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| − | <h2> Project Description - Cyanobacteria AND Gate (BioLogic Gate) </h2>
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| − | <p>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 <i> Synechocystis </i> 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 <i> Synechocystis </i> sp. PCC 6803 (herein referred to as <i> Synechocystis </i>) and the quorum sensing mechanism from <i> Vibrio fischeri </i>. </p>
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| − | <p> <i> Synechocystis </i> 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 <i> Synechocystis </i> 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 <i> LrtA </i>
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| − | as well as a green light induced promoter called <i> cpcG2 </i>. Both <i> LrtA </i> and <i> cpcG2 </i> promoters are found in the genome of <i> Synechocystis </i>. 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.
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| − | <p> 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 <i> Vibrio fischeri </i> uses quorum sensing to provide bioluminescence for the bobtail squid. Bioluminosity isn’t easily visible until the <i> V. fischeri </i> 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 <i> Synechocystis </i>. We chose to implement the <i> luxR-luxI </i> intergenic region from <i> V. fischeri </i> strain MJ1S because its promoters and RBS result in considerably high expression of bioluminescence (Bose et al. 2011). </p>
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| − | <p> 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 <i> luxR-luxI </i> quorum sensing system allowing product expression only at a threshold population density. </p> | + | <div class="clear"></div> |
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| − | </div> | + | <p style="float: right; padding-right: 215px"> What if the bacteria in this pond could be harnessed to create biofuels?</p> |
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| | + | <p>Lights, Quorum, Action! Boolean logic is used in computer processes by stipulating necessary inputs to |
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| | + | produce a desired outcome. We designed a logic gate in Synechocystis sp. PCC 6803 to optimize product |
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| | + | production. Utilizing the Boolean operator AND, gene expression accommodates the organism’s natural |
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| | + | metabolic regulation combined with the quorum sensing mechanism from Vibrio fischeri to create an |
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| − | <h2> Works Cited </h2>
| + | autoinduction system. With the cost of large scale production in mind, our system eliminates the need for |
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| − | <p> 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. </p>
| + | expensive induction molecules, such as IPTG. Under the control of light and a quorum of cells, the T7 |
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| − | <p> 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 </p>
| + | promoter, from T7 bacteriophage, drives production of a wide range of products from biofuels to |
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| − | <p> 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. </p>
| + | pharmaceuticals. CyanoLogic, coming soon to a lab near you!</p> |
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| − | <p> 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 </p>
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| − | <p> 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 </p>
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What if the bacteria in this pond could be harnessed to create biofuels?
Lights, Quorum, Action! Boolean logic is used in computer processes by stipulating necessary inputs to
produce a desired outcome. We designed a logic gate in Synechocystis sp. PCC 6803 to optimize product
production. Utilizing the Boolean operator AND, gene expression accommodates the organism’s natural
metabolic regulation combined with the quorum sensing mechanism from Vibrio fischeri to create an
autoinduction system. With the cost of large scale production in mind, our system eliminates the need for
expensive induction molecules, such as IPTG. Under the control of light and a quorum of cells, the T7
promoter, from T7 bacteriophage, drives production of a wide range of products from biofuels to
pharmaceuticals. CyanoLogic, coming soon to a lab near you!
