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From 2011.igem.org

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Module 3: Gene Guard

Containment is a serious issue concerning the release of genetically modified organisms (GMOs) into the environment. To prevent horizontal gene transfer of the genes we are expressing in our chassis, we have developed a system based on the genes encoding holin, anti-holin and endolysin. We are engineering anti-holin into the genome of our chassis, where it acts as an anti-toxin, and holin and endolysin on plasmid DNA. In the event of horizontal gene transfer with a soil bacterium, holin and endolysin will be transferred without anti-holin, rendering the recipient cell non-viable and effectively containing the Auxin Xpress and Phyto-Route genes in our chassis.

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Assembly

The assembly of this module shall be the most challenging. Not only are we starting it the latest, but we will be using standard Biobrick assembly. The assembly of this module will go through three stages which must be completed sequentially. First we must create the Anti-Holin construct that will contain the insulator and our strong RBS. Then we must insert the Anti-Holin insert into the CRIM plasmid and integrate the plasmid into the genome. Once the Anti-Holin is in the genome we will build the Holin-Endolysin construct that will only be able to be maintained within our strain of E. coli.

Stage 1: Anti-Holin construct assembly

The first step of assembly will require us to place the anti-Holin from the BBa_K112808 Biobrick under the J23100 promoter in BBa_K398500. In order to perform this step we will be using PCR to add our new RBS and insulator sequence into the construct. The primers were designed to have 15bp overhangs for In-Fusion. The PCR step was incredibly challenging due to the long non-homologous regions within the primers. This cost us almost two weeks. However, after a lot of hardship and numerous In-Fusion and CPEC attempts we managed to assemble this construct.

As we can see, colony 2 does not contain the insert we are looking for. However, colony 9 has an insert of the correct size. This stage has been completed successfully.

Stage 2: Integration of Anti-Holin into Escherichia coli genome

The next stage of the assembly is to integrate the insert from our Anti-Holin construct into the genome of E. coli. In order to do that we will ligate the insert into the CRIM plasmid. Once inserted we must transform the plasmid into pir⁺ cells in order to replicate the plasmid. Once we have a good yield of the plasmid we will co-transform the CRIM plasmid into cells containing the accessory plasmid and we will select for the colonies that contain the CRIM plasmid in the genome.

During this process we discovered that the SpeI site is non-functional due to the non-RFC10 insertion of a terminator. This was also discovered within our sfGFP construct which we had to repair through the use of PCR, phosphorylation and ligation of the plasmid. This has altered our plans to run two insertions in parallel (one in the suffix and one in the prefix) to see if there would be any difference in Anti-Holin expression levels.

Stage 3: Holin-Endolysin construct assembly

In order to assemble this construct we inserted the J23103 promoter onto the BBa_K093005. Then we PCR'd out the Holin and Endolysin genes from BBa_K112808 and added an XbaI site and suffix into the primers. The plan was to then ligate the J23103-RFP (BBa_K093005) construct into a pSB1C3 plasmid and then ligate the Holin-Endolysin PCR product after it. However, after sequencing the BBa_K093005 RFO construct we have discovered an error in the suffix that will be problematic.