Abstract
Here we present a modification of the widely used pET29 expression vector for use in rapid and straightforward parallel cloning via a gene replacement and Golden Gate strategy. The modification can be applied to other expression vectors for Gram-negative bacteria. We have used the modified vectors to clone large numbers of bacterial natural enzyme variants from genomic and metagenomic sources for applications in biocatalysis.
Highlights
The Golden Gate cloning strategy has been shown to be incredibly powerful in cloning DNA fragments with high efficiency [2,3,4,5]
It relies on the use of Type IIs restriction enzymes that cleave DNA outside of their recognition site, providing unique cohesive ends that enable directional and seamless cloning of the gene of interest
We amplified the 1903-bp region from B. subtilis containing 445 bp of upstream regulatory sequences, the sacB gene and the terminator sequence, and inserted it in the pET29, where it replaced the multiple cloning site in the opposite orientation to the T7lac promoter [10]. We found that this orientation resulted in efficient sacB expression and counterselection
Summary
NdeI and XhoI restriction sites are in bold; BsaI and SapI restriction sites are underlined. PCR products were gel-purified, ligated to form circular vectors and transformed into chemically competent E. coli Nova Blue cells. Vectors were isolated from transformants that were resistant to kanamycin and sensitive to sucrose Both constructed vectors, pET29:SacBBsaI and pET29:SacB-SapI, were further verified by DNA sequencing and their functionality was initially tested by cloning and expression of eGFP in E.coli BL21 (DE3) (data not shown). 2 μl of each reaction was added to 20 μl of Nova Blue E. coli chemically competent cells and incubated on ice for 30 min. >98% of clones positive for the desired recombinant vector were obtained after transformation This high efficiency eliminated the need for colony screening and allowed for a single colony per construct to be picked and verified by DNA sequencing. Examples of panels of recombinant enzymes constructed in this way include enzyme classes such as transaminases from a drain metagenome [13], ene-reductases from a drain metagenome [14] and epoxide hydrolases mined from sequenced bacterial genomes [15], demonstrating the versatility of the protocol
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
