Team:NKU China/protocols
Protocol
1. The λ/red technique for the construction of BcsA-knockout FY-07
The λ/red system is derived from the λ red bacteriophage and it is a method that can be used for cloning or genome engineering, which is based on homologous recombination. It allows for direct modification of genome DNA: insertion and deletion of selectable and non-selectable sequences, point mutations or other small base pair changes, and the addition of protein tags.
The λ red system has three components: Exo, Beta and Gam.
Gam: Gam prevents both the endogenous RecBCD and SbcCD nucleases from digesting linear DNA introduced into the bacterium.
Exo: Exo is a 5’→3’ dsDNA-dependent exonuclease. Exo will degrade linear dsDNA starting from the 5’ end and generate 2 possible products: a partially dsDNA duplex with single-stranded 3’ overhangs or a ssDNA whose entire complementary strand was degraded, if the dsDNA was short enough.
Beta: Beta protects the ssDNA created by Exo and promotes its annealing to a complementary ssDNA target in the cell.
Experimental Procedure:
●Amplify the upstream and downstream homologous arms of the targerted gene.
●Overlap the upstream and downstream homologous arms of the targeted gene and insert them into the vector pTsk1.
●Transform the recombinant plasmid into specific bacterium and select the positive clones.
●Culture the selected clones to get the double-crossover bacterium and expel the recombinant plasmid at 37℃.
2. Gibson Assembly Cloning for Plasmid Construction:
Regardless of fragment length or end compatibility, multiple overlapping DNA fragments can be joined in a single isothermal reaction through Gibson. With the activities of three different enzymes, the product of a Gibson Assembly is a fully ligated double-stranded DNA molecule,which has proven to be an efficient and effective method for the assembly of plasmids, and molecular biologists now use this method extensively.
T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each other.
Phusion DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments.
Taq DNA Ligase - covalently joins the annealed complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment.
Experimental Procedure:
●Design the primers (see figure below). Adjacent segments should have identical sequences on the ends (sequences A and B in the figures). One strategy is to design primers that are 60 bp long, with 30 bp matching the end of the adjacent fragment and 30 bp annealing to the target sequence. Avoid Hairpins in this region, which can significantly reduce the efficiency of two homologous ends annealing.
●Generate DNA segments by PCR.
●Combine segments in Gibson Assembly Reaction and incubate the mix for 1 hour at 50°C. Note: Yields will be best when the DNA fragments are present in equimolar concentrations. The Gibson Cloning Master Mix (purchased from NEB) consists of three different enzymes within a single buffer.
●Transform the recombinant plasmid into bacteria and screen for the correct plasmid Product by Restriction Digest.
●Sequence the important regions of your final plasmid, particularly the seams between the assembled parts.
3. Conjugative transfer for the introduction of plasmids into FY-07:
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. It is a mechanism of horizontal gene transfer as are transformation and transduction although these two other mechanisms do not involve cell-to-cell contact. During conjugation, the donor cell provides a conjugative or mobilizable genetic element that is most often a plasmid or transposon. Most conjugative plasmids have systems ensuring that the recipient cell does not already contain a similar element.
