Figure 3d. Fluorescence levels of iRFP 670 upon induction by a fdhf promoter differ significantly upon oxygen vs. no oxygen (hypoxic conditions); the iRFP has significantly higher fluorescence for the cultures grown in aerobic conditions.
Figure 3e. Fluorescence levels of iRFP 713 upon induction by a fdhf promoter differ significantly upon oxygen vs. no oxygen (hypoxic conditions); the iRFP has significantly higher fluorescence for the cultures grown in aerobic conditions.
**For materials and methods for hypoxia assay, see July and October lab notebook
Violacein
The current model is not able to show the expected dependence of violacein yield on promoter strength. After reevaluating our assumptions, we identified some potential flaws of the model that might cause the unexpected results.
One of the assumptions from our model is that the rate of production of L-tryptophan is constant and independent of the promoter strength. Jones el al. suggest that the L-tryptophan production rate may be affected by the metabolic burden of the production of the recombinant enzymes (VioA, VioB, etc.). This phenomenon may be caused by the depletion of essential metabolic resource, such as amino acids, mRNA and ATP. Therefore, the L-tryptophan production rate might need to be dependent on enzymes production rates.
Another effect that we didn’t consider is the saturation of the enzymes. To improve our model, we could include these effects by employing Michaelis-Menten Kinetics equations in our next step. Nevertheless, we have been cautious about including this in our model, since increasing the number of parameters, without increasing the number of data points usually causes the overfitting of the model.
Finally, since the violacein pathway has not been fully characterized, it is possible that we ignored some reactions in the complete pathway. Moreover, there may be feedback loops that regulate the pathway. We will need to investigate these possible components and incorporate them into our model if they prove to be present in the pathway.
Conclusion
Here we present a method to fit a model of violacein production in E.coli to experimental data of violacein yield with different promoters using nonlinear regression. Although it fails to calculate the dependence on promoter strength, our model is able predict the average violacein concentration. We expect that small changes on the model, such as including a L-tryptophan production dependence of the metabolic burden, would allow us to successfully predict the violacein production in response to the variation of promoter strength. Once the predictive model is complete, we will be able to find the strains that lead to optimal violacein yield computationally.
References
Carvalho, D. D., Costa, F. T. M., Duran, N., & Haun, M. (2006). Cytotoxic activity of violacein in human colon cancer cells. Toxicology in Vitro, 20(8), 1514–1521. http://dx.doi.org/10.1016/j.tiv.2006.06.007
Jones, J. A., Vernacchio, V. R., Lachance, D. M., Lebovich, M., Fu, L., Shirke, A. N., … Koffas, M. A. G. (2015). ePathOptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways. Scientific Reports, 5, 11301. http://doi.org/10.1038/srep11301
Lee, M. E., Aswani, A., Han, A. S., Tomlin, C. J., & Dueber, J. E. (2013). Expression-level optimization of a multi-enzyme pathway in the absence of a high-throughput assay. Nucleic Acids Research, 41(22), 10668–10678. http://doi.org/10.1093/nar/gkt809
Hypoxia induced fluorescence
In addition to the sinduction of iRFP fluorescence by nitric oxide, we also tested the induction of iRFP fluorescence with a hypoxia promoter. We expected iRFP fluorescence to increase with increased hypoxic conditions (less oxygen) when using NarK promoter and fdhf promoters, both characterized as hypoxia-inducible.
Transcription of the fdhf promoter is regulated by an RNA polymerase with sigma factor 54 whose binding is dictated by presence of an additional activator complex consisting of FhlA and formate. Only when the FhlA-formate complex is present will the sigma-54 polymerase initiate transcription. This process is induced by formate, but is also heavily repressed by presence of oxygen, giving it characterization as a hypoxia sensor.
Figure 3d. Fluorescence levels of iRFP 670 upon induction by a fdhf promoter differ significantly upon oxygen vs. no oxygen (hypoxic conditions); the iRFP has significantly higher fluorescence for the cultures grown in aerobic conditions.
Figure 3e. Fluorescence levels of iRFP 713 upon induction by a fdhf promoter differ significantly upon oxygen vs. no oxygen (hypoxic conditions); the iRFP has significantly higher fluorescence for the cultures grown in aerobic conditions.
