Team:ETH Zurich/Part Collection

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• About Us
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• Project
  • Project Description
  • Design
  • Applications
  • Achievements
  • Collaborations
  • Glossary
  • References
• Modeling
  • Overview
  • Lactate Module
  • AHL Module
  • Compartment Model
  • Reaction-Diffusion
  • Experiments Meet Modeling
  • Parameters
• Lab
  • Overview
  • Results
  • Chip Design
  • Experiments
  • Interlab Study
  • Safety
  • Notebook
  • Materials
• Human
  Practices
  • Overview
  • Medicine
  • Education
  • Awareness
  • Ethics
  • Business
  • Patents
• Parts
  • Overview
  • Basic
  • Composite
  • Part Collection
  • Characterization
• About Us
  • Team
  • Attributions
  • iGEM Team Roster

"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 (here represented as O_lldR in Figure 1). 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 to reach the highest possible ON/OFF ratio and a K_M value consitstent with the expected levels of lactate. If the K_M 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 K_M 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 et al, 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. 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
	BBa_K1847002
	BBa_K1847003
	BBa_K1847004
	BBa_K1847005
	BBa_K1847006
	BBa_K1847007
	BBa_K1847008
	BBa_K1847009

Nomenclature

O1: operon O1 of lldR. O2: operon O2 of lldR. R2oDNA: biologically neutral DNA. J23100, J23118 and J23117 make reference to the Anderson promoters

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. (represented as O_lldR-O_lac in Figure 1). Again, we tested our promoters for tehir response to various levels of lactate and IPTG and chose the promoter with the best K_M 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
	BBa_K1847011
	BBa_K1847012
	BBa_K1847013
	BBa_K1847014

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.

We would like to thank our sponsors