Team:Amsterdam/test/joeri/index

Detect

From conversations with Photanol we know that, in our quest to engineer fumarate-producing cyanobacterial factories, we need a way to quickly and easily determine if a given genetically engineered construct is actually producing fumarate. For this purpose, we constructed a biosensor, i.e. an Escherichia Coli strain that is able to sense and quantitatively report the presence of fumarate in the extracellular medium.

detect

Overview

Our fumarate biosensor is based on a well-characterized chimeric two-component system. When integrated in E. coli, this system has been reported to make the cells fluoresce when fumarate is in the environment. We constructed and investigated the functionality of different variants of this system in E. coli. This way we created several variants of our necessary fumarate biosensor, while deepening our fundamental understanding of chimeric two-component systems at the same time!

Highlights

  • Engineered at least 3 functional biosensors with a combined signal dynamic range of 0.1-20 mM fumarate.
  • Investigated the effect of linker length and kinase orientation on biosensor functionality.
  • Demonstrated real world applicability.

References

  1. Hazelbauer, G. L., & Lai, W. C. (2010). Bacterial chemoreceptors: providing enhanced features to two-component signaling. Current opinion in microbiology, 13(2), 124-132.
  2. Stock, A. M., Robinson, V. L., & Goudreau, P. N. (2000). Two-component signal transduction. Annual review of biochemistry, 69(1), 183-215.
  3. Ganesh, I., Ravikumar, S., Lee, S. H., Park, S. J., & Hong, S. H. (2013). Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system. Journal of biotechnology, 168(4), 560-566.
  4. Görke, B., & Stülke, J. (2008). Carbon catabolite repression in bacteria: many ways to make the most out of nutrients. Nature Reviews Microbiology, 6(8), 613-624.
  5. Bi, S., Pollard, A. M., Yang, Y., Jin, F., & Sourjik, V. (2016). Engineering hybrid chemotaxis receptors in bacteria. ACS synthetic biology, 5(9), 989-1001.
  6. Immethun, C. M., DeLorenzo, D. M., Focht, C. M., Gupta, D., Johnson, C. B., & Moon, T. S. (2017). Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803. Biotechnology and Bioengineering.
  7. Batchelor, E., Silhavy, T. J., & Goulian, M. (2004). Continuous control in bacterial regulatory circuits. Journal of bacteriology, 186(22), 7618-7625.
  8. Bachmann, H., Fischlechner, M., Rabbers, I., Barfa, N., dos Santos, F. B., Molenaar, D., & Teusink, B. (2013). Availability of public goods shapes the evolution of competing metabolic strategies.Proceedings of the National Academy of Sciences, 110(35), 14302-14307.