Biomedical subjects

Shan-Hu Li

Publications and source records attributed to Shan-Hu Li.

4 recordsLinked to original sources

PubMed2006

[pBR322-Red mediated gene knockin, sites and expression in E. coli chromosome].

Genes lacZ, lacY and lacA in the lac opron of E. coli chromosome were respectively substituted with gene luc by using plasmid pBR322-Red, selection-counterselection system kan/sacB and various strategies of Red homologous recombination including Red mediated linearized double-stranded DNA homologous recombination and Red mediated recombineering with overlapping single stranded DNA oligonucleotides. Then, a series of new strains, CWL2, CWL4 and CWL6, were constructed and we found that they can express protein Luc efficiently. To further study the expression of exogenous genes at the site of lacZ, we have constructed a strain named CWD1 by knockin the cholera toxin B subunit(ctxb) gene at the lacZ site, then we found that CWD1 can express protein CTB efficiently and CTB was secreted out of the cell. So we assured that the sites of structure genes in the lac operon of Escherichia coli chromosome were suitable for expressing foreign genes.

Cholera Toxin↗

PubMed2005

[Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system].

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.

Bacteriophage lambda↗

PubMed2005

[Development of a new recombineering system by gap repair].

Using lambda phage Red recombinase mediated in vivo homologous recombination system, a 6.7 kb lambda PL operon sequence including the Red encoding genes was subcloned into pBR322 by gap repair technique, and generated a pBR322-Red recombinant plasmid that can provide the Red recombination function and can be transferred into many kinds of bacteria. To confirm the recombination functions of pBR322-Red, a single-stranded 70-bases oligo was introduced into W3110 by electroporation to create a single base T-->G mutation in galK gene on the bacterial chromosome. The result demonstrated that a new lambda Red-mediated recombineering system based on pBR322-Red was successfully established.

Bacteriophage lambda↗

PubMed2005

[Construction of recombinant plasmid using Neo/E Technology].

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.

Bacteriophage lambda↗