Part Collection

Overview

Figure 1: Lactate sensor design

Our whole cancer sensor depends on the sensitivity of the promoter regulated by LldR. LldR is a regulatory protein that binds to O1 and O2 (here represented as O_lldR in Figure 1)and forms a loop, avoiding any further transcription. If the promoter is too strong it will supress the transcription even in the presene of lactate, which will result in a reduced sensitivity of our system. If the promoter is too weak, it can result in an increased number of false positives. To decide the optimal arrangement of our regulatory system, we designed nine promoters by rearranging O1, O2 and a promoter.

Design of synthetic lldR-promoters

In the design of our synthetic promoters, we wanted to keep several things in mind. According to literature ref the repression of gene expression via lldR is based on DNA looping. We therefrore tried to keep the distance between the two operators O1 and O2 constant. The first class of promotes we designed by replacing the natural lldR promoter by three biobrick anderson promoters of different strenghts (J23100, J23117, and J23118), leaving the general architecture constant. In addition to that, we removed the ArcA binding site because we wanted to. Since the actual mechanism of action of lldR is not described yet, we wanted to test how important the arrangement of operators and promoter is. We therefore designed a secon and a third group of promoters where the promoter it self is not flanked by the operator, but is positioned upstream of the operators. DNA looping might in that case still allow to block transcription simply by blocking progression of the RNA Polymerase. In the second set of promoters we kept the disctance between the operators O1 and O2 at the same lenght as they are in the natural verison. To this end we introduced the spacer R2oDNA, which we optimized for lack of restriction or interaction sites. In the third and last group of promoters we removed the spacer between the two operators, having them next to eachother without and space in between them. With this setup we wanted to investigate wether or not the mechanism of repression by lldR is really based on DNA looping or just of binding to the operators. check this:The only thing that is really known is that two molecules of lldR bind to the two operators. The model for DNA looping was developped in analogy to other operators of the same family. From this point of view, it might well be possible to conserve repression even without proper spacing.

Nomenclature

lldRO1: operon O1 of lldR. lldRO2: operon O2 of lldR. R2oDNA: biologically neutral DNA.

The second step of our system is a GFP protein inhibited both by LldR and LacI. To do that, we designed a series of regulators in which we combined lldR and lacI operators to find the optimal path for our system (represented as O_lldR-O_lac in Figure 1).

Nomenclature

lldRO1: operon O1 of lldR. lldRO2: operon O2 of lldR. R2oDNA: biologically neutral DNA. plac: lac promoter. lacO: lac operon. placUV5: enhanced lac promoter with strong expression in the case of lack of activation by cAMP.

Design of synthetic lldR-promoters

We designed these promoters keeping in mind a lot of things like spacing and stuff.