Introduction

In the native DCM-degrading bacterium the following genetic construct exists: Oxford iGEM has isolated this intergenic region that contains a bidirectional promoter. We have termed these "PdcmA" acting towards the right in the direction of the gene encoding DcmA, and "PdcmR" acting towards the left in the direction of the gene encoding DcmR. The literature claims DcmR to be a regulatory protein acting to control the expression genes encoding DcmA and DcmR. Little is known about the different parameters in each direction.
We have observed and characterised the effect of DcmR regulating both promoters; in both directions under the influence of DcmR.

This is a new tool for molecular biologists wishing to manipulate stoichiometries of genetic products in the same construct.

We have also characterised PdcmA repression as a double repression system with DCM modulating DcmR activity resulting in derepression of genes downstream of PdcmA. This DCM sensitivity is not observed in the PdcmR direction. Wetlab Results:

Is DcmR a repressor or an activator at PdcmA/PdcmR promoters?

Conclusion: DcmR is a repressor of both the PdcmA and PdcmR promoter.

DcmR at the bidirectional promoter - A new tool for molecular biologists

We can see from these results that at both growth stages the relative expression is weighted towards PdcmR. An interesting result is that in the presence of DcmR this ratio is reduced such that expression through PdcmR is relatively increased while expression through PdcmA is relatively decreased. This result is observed as a decrease in the PdcmA/PdcmR expression ratio. From our results this appears more statistically relevant in stationary phase. The cut off for exponential/stationary phase was set at 600 minutes as seen in the above graph.

Through analysing this new bidirectional promoter we have discovered a new tool for molecular biologists. Our new intergenic region is able to modulate different stoichiometries between genes inserted either side of it in response to the presence or absence of DcmR (available in our BioBrick BBa_K1446003). The relative stoichiometry (PdcmA/PdcmR activation) of ~0.9 being reduced to ~0.3 upon DcmR addition.

DCM interacting with DcmR - the basis for our biosensor

For the PdcmA promoter both with and without DcmR:



For the PdcmR promoter both with and without DcmR:

From these results we can see that in the direction of PdcmA DCM has a modulating effect of relieving DcmR repression thus DCM acts through depression. Interestingly, DCM appears to decrease sfGFP fluorescence in the absence of DcmR. In the direction of PdcmR DCM appears to have no significant effect on the expression of sfGFP. Therefore in the native system we suggest that DCM has a modulating effect through depression of dcmA expression. Additionally DCM shows no effect on the auto regulation of dcmR expression.

In the context of our biosensor this means that DCM is capable of modulating expression of sfGFP through interaction through the PdcmA repressor DcmR.

Ultimately we can conclude that DCM acts to encourage expression of sfGFP through derepression: consistent with our double repression model.

Discussion:

The results displayed here demonstrate the repression nature of DcmR on PdcmA and PdcmR bidirectional promoter. The modulation by DCM on the PdcmA promoter we have observed offers a constructive route towards a Dichloromethane biosensor. In this way we are able to use a reporter gene downstream of PdcmA to respond to the concentration of DCM.

Additionally the nature of the bidirectional promoter offers a uniquely useful tool to molecular biologists. The relative stoichiometry of two genes inserted either side of the bidirectional promoter can be can be modulated through the addition of DcmR (BBa_K1446003 - Oxford iGEM). This can be utilised easily for investigations into the effect of protein stoichiometries between two interacting proteins.

For the bioremediation aspect of DCMation, we managed to achieve the following:

DCMA-sfGFP is expressed in E. coli

Figure 1
(A)A western blot showing the expression of a protein at 62 kDa (the calculated molecular weight of DCMA-sfGFP). The existence of some protein product at 0 mM IPTG suggests that the promoter is slightly ‘leaky’ and allows a basal expression level when no inducer is present.
(B) The image shows the expression of GFP under blue LED light in E. coli at various concentrations of the inducer IPTG. The graph quantifies the observed fluorescence, correcting for differences in optical density at 600 nm. Expression significantly increases with IPTG concentration as expected well above the basal level at 0 mM IPTG

Proteins can be targeted to microcompartments

Proteins can be inserted into the microcompartment when they have an N-terminal targeting sequence. In order to show that this targeting mechanism works for our constructs, we have constructed a plasmid with sfGFP fused to the N-terminal tag. When transforming this vector into E.coli along with the pUNI-ABTUNJK vector, we observed high-density fluorescence spots in those cells that expressed both the microcompartment and microcompartment-tagged sfGFP. This is shown in the image on the left, whereas the image on the right shows a control sample without the pUNI-ABTUNJK vector, thus expressing sfGFP evenly in the cell.





The details for the experimental procedure can be found here.

1. Express ABTUNKJ in E. Coli
3. Proof of successful mechanism for targeting into microcompartments by fluorescence microscopy
4. Assay design - successful assay set-up

We also developed the following models:

1. Microcompartment Shape Model (see 'Predicting the microcompartment structure')
2. Prediction of number of enzymes per microcompartment (see 'Modelling the number of enzymes in a microcompartment')
3. Effect of microcompartments on collision rates (see 'Microcompartment rate of collision model')
4. Stochastic diffusion of formaldehyde within microcompartments (see 'Modelling the diffusion of formaldehyde inside the microcompartment')
5. Star-peptide model (see 'The Star-Peptide Model')

Realisation Results:

For the containment of our bacteria, we have managed to:

1. synthesise novel agarose beads that have a polymeric coating which limits DCM diffusion into the beads. This allows optimum degradation by the bioremediation bacteria, while physically containing the bacteria for safety reasons
2. verify the functioning of the biopolymeric beads by measuring diffusion using indigo dye 3. use computer-aided modelling to design a prototype of the DCMation system, and physically constructed this container
4. 3D print a cartridge to hold our biosensor bacteria, which can easily be replaced by the user
5. construct a prototype circuit that lights up when the photodiodes detect light emission from our biosensing bacteria that are contained in the cartridge. This lets the user have a simple yes/no response to whether the contents of the container are safe for disposal.