Propionibacteria-E, Coli Shuttle Vector Construction And Expression

Propionibacteria, which have a wide range of probioticactivity, are used in themanufacture of dairy products such as cheese, in the productions of vitamin B12,tetrapyrrole compounds, and propionic acid. In bread baking, it is used as starters forensilage, and in some pharmaceutical preparations.Because of the knowledge lack of the regμLatory mechanism for Propionibacteria,their molecμLar research is in its infancy. It is necessary to construct P. shermanii-E.colishuttle vector for genetic manipμLation. In this research a shuttle vector betweenP.shermanii and E.coli was constructed and used to study the electroporation condition.The main conclusions are as follows.1. Constructing of P.shermanii-E.coli shuttle vector pTD-PcsAF. In this study,vector pTD-PcsAF was constructed with a promoter trapping vector pTD210and acloning vector T-PcsAF which contains the promoter PcsAF. The res μ Lt thatrecombinant vector pTD-PcsAF can replicate in E. coli and P. shermanii shows that theconstructed vecor is a shuttle vector between P.shermanii and E.coli.2. The electroporation procedure for Propionibacteria was optatimized based on thebacteria growth phase, washing buffer and voltage. The resμLts show that it is better touse the Propionibacteria cells in the mid-logarithmic phase as the competent cells andthe washing buffer is10%glycerol. The optimized condition is1.0kV/cm,250and25μF.3. The ALA sythesase (ALAS) gene, hemA, from Rhodobacter sphaeroides wascloned into pTD-PcsAF to develop the application of expression vector. The constructedpTD-hemA was transformed into P. shermanii. The HemA gene expression wasdetermined by RT-PCR. The results showed that the HemA gene directed by PcsAFpromoter did express, however the ALA level has no obvious fifference between mutantpropionibacteria(pTD-hemA) and mutant propionibacteria(pTD-PcsAF).