Team:OUC-China

Plasmid transfer

We are working on building different kinds of plasmid transfer system. The Special plasmid that we constructed is transferred to the different cell by different ways. Here are two project for plasmid transfer.

For prokaryote:

Previous studies have shown that DNA transfer by bacterial conjugation requires a mating pair formation (Mpf) system that specifies functions for establishing the physical contact between the donor and the recipient cell and for DNA transport across membranes. During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. For example the IncPa plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins, both of which contribute to Mpf.

Our project aims to construct a system of double plasmids, one is a mini plasmid carrying the oriT sequence and the other is a plasmid carrying the transfer regions of the broad-host-range IncP plasmid RP4, moreover, the two plasmids we construct are compatible. In the transfer system, the large one doesn’t carry oriT region while it carry the transfer regions tra and trb, due to lacking the sequence of oriT, the large plasmid can’t transfer between cells, thus the recipient strain don’t have the ability of conjugation. In the meantime, the other plasmid carrying oriT can transfer under the induction of the tra1 and tra2.

For eukaryote:

Fish diseases cause billion loss in the aquaculture filed every year. Traditional therapeutic method may do harm to the fishes and environment. However, using plasmid that contains the antigen genes as a new kind of vaccine can cause the immune reaction.

To achieve that goal, we will construct the recombinant pet32a (+) - TAT :: H4 plasmid, and transform into competent cells BL21 (DE3) in order to produce TAT :: H4, the products were purified and finally wewant to gain the fusion protein. Meanwhile for transfection of fish cells, we are going to construct carrying EGFP and CMV promoter plasmid pcDNA3.1 (+) - EGFP plasmid for detecting the expression and effect of transfection in eukaryotic cells.

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