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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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Testing

1. Anti-holin expression

We have completed stage 1 of the assembly of the Gene Guard. A protein gel showed a clear band of the appropriate size right at the bottom of the gel when compared to a control cell. Therefore, we have sequence verified and shown that the BBa_K515104 expresses a protein of the appropriate size (Fig. 1).

2. Escherichia coli survivability and plasmid retainment in soil

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We set up a soil experiment to test how long our E. coli chassis can retain its plasmid in soil. We initially transformed chemically competent E. coliDH5α cells with superfolder GFP. These cells were inoculated on small filter discs (about 0.5 cm diameter), which were placed in autoclaved and non-autoclaved soil. We periodically grew up cultures from these filter discs over the course of six weeks.

After six weeks, we were able to recover fluorescent bacteria from sterilised and non-autoclaved soil (Figure 2).

As is visible from these plates, fluorescence was present in bacteria recovered from both sterile and non-sterile soil. A control plate grown from a filter disc inoculated in non-sterilised soil without fluorescent bacteria showed that there was no contamination with other fluorescent lab bacteria. In order to investigate whether the fluorescence observed was due to the presence of the original sfGFP construct and whether the E. coli-like colonies from the non-sterile sample had retained a plasmid, we extracted plasmid DNA using a miniprep kit and did a digest with EcoRI and PstI and with EcoRI on its own to check for presence of the original insert and size of the unfolded vector, respectively (Figure 3).

The insert is very clearly visible at just below 2 kb. This confirms the presence of superfolder GFP in both cultures. Sequencing of the GFP insert revealed that no mutations had taken place in the superfolder GFP gene contained in the bacteria inoculated in non-sterile and sterile soil.This result was obtained in three separate replicates.

In addition, small colonies appeared on the non-sterile plate that had very different colony morphology. We grew this colony up in LB medium containing selective antibiotic and subsequently performed a separate miniprep. No DNA was yielded in this miniprep. It is therefore likely that the plasmid was not transferred to these bacteria but that they either possess natural antibiotic resistance or were able to survive on plates that whose antibiotics had already been depleted by the presence of resistant engineered bacteria.

All three samples, the sample from the sterile plate, and the two from the non-sterile plate showing E. coli-like and non-E. coli-like morphologies will be used for 16S ribosomal RNA sequencing (using commonly used primers ^[1]) to determine the bacterial species.

This result is extremely important as it shows that plasmids can be retained in E. coli for a very long period of time even in the presence of competition when inoculated in soil. This gives us an indication of the life-span our chassis would have in the soil in its implementation stage. In addition, long retainment of the plasmid means that the chance for horizontal gene transfer increases, rendering this result very important for the Gene Guard module.

Gene integration of the Anti-holin

Once we knew that the anti-holin construct was alright and seemed to be producing a small protein we had to excise the insert and ligate it into the CRIM plasmid. This was complicated as the high copy number plasmid was placing a heavy metabolic burden on the cells making them grow incredibly slowly (something that is not desirable for when you have to be working for a deadline). Not only that, the cells seemed to be losing the plasmid after four days on a plate in a cold room and some of the colonies managed to alter the plasmid in a way that made it lose the sfGFP and allowed the cells to grow quickly and out-compete any cells with the correct plasmid. However, after a lot of trial and error we managed to isolate eight plasmids that were possible candidates for the plasmid we wanted to integrate.

In order to test these eight colonies we first did an EcoRI and PstI digest and then looked at the size of the insert compared to a control which only contained sfGFP in the CRIM. According to our results, three of the colonies contained an insert that was larger than that of the control. However, the gel red we used to stain the gel seems to do something funny with the buffer we were using to digest making all the bands run higher than they should be running. Therefore, to conclude that the plasmid contained the inserts we wanted we looked for a restriction site that is unique on the anti-holin and the CRIM. We found that the anti-holin construct contains a ClaI site that is also present on the CRIM vector. We performed this digest in another buffer and obtained the expected bands for the three colonies confirming that they contain the anti-holin on the CRIM.

Then we performed the genome integration step

References

[1] Tanner M et al. (2000) Molecular phylogenetic evidence for noninvasive zoonotic transmission of Staphylococcus intermedius from a canine pet to a human. Journal of Clinical Microbiology 38(4): 1628-1631.

[2] Haldimann and Wanner, 2001. Conditional-Replication, Integration, Excision, and Retrieval Plasmid-Host Systems for Gene Structure-Function Studies of Bacteria. Journal of Bacteriology 183(21) p. 6384-6393.

M3: Assembly M3: Future Work