Catechewing coli is a paan stain degrading E. coli that not only munches on the stains but also takes care of its own biocontainment issue.
Degradation module 1
Degradation module 2
Why two enzymes?
Keeping in mind that the degradation product after breakdown of catechu is not colourless but yellow, due to the formation of gallic acid, we have incorporated two enzymes in the degradation module to ensure complete removal of the coloured components. We present two such pairs of enzymes that can degrade the stains.
To ensure that the enzymes have better access to their substrates we have decided to make them extracellular by adding the ompT tag. We have incorporated the ompT tag to secrete out enzymes so that they are extracellular and are present in the environment even after the death of the GMO.
Binding domainCalcium is present as an integral component of paan which is why, to increase the affinity of our enzyme towards the stains, we have incorporated a calcium binding domain in our enzyme complex. Moreover, public places in India are often painted with slaked lime based paints which means our enzyme will have high affinity to these surfaces.
We have also ensured that the byproducts of the reactions are completely non toxic and safe for the environment.
While it was important to come up with a solution keeping in mind the problem of water consumption and health hazard, it was also equally important to make sure that we do not introduce GMOs rampantly in the environment. Paan stains are present ubiquitously in the country; on the roads, on the walls, on railway stations, there is no public place which is devoid of paan stains. This suggests that the way the product would be used, would expose the environment to a large number of GMOs which is not safe due to multiple reasons, some of them being the risk of horizontal gene transfer and rise of drug resistant pathogens. To tackle this issue we have come up with the idea of incorporating a kill switch in our design such that the by products of stain degradation would signal the bacteria to induce death.
KILL SWITCH “ON” STATE
KILL SWITCH “OFF” STATE
Why DpnC as a killing enzyme?
Most of the kill switches in the registry included proteins like holin which lead to cell wall damage and cause death. However, the DNA of the cells remains intact and can be taken up by other bacterial cells in the environment leading to horizontal gene transfer. We have used DpnC under the control of arabinose regulated promoter. DpnC is a nuclease which will shred the DNA of the cells as soon as it is expressed. Our construct is such that it will also take care that the GMO does not disperse in the environment since absence of arabinose in the environment will immediately trigger the expression of DpnC.