Biosafety

We implemented safety measures in order to control our engineered organisms on two different levels: the leak of DNA into the environment and the leak of bacteria into the environment. To prevent the leak of DNA we utilised an extracellular nuclease developed by UCL iGEM 2012 (Plastic Republic) that cleaves DNA in the environment and we tested its functionality in the presence of Azo Dyes. The purpose of this experiment was to make sure that this biosafety strategy is still viable in our system.
The second part of our strategy investigates the first stage of a Xenobiological approach of biosafety. We tested an antisense RNA gene silencing system to knock-down the expression of ispB, a gene used in a key step of the biosynthesis of ubiquinone and menaquinone. This will allow us to substitute the natural quinones with a synthetic equivalent of our design, derived from azo dyes, to which E. coli will be auxotrophic.

Biological strategy

DNase agar assay with Azo-dyes

The BBa_K729004 periplasmic nuclease that UCL iGEM 2012 submitted and characterised was able to demonstrate positive DNase agar assays. This assay involves washing DNA-containing agar with HCl after streaking bacteria. After this wash, a ‘halo’ surrounding the streaked bacteria indicates that extracellularly secreted DNase digested the DNA surrounding the colonies within the agar. In order to further characterise this BioBrick and incorporate it into our project as a biosafety method of minimising the transfer of extracellular DNA, we decided to test whether BBa_K729004 would function in the presence of Azo-Dyes.



Figure 1 - BBa_K729004 BBa_K729004 periplasmic nuclease enzyme shows functionality in the presence of multiple Azo-dyes. Figure showing that the BBa_K729004 periplasmic nuclease enzyme is still able to digest the surrounding DNA in the DNase agar. Figure 1a and 1b demonstrate the presence of halos around colonies on plates with and without Acid Orange 7 (AO7) azo-dye. Figure 1c and 1d demonstrate the presence of halos around colonies on plates with and without Reactive Black 5 (RB5). All azo-dye agar plates were made with a 1:500 dilution of 0.5mgml-1 dye.

Since the azo-dye degradation would be taking place in industrial environments, being the products of the bioreaction at one point dumped into other channels of water-treatment or into the environment, it is essential that there are certain barriers that stop the DNA from our genetically modified organisms from potentially being transferred into wild-type strains. On a basic first-level defense, this assay suggests that horizontal gene transfer could be inhibited in an azo-dye contaminated environment byBBa_K729004 , as it has proven effective in degrading extracellular bacterial DNA.

Xeno-biological strategy

The protocols for the experiments in this page can be found here.

Effect on growth of ispB gene silencing antisense RNA

As an initial proof of concept for the possibility of replacing natural quinones with synthetic equivalents derived from azo dyes, we investigated an antisense RNA method to silence ispB, the key gene involved in ubiquinone and menaquinone biosynthesis. This gene has been proved to affect the growth of E. coli and we assayed the effect of induction of the asRNA on the bacterial growth in different media via spectrophotometer.























Figure 3 - BBa_K1336006 LEC-ispB xenobiological module is compatible with Reactive Black 5 and Acid Orange 7 dye-contaminated waste waters. Graph showing that E.coli transformed with BBa_K1336006 ispB shows comparable growth to the plasmid-free control in LB media with AO7 and RB5 dyes. OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2. Figure 3 - BBa_K1336006 LEC-ispB xenobiological module effect on E. coli's growth in LB media after induction. . OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.























Conclusions: The ispB antisense RNA silencing tool hasn't proved effective in our initial research in LB media. We decided to characterise the part in M9 minimal media to confirm that quinones were not externally provided in LB media, which would detriment the usefulness of a knock-out of quinones biosynthesis. We are in the phase of development of further silencing tools, in collaboration with the Edimburgh team in order to test if we can complement ubiquinone and menaquinone with synthetic equivalents after silencing ispB the key gene for their biosynthesis.