Combination of novel counter-selection system kil and pSim6 plasmid in recombination engineering / 生物工程学报

Seamless modification is a popular genomic manipulation technique in genetic engineering. Selection stringency of the counter-selection system determines the efficiency of the seamless modification. Recently, a novel counter-selection system, kil, was constructed. It is reported that the selection selectivity of kil is higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, indicating that recombinants could be selected out more efficiently by combining kil counter-selection system and plasmid pSim6. In order to confirm this speculation, four different loci (lacI, dbpa, ack, glk) in Escherichia coli strains W3110, MG1655 and DH10B were selected for testing: dsDNA fragments of different sizes (500 bp, 1 000 bp, and 2 000 bp) were used to substitute tet/kil. As expected, recombination efficiency was higher in host bacteria harboring plasmid pSim6 than that harboring pKD46, and the results were more obvious with the length of dsDNA increasing. Specifically, recombination efficiency was 1.2 to 2 fold higher in pSim6 harboring bacteria than in pKD46 harboring bacteria when dsDNA fragments were 1 000 bp in length. With the length of dsDNA increasing up to 2 000 bp, the gap increased to 2.2-5 fold. In conclusion, it is easier to perform seamless modification by combining kil counter-selection system and plasmid pSim6 than combining kil and pKD46. An alternative tool in genomic engineering is provided in this study.

A highly efficient approach to introduction of the single-base mutation in a plasmid containing adenovirus genome through Red recombination / 军事医学

Objective To construct a highly efficient approach to the introduction of the single-base mutation in a plasmid containing the adenovirus whole genome larger than 40 kb.Methods The target DNA with a mutation site was achieved by over-lapping PCR.The large plasmid with adenovirus genome and target DNA were co-transformed into Escherichia coli strain DY330 carrying a high rate Red recombination system.The positive clone was selected via colony PCR in combination with enzyme identification.The site-mutation large plasmid was transformed into E.coli strain DH10B in which the backbone of the large plasmid remained was stable.Results Two mutations were continuously introduced into the adenovirus genome,the location of which was pos.9171 and pos.24410 respectively.The integrality and stability of the plasmid backbone were verified by enzyme cutting identification.The two mutations on the plasmid were verified by DNA sequencing.Conclusion An efficient approach to the introduction of the single-base mutation in positions 9171 and 24410 from the adenovirus genome which was integrated into a plasmid is successfully established.The positive selection efficiency ranges from 5%to 15%.The construction of the approach will facilitate the study of adenovirus infection mechanism.

Construction of a qseC-deleted mutant of Escherichia coli and evaluation of biofilm formation of the mutant / 中华微生物学和免疫学杂志

Objective To construct a qseC-deleted mutant strain of E.coli by Red recombination and to study the effect of qseC gene on biofilm formation in the mutants.Methods The chloramphenicolresistant gene flanked by homologues of target genes was amplified by PCR and electro-transformed into E.coli MC1000.When induced by L-arabinose,the plasmid pKD46 could express three recombinant proteins of λ-prophage,which led to the replacement of target gene(qseC) with chloramphenicol-resistant gene.Then the chloramphenicol-resistant gene was eliminated by FLP-promoted recombination events.The biofilm formation of wild-type and mutant strain was detected by crystal violet staining.Results The qseC-deleted mutant of E.coli was confirmed by various PCR and DNA sequencing.Gene qseC was completely deleted.There was no significant difference in growth ability between the qseC mutant strain and the wild-type strain MC1000.The biofilm formation of wild-type and mutant strain was quantified by crystal violet staining.The absorbance determined with a plate reader at 570 nm was 1.00±0.15 and 0.47±0.10 respectively.Conclusion The qseC-deleted mutant of E.coli was constructed successfully.And the qseC gene plays an important role in regulation of biofilm formation in E.coli.

Advances in Red Recombination Applied in Knockout of Bacteria Chromosomal Gene / 中国生物工程杂志

Traditional recombination technology of bacteria chromosome and its limitation were introduced. The definition of Red recombination technology is put forward: a method of homologous recombination between foreign linear DNA and the target gene in chromosomes mediated by ? phage Red system. The linear DNA referred here is general PCR product or oligonucleotide, which has a 36~50bp homologous sequence with the target gene in chromosome at both flanking. Red recombination technology leaves out the in vitro DNA restriction enzyme digestion and link process, which makes the knockout and alternation of target gene in bacteria chromosome relatively easier, and becomes an effective method to exploring genes and constructing new strains gradually. The gene inactivation and alternation method aiming at bacteria chromosome applied to Red recombination system was summarized by the structure element, action mechanism, and strategy of recombination, advantage and developing prospect. The Red system includes three genes: bet (aka?), exo and gam (aka ?). Exo is a 5′→3′ exonuclease, which degrades the 5′ ends of linear DNA molecules. Bet is a single-stranded DNA binding protein that binds to the single stranded 3′ ends generated by Exo and promotes annealing to complementary DNA. Gam binds to the host RecBCD complex and inhibits its exonuclease activity. Red recombination system may be constructed in such plasmids as pKD20 and pKD46 or in chromosome of bacteria. Most bacteria are not readily transformable with linear DNA because of the presence of intracellular exonucleases that degrade linear DNA. But when bacteria cells are transformed with pKD20 or pKD46 plasmid, or integrated with a detective ? prophage, Red recombination enzymes may be expressed in host cells, which make linear DNA with 36~50bp extensions that are homologous to both flanking of target genes transform E.coli readily and knock-out or alternate target gene. The Red recombination method is not only useful in chromosomal gene inactivation in E.coli, but also in other bacteria or virus, such as Salmonella, Shigella flexneri and virus HaSNPV. With the proceeding research, Red system will be applied for more and more purposes, and contribute a lot for gene improvement and gene function investigation in the coming Postgenome Era.

Epitope Tagging of the rpoS gene of Y. pestis by Recombineering Technique / 中国生物工程杂志

Objective: To facilitate the functional analysis of chromosomal genes and their products, the recombineering technique to epitope tagging of chromosomal genes of Y. pestis was adapted. Methods: The epitope tag was generated by primer annealing and then fused with resistance gene by fusion PCR. The epitope-resistance cassette was inserted into pBluecript, resulted in the template plasmid, pBS-MH. The tagging cassette for rpoS was obtained by PCR amplification from pBS-MH with primers containing homology specific to the target gene. PCR products were transformed into recombination competent cells and recombinants were selected. PCR and DNA sequencing were used to confirm the correct tagging event. The expression of the tagged protein was detected with Western blot by using monoclonal antibody to the epitope. Results: The template plasmid containing fusion of epitope and resistance gene was successfully constructed. The sigma factor gene, rpoS, was tagged with a myc-his tag at the COOH terminus. Expression of the tagged rpoS was successfully detected indirectly by the antibody against His tag. Conclusion: The chromosomal gene tagging by recombineering technique represents a powerful tool in the functional study of bacterial genes and their products.

Utilization of ?-Red Recombination System and Balanced Lethal System to Reconstruct the Plasmid with Antibiotic Resistant Gene Using in Live Vaccine / 微生物学通报

Recombination plasmid pMM085 possessed both immunogens heat-labile enterotoxin(LT) and fimbriae antigen K88 of enterotoxigenic Escherichia coli (ETEC). Althouth vaccine strain MM-3 carrying pMM085 had good effect to protect piglets against diarrhea due to ETEC infections,it was not ideal live vaccine for pMM085 bringing chloramphenicol resistance gene (cat). To solve the problem,the host-plasmid balanced lethal system was introduced which including the replacement of cat gene by asd gene and transformation the new plasmid to the strain X6097 which asd gene was knocked out in its chromosome. Considering pMM085 was a big plasmid (23kb) and traditional genetic manipulations was not easy to carry on,?-Red recombination system was adopt in this work to realize the replacement of cat gene by asd gene. The results indicated that ?-Red recombination system was convenient and efficient to reconstruct big plasmid.