Team:UCL/Science/Results/Xeno

Biosafety

Edo do the brief intro into Biosafety

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 the stated 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

Figure 2 - BBa_K1336005 ispB xenobiological module is compatible with Reactive Black 5 dye-contaminated waste waters. Graph showing that E.coli transformed with BBa_K1336005 ispB shows comparable growth to the plasmid-free control in LB media with RB5 dye. 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 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 4 - Growth of BBa_K1336005 ispB and BBa_K1336006 LEC-ispB xenobiological module in M9 minimal media Graph showing WHAT?? EDO TO FILL OUT! . OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2.



Figure 5 - Growth of BBa_K1336005 ispB and BBa_K1336006 LEC-ispB xenobiological module in M9+CAS minimal media Graph showing WHAT?? EDO TO FILL OUT! . OD measured at 680nm and Time is shown in hours after incubation. Error bars indicate SEM, n=2. Conclusions: DANIEL/EDO TO FILL OUT