Toxic protein gene leakage expression cloning method

Abstract

The invention discloses a toxic protein gene leakage expression cloning method. The method comprises the following steps: S1, adding an SsrA polypeptide sequence to a C terminal of CcdB protein; S2, obtaining a gene segment through a PCR amplification method, and recombining the gene segment to a target vector PUC57 by utilizing Gibson to construct a complete sequence; and S3, connecting and cloning a target gene sequence into an expression vector, transforming escherichia coli, culturing for 12-16 h, observing a spot growing condition, carrying out PCR bacterial colony screening positive cloning, extracting plasmids, sequencing, and verifying the sequence. The gene can be cloned in yeast, the yeast belongs to eukaryotes, most toxic proteins can be cloned, the gene is cloned in the yeast firstly, then yeast plasmids are extracted, the plasmid concentration is improved, escherichia coli is transformed for plasmid amplification, and downstream experiments are carried out.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for cloning expression due to the leakage of toxic protein genes. Background technique [0002] Plasmids in E. coli are small DNA molecules that replicate independently of chromosomes. Essentially the plasmid is a burden for E. coli. It uses the internal machinery of E. coli (replication system, ribosome, transfer tRNA, etc.) and raw materials (A / T / C / G / U, amino acids) to replicate itself and produce RNA and proteins that E. coli itself does not need to survive. E. coli dies when the replication of the plasmid takes too many resources or when the RNA or protein expressed by the plasmid interferes with the viability of the host itself. This is how toxic genes work. Common proteins that are lethal to E. coli include nucleases, cell division and replication-related proteins, ion channel proteins, signal regulation proteins, kinases, toxins, transposases, integrases, proteins ...

AI Technical Summary

Problems solved by technology

Sometimes, although SsrA 11 amino acid polypeptides are added to the C-terminus of the toxic protein, it may be due to the high copy number of the plasmid in the bacteria, the protein leakage expression is serious, so that it is too late to degrade, resulting in the death of the host bacteria. Faced with this situation, we The gene can be cloned into the low copy number and reduced leakage expression vector PCCI-YAC designed by this invention

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