Team:CSU Fort Collins
Project Description - Cyanobacteria AND Gate (BioLogic Gate)
Boolean logic is largely used in computer science but can be applied to various other disciplines. It regulates the occurrence of desired outcomes by stipulating necessary inputs. One operation from this system is AND. In its simplest form, an AND gate requires two simultaneous inputs in order for an output to be produced. An example of this operation is a driver moving a car forward. For this situation, the engine must be on AND the gearbox must be in drive before pushing the gas pedal will allow the car to move forward. Adhering to Boolean logic, our goal is to create a modular genetic circuit in the cyanobacterium Synechocystis sp. PCC 6803 to optimize expression of a desired product. Additionally, we want to eliminate the need for expensive induction molecules such as IPTG. To accomplish these goals, we selected two conditions to implement in an AND gate and regulate gene expression: light and a quorum of cells. These conditions make use of natural mechanisms of Synechocystis sp. PCC 6803 (herein referred to as Synechocystis ) and the quorum sensing mechanism from Vibrio fischeri .
Synechocystis is a photosynthetic bacterium which has the ability to react diurnally (Labiosa 2006, Beck 2014), suggesting that it is able to change central metabolic flux following a circadian clock. In fact, studies using constant light found that about 2% to 9% of the organism's genes change following a circadian rhythm (Kucho 2005). Using data collected by Beck et al. about the relative abundance of transcripts during light-dark cycles, we identified two loci in the genome of Synechocystis that show peak abundance during the dark cycle. From these two loci, we aim to extract the promoter region and compare them to a known dark induced promoter called LrtA as well as a green light induced promoter called cpcG2 . Both LrtA and cpcG2 promoters are found in the genome of Synechocystis . After characterizing the four promoter regions, we will chose the one that expresses best during a dark cycle for further use in our AND gate.
According to Merriam-Webster, a quorum is defined as ”the smallest number of people who must be present at a meeting in order for decisions to be made.” For our project, we are construing a quorum as the lowest density cell culture at which a product can be optimally produced. Quorum sensing is a communication mechanism bacteria may use to assess population density before allowing gene expression to occur. The bacterium Vibrio fischeri uses quorum sensing to provide bioluminescence for the bobtail squid. Bioluminosity isn’t easily visible until the V. fischeri population reaches a certain density, so to conserve resources, the population will act as a group and drive gene expression only when a quorum is met (Waters and Bassler 2005). Similarly, we want to optimize production in Synechocystis . We chose to implement the luxR-luxI intergenic region from V. fischeri strain MJ1S because its promoters and RBS result in considerably high expression of bioluminescence (Bose et al. 2011).
The first portion of our AND gate will consist of a dark induced promoter driving expression of an antisense RNA (asRNA) that is complementary to our desired product. The asRNA interferes with translation of the product if light is not present. In the second portion, we have the luxR-luxI quorum sensing system allowing product expression only at a threshold population density.
Works Cited
Beck C, Hertel S, Rediger A, Lehmann R, Wiegard A, Kölsch A, Heilmann B, Georg J, Hess WR, Axmann IM. 2014. Daily expression pattern of protein-encoding genes and small noncoding RNAs in Synechocystis sp. strain PCC 6803. Appl Environ Microbiol 80:5195–5206. 10.1128/AEM.01086-14.
Bose, J. L., Wollenberg, M. S., Colton, D. M., Mandel, M. J., Septer, A. N., Dunn, A. K., & Stabb, E. V. (2011). Contribution of Rapid Evolution of the luxR-luxI Intergenic Region to the Diverse Bioluminescence Outputs of Vibrio fischeri Strains Isolated from Different Environments. Applied and Environmental Microbiology, 77(7), 2445-2457. doi:10.1128/aem.02643-10
Kucho K, Okamoto K, Tsuchiya Y, Nomura S, Nango M, Kanehisa M,Ishiura M. 2005. Global analysis of circadian expression in the cyanobacterium Synechocystis sp. strain PCC 6803. J. Bacteriol. 187:2190 –2199. http://dx.doi.org/10.1128/JB.187.6.2190-2199.2005.
Labiosa, R. G., Arrigo, K. R., Tu, C. J., Bhaya, D., Bay, S., Grossman, A. R. and Shrager, J. (2006), EXAMINATION OF DIEL CHANGES IN GLOBAL TRANSCRIPT ACCUMULATION IN SYNECHOCYSTIS (CYANOBACTERIA). Journal of Phycology, 42: 622–636. doi: 10.1111/j.1529-8817.2006.00217.x
Waters, C. M., & Bassler, B. L. (2005). QUORUM SENSING: Cell-to-Cell Communication in Bacteria. Annual Review of Cell and Developmental Biology Annu. Rev. Cell Dev. Biol., 21(1), 319-346. doi:10.1146/annurev.cellbio.21.012704.131001
