Our Design
We utilized the existing quorum sensing system in bacteria to create a signal that propagates through a bacterial population of E. Coli DH5a upon detection of a mycotoxin produced by Fusarium oxysporum.
F. oxysporum produces a mycotoxin known as fusaric acid, and it can be detected by a fusaric acid inducible promoter (BBa_K1493002), which is composed of the PP_1262 gene and a promoter. The PP_1262 gene inhibits the binding of RNA polymerase to the promoter, subsequently preventing transcription. But fusaric acid enables transcription by blocking the inhibitory activity of the PP_1262 gene. Hence, in the presence of fusaric acid, the LuxI gene (BBa_K4952000) is transcribed and translated to produce 3-oxo-hexanoyl-HSL, an N-acyl-homoserine lactone (AHL). The 3-oxo-hexanoyl-HSL molecule produced is transported to the cell membrane by a signal peptide (BBa_K4952001) and diffuses out of the cell. This sequence also contains a his tag for protein purification purposes.
When sufficient 3-oxo-hexanoyl-HSL accumulates in the environment, it diffuses back into the R. leguminosarum cells down the concentration gradient. In the bacterial cells, the LuxR protein ( BBa_C0062) is being produced continuously by a constitutive promoter (BBa_J23119).
When 3-oxo-hexanoyl-HSL enters the bacterial cells, it binds to the LuxR protein to form a transcription factor complex that then activates the Lux pR promoter. (BBa_R0062)
This promotes the transcription and translation of the LuxI gene, which leads to more production of the 3-oxo-hexanoyl-HSL molecule in a positive feedback loop.
This eventually results in the propagation of the cell signal initiated by the detection of F. oxysporum. As this experiment isn’t being done in vivo, there is no risk of the AHL triggering pseudomonas virulence or horizontal gene transfer.
Downstream of the Lux I gene, 2 possible mutations have been engineered to act as reporter proteins for the hardware. One of them is through bacterial luciferase genes Lux A and Lux B. These are favored for a hardware application as they are chemiluminescent reactions that don't require much additional energy apart from the catalyst to oxidize the substrates. The second version of the circuit uses BRET + a circularly permuted protein( BBa_K5394228) to improve brightness along with mutations to make it less sensitive to environmental factors like pH and high temp.
In order to do tests with this circuit in the plant ecosystem ( soil) the following choices would have to be made:
- A eukaryotic chassis like yeast could be used to prevent the possibility of 3-oxo-hexanoyl-HSL horizontal gene transfer.
- However, as high concentrations of AHL have to build up in order for the circuit to work, it would be better to use an oligo peptide instead (gram-positive autoinducers)
- In order to keep balance in the ecosystem there could be an RNA thermometer at the end of the Circuit 2. This could act as a negative confirmation system. Foc’s fatality rate increases once it crosses a threshold of 37 degrees Celsius so an RNA thermometer (BBa_K115002) that only allows for transcription at temperatures above this would be expressing genes when there is less likelihood that Foc is there.
Therefore, expression downstream should act as negative feedback for the quorum sensing system. This could be done through a quorum quenching enzyme for an auto-inducer peptide.