Deletion

Most bacteria have an innate mechanism of effluxing arsenic ions which make them resistant to the heavy metal ion. The efflux system is encoded by the ars operon located in the chromosome (genome) of the bacterial cells and in our case, it needs to be deleted as else it would hamper our design which aims to sequester arsenate and arsenite ions within the bacterial cell.

Ars Operon has genes ArsRBCDA in tandem. We designed primers against ArsR and ArsB such that a 600 bp region between gets amplified.

Thus appearance of 600 bp band would confirm the presence ars operon in the genome of the bacterial cell (E. coli BL21 strain). It was confirmed that the primers dont show non-specific annealing elsewhere in the genome using NCBI Blast.

Now that the presence of ars operon was evident, we started researching on the techniques that can be used for deleting the operon. GeneBridges, a leading company in recombineering agreed to sponsor their RedE/T gene deletion kit for the team.

Image Ref: GeneBridges RedE/T Kit Manual

The Kit makes use of Lambda Bacteriophage recombination techniques to the desired region on the bacterial genome with a nonsense stretch of nucleotides along with a selectable marker.

A small video clip will help understand the mechanism of deletion better.

In order to carry out the deletion process a cassette provided in the kit needs to be amplified using primers that have ~50 bp long overhangs specific to the neighbouring region of the dene being deleted. Thus a cassette that can now be recombined with the bacterial genome to delete your desired gene is obtained.

We obtained this cassette by PCR amplification using primers that contain 50 bp overhangs specific to the neighbouring sites of ars operon.

Then this linear cassette needs to be transformed into the bacteria whose genome contains the gene to be deleted. This transformation step needs electroporation.

Along with the linear cassette, pRED plasmid that codes for recombination enzymes needs to be co-transformed into the bacteria to cause gene deletion.

We accomplished transformation of the bacteria with ars operon specific deletion cassette but could not proceed further to co-transform with pRED plasmid within the iGEM project deadline. We however are determined to continue working on the same until we accomplish what we set to achieve, proof of concept that BacMan works, at least in E. coli if not the ambitious aim of showing results in probiotic Lactobacillus spp.