escherichia coli strains such as MG1655 and the like are taken as starting strains, and related genes such as a defense system of the wild-type escherichia coli strains to foreign DNA and the like are knocked out by utilizing a novel accurate traceless genome editing technology, so that the transformation potential can be improved while the advantages of high growth speed, strong stress resistance and the like are kept, and a better host cell is provided for plasmid cloning.

Disclosure of Invention

The invention aims to provide a strain which is high in growth speed and stress resistance and suitable for plasmid cloning, is obtained by performing five times of iterative editing on the basis of an escherichia coli MG1655 strain by using a CRISPR/Cas9 gene editing technology, has the advantages of high conversion efficiency, high growth speed and the like, and provides new host selection for plasmid cloning.

Compared with the original strain, the engineering strain modified based on MG1655 provided by the invention has the newly added genotypes as follows: Δ (mcrCB-hsdSMR-mrr), mcrA-, endA, recA, Δ (araD-araA-araB): tet^R. The details of these genotypes are as follows:

the mcrCB-hsdSMR-mrr and mcrA form a restriction system of the escherichia coli, the generated restriction enzyme can specifically cut the exogenous DNA, the exogenous DNA is promoted to be further degraded by other nuclease in the cell, the restriction system of the escherichia coli is knocked out, the defense of the exogenous DNA can be relieved, and the stable existence of the circular DNA or the linear DNA in the cell is facilitated.

Enda expresses DNA specific endonuclease I, which is located in the membrane gap (periplasm) of cell wall, can crack double-stranded DNA into oligonucleotide of about 7bp, is the "gatekeeper" of cell for resisting exogenous DNA, and knocking out the gene is favorable for circular DNA or linear DNA to pass through cell membrane to enter cell.

recA expression DNA recombinant protein RecA can catalyze DNA to carry out homologous recombination, can also be used as regulatory protein to induce cell stress reaction, is not beneficial to the stable existence of exogenous DNA in cells, and can improve the stability of the exogenous DNA in the cells by knocking out the gene.

P induced by L-arabinose_BADThe promoter is an inducible promoter commonly used in Escherichia coli, and P is used_BADThe promoter is often induced by adding high-concentration L-arabinose, araD-araA-araB is an L-arabinose degradation gene, and the araD-araA-araB can be knocked outThe degradation of the Escherichia coli to the L-arabinose is blocked, the induction intensity of the L-arabinose is effectively enhanced, and the induction time of the L-arabinose is prolonged.

Tetracycline is a broad-spectrum antibiotic, tet^RThe gene is a tetracycline resistance gene, and the gene is inserted into a genome to ensure that the gene obtains resistance to tetracycline, so that the strain can be effectively prevented from being polluted by mixed bacteria in the environment.

And (3) measuring the growth curves of the strains before and after modification.

The transformation efficiency of the strain before and after transformation was determined.

Conditions (competence manufacturing method and transformation method) are optimized to improve transformation efficiency of the transformed strain.

The growth rate of the engineered strain on different resistant plates was determined.

Drawings

FIG. 1 is a comparison of the growth curves of MG1655 with currently common strains;

FIG. 2 is a comparison of growth curves before and after modification of MG 1655;

FIG. 3 shows a comparison of conversion efficiencies before and after modification of MG 1655.

The specific implementation mode is as follows:

the experimental procedures used in the following procedures are all conventional ones unless otherwise specified.

Materials, reagents and the like used in the following operations are commercially available unless otherwise specified.

1. Measuring growth curves of the strains before and after modification:

single colonies were picked in 5mL LB medium, cultured at 37 ℃ for 12h, and cultured at a temperature of 1: 100 portions of the culture were inoculated into 100mL of LB medium and cultured at 37 ℃. Samples were taken at 1h intervals and stored at 4 ℃. The samples were taken 13 times, 0-12h and 24h, and after the sampling was completed, the OD600nm values were measured together.

Composition of LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl

2. Measuring the transformation efficiency of the strain before and after modification:

MG1655 and the strain of the present invention were made into transformation competence and electrical transformation competence, 10ng pUC19 was transformed into each 100. mu.l competence, ice-washed for 30min, heat-shocked at 42 ℃ for 1min, ice-washed for 2min, added with 1mL SOC, cultured at 37 ℃ for 40min, 100. mu.l coated Amp plate (Amp: 0.1MG/mL), CFU was counted and transformation efficiency was calculated.

3. Optimizing conditions:

3.1 transformation competent preparation: single colonies were picked in 5mL LB medium, cultured at 37 ℃ for 12h, and cultured at a temperature of 1: inoculating 100% of the extract in fresh LB medium, culturing at 30 deg.C until OD600nm is 0.5, ice-cooling for 30min, centrifuging at 4000rpm at 4 deg.C for 10min, removing supernatant, and adding 0.1M CaCl₂Eluting twice, standing at 4 deg.C for 5min, and adding 0.1M CaCl₂50% glycerin is 3: 2 and resuspended and dispensed into 100. mu.l/tube.

3.2 optimized transformation procedure 10ng pUC19 was transferred per 100. mu.l competence, 3. mu.l PEG8000 (40%), ice bath 30min, 42 ℃ heat shock 1min, ice bath 2min, 1mL SOC, 37 ℃ culture 40min, 100. mu.l Amp coated plate (Amp: 0.1mg/mL), CFU counted and transformation efficiency calculated.

4. Determination of growth rates of the engineered strains on different resistant plates:

plasmids with different resistance are transferred into the competence of the modified strain, and the transformation operation is the same as [0024 ]. The colonies can be seen in 7h on the nonreactive plate, and the colonies can be seen in 9h on the Kan plate, the Amp plate and the Cm plate. (Kan:50mg/mL, Amp:100mg/mL, Cm:25 mg/mL).

Claims

1. An engineering strain based on escherichia coli modification is characterized in that wild escherichia coli with obvious growth advantages is used as a starting strain to carry out a series of genome editing, and finally, the growth advantages are retained while the transformation potential is greatly improved.

2. The wild-type E.coli strain according to claim 1 includes, but is not limited to, MG1655 and the like which grow fast.

3. Genome editing according to claim 1, characterized in that the genome editing technology used comprises, but is not limited to, CRISPR/Cas9 technology and the like, which enables precise traceless editing.

4. The genome editing of claim 1, including but not limited to: knocking out mcrCB-hsdSMR-mrr gene cluster, knocking out recA gene, knocking out endA gene, knocking out mcrA gene, replacing araD-araA-araB gene cluster with tet^RA gene.