Introduction: how we constructed our biosensor

In order to be able to use our model and to determine whether DcmR acts as a repressor or activator in the presence of DCM we designed and constructed the following two plasmid system. We primarily used Gibson assembly methods and source most of the necessary DNA from gblocks(synthesised oligonucleotides) we had designed based in the sequenced genome of Methylobacterium DM4. This system will also form the DCM biosensor and will be integrated with an electronic circuit to complement this genetic one:

pOXON-2-dcmR-mCherry

Oxford iGEM 2014

pSRK-Gm-pdcmAsfGFP

Oxford iGEM 2014

Why these two plasmid backbones?

Predicting the sfGFP fluorescence

Introduction

To allow us to characterize the second half of the genetic circuit, we needed to be able to predict the difference in response. To do this, we constructed models by cascading the differential equations according to the respective circuit structures thereby producing two different potential system responses.

To achieve this, we constructed simplified equivalent circuits that were linked by two potential activation-repression relationships.

It is important to understand that these simplified equivalent circuits will not give the correct mCherry response but they will give the correct GFP response after correct parameterisation.

We then set up the differential equations necessary to solve this problem in Matlab. The method and results are as detailed below:

Conclusion

The bottom graphs illustrate the predicted response of each system to a simultaneous step input of both DCM and ATC. As you can see, there is little difference in the predicted steady-state value of the fluorescence, however, providing the basal transcription rate of GFP is relatively low, there should be a clear difference in the level of fluorescence before either of these inputs are added. This very easily identifiable difference between the two systems will enable us to characterize the genetic circuit present in our particular system.