Construction and Expression of β-galactosidase Genetically Engineered Lactococcus lactis

Our objective is to solve the lactose malabsorption and intolerance of human beings by combining micro-ecology path with genetic engineering technique. Plasmid pMG36e was used to clone and express a β-galactosidase gene from L. delbrueckü bulgaricus strain 1.1480 in the Lactococcus lactis subsp. cremoris MG1363 and Lactococcus lactis subsp. lactis IL1403. The recombinant plasmid was preserved and proliferated in Escherichia coli ( E. coli) JM109, and transformed into MG1363 and IL1403 by electroporation. The protein expression was studied. ( 1 ) The bifidobacterium culture medium ( BBL) was suitable for the growth of the strain 1.1480. (2) With 13 amino acids at the N-terminus from the vector, β-gal- actosidase fusion protein (which retained the enzyme activity) could be successfully expressed in E. coli JM109, MG1363 and IL1403, but the expression quantity was larger in the former than in the latter two. (3) The SD sequence designed could be successfully recognized by both the E. coli and the Lactococcus lactis, but the expression level of the non-fusion β-galac- tosidase protein was lower than that of the fusion protein in the same host. The β-galactosidase genetically engineered E. coli JM109 is a useful tool to produce this enzyme in vitro . The signal peptide of the usp45 protein from the Lactococcus lac- tis can be added before the promoter sequence to promote β-galactosidase secretion from Lactococcus lactis . The potential ap- plication of the β-galactosidase genetically engineered MG1363 and IL1403 to cure the lactose malabsorption and lactose in- tolerance in both health food and medicine is promising.

Construction of the Plasmid Reference Molecule for Detection of Transgenic Soybean MON89788

[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant DNA integrated DNA of MON89788 soybean were amplified independently,and the three fragments were cloned into the cloning vector pMD18-T in order through molecular manipulation method to construct pMD-LM3M5,the applicability of the constructed novel PRM was tested. [Result] Sequencing confirmation result showed that the PRM was 3 700 bp in length,containing 1 029 bp of recombined DNA fragment. The limits of qualitative detection of the PRM were 10 copies. [Conclusion] The PRM constructed in this study was suitable for the identification of MON89788 event.

A Stably Transgenic INA Enterobacter cloacae for Control of Insect Pests

Using the minitransposon pMini-Tn5 and the ice-nucleation active (INA) gene of iceA, a suicide recombinant plasmid pTnice1 was constructed, which has the ability of broad-host-range conjugal mobilization and integration of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition. We used this plasmid to integrate the iceA into chromosomal DNA of Ent. cloacae and obtained the transgentic strain Enc1.2022 ina. In this transgenic Ent. cloacae, iceA would never be transferred elsewhere through transposition, and constantly expressed high ice nucleation activity even in the absence of antibiotic pressure. The transgenic strain was ingested by corn borer larvae. Over the 7 d after ingestion, the mean supercooling points (SCPs) of the larvae was about 10℃ higher than those of larvae treated with distilled water (control). The maintenance of these high SCPs was related to the stable gut colonization of transgenic strain. At 6th day post ingestion, the larva was exposed at 5 or 7℃ for 12 h, the percentages of larvae frozen to death were 85 and 100%, respectively. In contrast, none or a small proportion of control larvae was frozen to death under the same conditions. Further studies demonstrated that this transgenic strain bore weak epiphytic ability. Therefore, this genetically engineered strain may be a promising candidate for control of insect pests in agricultural fields.

Acquisition and dissemination mechanisms of CTXΦ in Vibrio cholerae : New paradigm for dif residents

Vibrio cholerae(V. cholerae) genome is equipped with a number of integrative mobile genetic element(IMGE) like prophages, plasmids, transposons or genomic islands, which provides fitness factors that help the pathogen to survive in changing environmental conditions. Metagenomic analyses of clinical and environmental V. cholerae isolates revealed that dimer resolution sites(dif) harbor several structurally and functionally distinct IMGEs. All IMGEs present in the dif region exploit chromosomally encoded tyrosine recombinases, Xer C and Xer D, for integration. Integration takes place due to site-specific recombination between two specific DNA sequences; chromosomal sequence is called att B and IMGEs sequence is called att P. Different IMGEs present in the att P region have different attP structure but all of them are recognized by Xer C and Xer D enzymes and mediate either reversible or irreversible integration. Cholera toxin phage(CTXΦ), a lysogenic filamentous phage carrying the cholera toxin genes ctx AB, deserves special attention because it provides V. cholerae the crucial toxin and is always present in the dif region of all epidemic cholera isolates. Therefore, understanding the mechanisms of integration and dissemination of CTXΦ, genetic and ecological factors which support CTXΦ integration as well as production of virion from chromosomally integrated phage genome and interactions of CTXΦ with other genetic elements present in the genomes of V. cholerae is important for learning more about the biology of cholera pathogen.