Team:NTNU Trondheim/Project

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In order to have the presence of lactate activate the production of LuxI, we need a lactate-sensitive [http://en.wikipedia.org/wiki/Promoter_%28genetics%29 promoter]. We will attempt to isolate the lactate-sensitive promoter found in the [http://ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU164 lldPRD] operon of ''E. coli'' by PCR. However, the available literature indicates that this promoter is repressed under low-oxygen conditions [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC205257/ due to the action of the ArcA regulatory protein]. A possible solution is using site-directed mutagenesis to change the nucleotide sequence at the probable binding site of ArcA on the promoter to reduce its binding affinity and abolish its regulatory effect.
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In order to have the presence of lactate activate the production of LuxI, we need a lactate-sensitive [http://en.wikipedia.org/wiki/Promoter_%28genetics%29 promoter]. We will attempt to isolate the lactate-sensitive promoter found in the [http://ecocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU164 lldPRD] operon of ''E. coli'' by PCR. However, the available literature indicates that this promoter [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC205257/ is repressed under low-oxygen conditions] due to the action of the ArcA regulatory protein. A possible solution is using site-directed mutagenesis to change the nucleotide sequence at the probable binding site of ArcA on the promoter to reduce its binding affinity and abolish its regulatory effect.
==Challenges==
==Challenges==

Revision as of 20:21, 1 July 2012

NTNU IS B.A.C.K.
Bacterial Anti-Cancer-Kamikaze

Project description

Contents


Overall project - BACK: "Bacterial Anti-Cancer Kamikaze"

Our goal for this years competition is to create a genetic circuit which enables E. coli cells to detect and attack cancer cells. To do this, the cells should produce a tumor inhibitor or toxin effective at killing cancer cells, and respond to environmental cues indicating the presence of cancer cells by undergoing lysis, releasing the anti-cancer agent into the surrounding environment. In principle, bacteria could be used in "search and destroy" missions against cancer inside the human body. We wish to develop our genetic circuit as a proof-of-concept of one such strategy.


A sketch of our current planned design for the circuit is shown below.


A sketch of our planned genetic circuit

Ideally, the toxin should only be released if cancer cells are actually encountered, and never otherwise. To achieve this, the lysis-inducing part of the circuit should be regulated such that it is highly unlikely to activated in other situations. One possible way is to use a signal molecule that is solely associated with cancer cells to activate the lysis device. Another possibility is to use a combination of environmental cues that together indicate a high likelyhood of cancer presence. We have chosen the latter strategy, and the environmental cues we aim to use are low O2 and high lactate concentrations.

Each of the two cues activates a separate gene, which encode different proteins. Together, these two proteins should activate the unit responsible for lysis. Therefore, if only one of the cues are present, the cell does not lyse, and toxin is not released in high levels. (Some leakage of toxin must be expected even under normal conditions with no activation of the circuit). Selective activation in the presence of cancer cells would be crucial for the concept to be effective in a medical situation.

As shown in the sketch, we plan to express the toxin-producing gene with a constitutive promoter. This means that the toxin production always be on. Ideally, the toxin should be maximally effective against cancer cells and minimally toxic to the toxin-producing cell. We have evaluated several candidate molecules. At the moment, we are leaning towards using the protein Colicin E1.

Project Details

Below is a more detailed sketch of the circuit design (legend). The environmental cues oxygen and lactate regulate the production of two regulatory proteins, LuxR and LuxI. Oxygen inhibits the production of LuxR, while lactate activates the production of LuxI. LuxI in turn catalyzes the formation of a homo-serine lactone (HSL) compound from S-Adenosyl Methionine (SAM) and Hexanoyl-ACP (Hex). When both LuxR and HSL is produced, they combine irreversibly to form a complex which activates the promoter in front of the lysis device (BioBrick part <partinfo>BBa_K112808</partinfo>, designed by theUniversity of California Berkeley iGEM 2008 team).

NTNU sketch1b.jpg

In order to have the presence of lactate activate the production of LuxI, we need a lactate-sensitive promoter. We will attempt to isolate the lactate-sensitive promoter found in the lldPRD operon of E. coli by PCR. However, the available literature indicates that this promoter is repressed under low-oxygen conditions due to the action of the ArcA regulatory protein. A possible solution is using site-directed mutagenesis to change the nucleotide sequence at the probable binding site of ArcA on the promoter to reduce its binding affinity and abolish its regulatory effect.

Challenges

The goal of making bacterial cells detect and attack cancer cells present several challenges. First, how can cancer cells be reliably and effectively detected? Early in the process after deciding on the goal, we considered using several human signalling molecules such as HGF and VEGF as cancer indicators, as these are known to be over-produced by tumors. However, we were unable to determine quickly if these would be able to enter and affect E. coli cells, due to their large size. In contrast, O~2 and lactate are small molecules which are easily taken up by the cells, and are known to directly regulate the expression of various genes.

The parts

The promoters

To allow our system to work correctly, we need several different promoters. Of these, the most simple is a constitutive (always on) promoter in front of the toxin-producing gene. Second, a promoter having higher activity at low oxygen levels, similar to those found in tumors. Thirdly, a promoter that is activated by lactate, a possible indicator of tumor cells in the vicinity (or strenous exercise!). Last, a promoter that is activated by a compound resulting from the activation of the previous two units. In our sketch, this compound is a LuxR-HSL complex.


The genes

The lysis device was made by the UC Berkeley iGEM 2008 team. We will use it as-is, placing a self-chosen promoter in front of the part to control the initiation of cell lysis.

Other

For Ribosome Binding Sites and transcriptional terminators, we plan to use the standard BioBrick parts <partinfo>BBa_B0034</partinfo> and <partinfo>BBa_B0015</partinfo>


The Experiments

The coming week, starting July 2, we will begin assembling test constructs in order to test the promoters and other parts we plan to use. By testing the components by themselves before assembling the system as a whole, we will hopefully have less problems and surprises later on.

Results

References

Lactate sensitive promoter:

Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli.

LuxI:

LuxR:

Lysis device:

https://2008.igem.org/Team:UC_Berkeley/LysisDevice

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