Team:ETH Zurich/Part Collection

"What I cannot create I do not understand."
- Richard Feynmann

Part Collection

Synthetic promoter collection responsive to L-lactate

Figure 1: Lactate sensor design

One central point of our system, the detection of lactate produced by cancer cells, depends on the sensitivity of the LldR-promoter which is responsive to L-lactate. The LldR-promoter is part of the E. Coli LldPRD operon, which is used by the bacteria for the metabolic utilization of lactate. LldR is a regulatory protein that binds to the two operators of the LldR-promoter, O1 and O2. This interaction leads to formation of a DNA loop, preventing any transcription from the following open reading frame.

To tune our system towards an optimal response to L-lactate in the surroundings of our bacteria, we decided to modify the natural version of the LldR-promoter (BBa_K822000) to reach the highest possible ON/OFF ratio and a KM value consitstent with the expected levels of lactate. If the KM value of the promoter is too high, transcription will always be supressed even in the presene of lactate, which would result in a reduced sensitivity of our system. If the promoter KM value is too low, already basal levels of lactate will be able to trigger gene expression from the regulated ORF, resulting in an increased number of false positives. To decide the optimal architecture of our regulatory system, we designed nine promoters suposedly regulated by LldR and lactate. We based our design on the natural version of the LldR-operon promoter which was already published as a Biobrick (BBa_K822000).

Design of synthetic LldR-promoters

In the design of our synthetic promoters, we wanted to keep several things in mind. According to literature [Aguilera, 2008] the repression of gene expression via LldR is based on DNA looping between O1 and O2. We therefrore tried to keep the distance between the two operators constant in respect to the natural version (BBa_K822000). We designed the first class of promotes 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 to avoid a regulation by other molecules.

Since the actual mechanism of action of lldR is not described in too much detail in the literature yet, we wanted to test how important the arrangement of operators and promoter is. We therefore designed a second and a third group of promoters where the promoter itself 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 [Aguilera, 2008]. There is evidence that two molecules of lldR bind to the two operators, but the model for DNA looping was developped in analogy to other operators of the same family [Aguilera, 2008]. From this point of view, it might well be possible to conserve repression even without proper spacing.

Regulatory system design

BioBrick

Comments
lldRO1-plldR-lldRO2 BBa_K822000 Wild-type promoter found in E. coli, published in 2012 by NTNU Trondheim. Check our characterization of this promoter!
BBa_K1847007 Promoter designed keeping the original architecture and changing the wild-type promoter by a strong Anderson promoter (BBa_J23100)
BBa_K1847008 Promoter designed keeping the original architecture and changing the wild-type promoter by a weak Anderson promoter (BBa_J23117)
BBa_K1847009 Promoter designed keeping the original architecture and changing the wild-type promoter by a medium Anderson promoter (BBa_J23118)
BBa_K1847005 Promoter designed substituting the promoter by a non-functional DNA sequence, with an Anderson promoter placed in front of the operators (BBa_J23117)
BBa_K1847006 romoter designed substituting the promoter by a non-functional DNA sequence, with an Anderson promoter placed in front of the operators (BBa_J23118)
BBa_K1847002 In this design, the spacing between the two operator sites was removed, and the Anderson promoter was placed in front of the operators (BBa_J23100)
BBa_K1847003 In this design, the spacing between the two operator sites was removed, and the Anderson promoter was placed in front of the operators (BBa_J23117)
BBa_K1847004 In this design, the spacing between the two operator sites was removed, and the Anderson promoter was placed in front of the operators (BBa_J23118)

Nomenclature

O1: operator O1 of lldR-promoter. O2: operator O2 of lldR-promoter. R2oDNA: biologically neutral DNA spacer. J23100, J23118 and J23117 are part of the Anderson promoter collection.

Double responsive promoter

The second step of our system, which is the basis of our fold change sensor, is a combined promoter inhibited both by LldR and LacI. To establish this, we enhanced our promoter collection applying the same principles, but instead of introcucing an Anderson promoter, we placed different versions of plac and the LacO operator between the two LldR-operators. Again, we tested our promoters for tehir response to various levels of lactate and IPTG and chose the promoter with the best KM value for our final system. To establish a complete contribution to the registry that will help future iGEM teams with the establishment of their systems, we also include the LacI responsive single promoter in our collection.

Regulatory system design

BioBrick

Comments
BBa_K1847010 The original PlldR was substituted by Plac and lacO.
BBa_K1847011 The original PlldR was substituted by PlacUV5 and lacO.
BBa_K1847012 The original PlldR was substituted by a spacer and Plac and lacO were located in front of the promoter.
BBa_K1847013 Plac and lacO
BBa_K1847014 PlacUV5 and lacO

Nomenclature

O1: operator O1 of lldR-promoter. O2: operator O2 of lldR-promoter. R2oDNA: biologically neutral DNA spacer. plac: lac promoter. lacO: lac operator. placUV5: enhanced lac promoter with strong expression in the absence of activation by cAMP.

Impact on future iGEM Teams

With this part collection we did not only manage to characterize the mechanism of repression and activation of the lldR-operator promoter in response to LldR and L-lactate, but we also provide future iGEM teams with a functionning and well characterized system for lactate detection by E. coli. We engineered an lldR-promoter with a higher ON/OFF ratio (15.26) and KM value (1075 μM) in comparison to the wild type version's ON/OFF ratio of 10.35 and KM value of 955 μM. In the analysis of our synthetic promoter's response to lactate, we realized that for the optimal functionning of the lldR-promoter, the promoter flanked by the two operators has to be weak, to allow activation next to derepression. The introduction of a strong or medium strong Anderson promoter did not allow for activation above levels of derepression, whereas the introduction of a weak promoter lead to the desired Results. Keeping this in mind, we suggest that the ON/OFF ratio and the sensitivity of the lldR-promoter can be further adapted to the values optimal for a different system, simply by introduction of weaker or slightly stronger promoters.

We would like to thank our sponsors