Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
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This page is different to the "Applied Design Award" page. Please see the <a href="https://2017.igem.org/Team:AQA_Unesp/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
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Since we are working with insulin and diabetes, we wanted our bacteria to produce insulin in response to the glucose concentration in the media. To do that, we designed a control system using the carbon catabolite repression of <i>Lactococcus lactis</i> and a sRNA regulation.
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Carbon catabolite repression (CCR) is a regulatory mechanism by which bacteria regulate the expression of functions for the use of secondary carbon sources in the presence of a preferred carbon source. Whereas the transcription factor cyclic AMP receptor protein (CRP) is responsible for CCR in gram-negative bacteria, in gram-positive bacteria the CCR is mediated by the carbon catabolite control protein A (CcpA). In <i>L. lactis</i>, a transcriptomic analysis revealed that Ccpa regulates genes of carbon metabolism and also its own expression [1].
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<h5>What should this page contain?</h5>
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To cause CCR, CcpA must bind to a specific palindromic sequence in the promoters regions of catabolic operons, called catabolite responsive elements (cre sites) [2]. Figure 1 shows the general CCR in gram-positive bacteria.
<p class="figure-caption">Figure 1. Phosphorylation of HPr occurs when intracellular concentrations of fructose-1,6-bisphosphate (FBP) and ATP are high, which reflects the presence of preferred carbon sources. The phosphorylated HPr(His-P) binds to the CcpA protein and this complex binds to cre site on the DNA and then represses the transcription of catabolic genes [2].</p>
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The CCR mechanism in <i>L. lactis</i> works as an inverter (NOT gate): when there is glucose, the gene expression is repressed, and when there is no glucose, the gene expression is activated. In order to regulate the expression of insulin in our genetically engineered machine by glucose we needed to invert this system, so we designed a circuit using the CCR mechanism regulating the expression of a small RNA (sRNA) that blocks the translation of insulin. That way, when there is glucose, the sRNA will not be expressed and the insulin will be expressed. When there is no glucose, the sRNA will be expressed and it will repress the expression of insulin.
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Our designed system uses the sfGFP(Bs) [colocar codigo BB] as gene reporter, previously inserted into the pSEUDO plasmid [ref], which was used in all of our constructions. So, we designed the sRNA to target the sequence of the sfGFP mRNA that goes from the RBS to the start codon to ensure its specificity of to its target and an efficient hybridization.
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To design the sRNA, we used the software RiboMaker [ref], which gave us the sequence, hybridization energy values and the possible secondary structure of the sRNA. Analyzing the data, we selected the best two results and then performed a new analysis using the software NUPACK [ref]. By this new analysis we were able to choose the best sRNA and build our final construction. To learn more, see our modeling section [link].
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Our final construction was then composed by:<br>
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<li>Explanation of the engineering principles your team used in your design</li>
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<li>the gal operon promoter, which contains the cre site and has already been well studied [3]** explicar melhor??</li>
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<li>Discussion of the design iterations your team went through</li>
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<li>the designed sRNA</li>
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<li>Experimental plan to test your designs</li>
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<li>the terminator T1 from E. coli rrnb [BB code] and its optimized version for B. subtilis [BB code]</li>
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Figure 2 shows our construction and its mechanism of action.
[1] Zomer et. al. Time-Resolved Determination of the CcpA Regulon of Lactococcus lactis subsp. cremoris MG1363. Journal of Bacteriology. vol. 189, p. 1366-1381. American Society for Microbiology, 2007.<br>
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[2] Görke, B.; Stülke, J. Carbon catabolite repression in bacteria: many ways to make the mos out of nutrients. Nature Reviews: Microbiology. vol. 6, p. 613-624. Macmillan Publishers Ltd, 2008.<br>
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[3] Luesink et. al. Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Molecular Microbiology. vol. 30, n. 4, p. 789-798. Blackwell Science Ltd, 1998.
Since we are working with insulin and diabetes, we wanted our bacteria to produce insulin in response to the glucose concentration in the media. To do that, we designed a control system using the carbon catabolite repression of Lactococcus lactis and a sRNA regulation.
Carbon catabolite repression (CCR) is a regulatory mechanism by which bacteria regulate the expression of functions for the use of secondary carbon sources in the presence of a preferred carbon source. Whereas the transcription factor cyclic AMP receptor protein (CRP) is responsible for CCR in gram-negative bacteria, in gram-positive bacteria the CCR is mediated by the carbon catabolite control protein A (CcpA). In L. lactis, a transcriptomic analysis revealed that Ccpa regulates genes of carbon metabolism and also its own expression [1].
To cause CCR, CcpA must bind to a specific palindromic sequence in the promoters regions of catabolic operons, called catabolite responsive elements (cre sites) [2]. Figure 1 shows the general CCR in gram-positive bacteria.
Figure 1. Phosphorylation of HPr occurs when intracellular concentrations of fructose-1,6-bisphosphate (FBP) and ATP are high, which reflects the presence of preferred carbon sources. The phosphorylated HPr(His-P) binds to the CcpA protein and this complex binds to cre site on the DNA and then represses the transcription of catabolic genes [2].
The CCR mechanism in L. lactis works as an inverter (NOT gate): when there is glucose, the gene expression is repressed, and when there is no glucose, the gene expression is activated. In order to regulate the expression of insulin in our genetically engineered machine by glucose we needed to invert this system, so we designed a circuit using the CCR mechanism regulating the expression of a small RNA (sRNA) that blocks the translation of insulin. That way, when there is glucose, the sRNA will not be expressed and the insulin will be expressed. When there is no glucose, the sRNA will be expressed and it will repress the expression of insulin.
Our designed system uses the sfGFP(Bs) [colocar codigo BB] as gene reporter, previously inserted into the pSEUDO plasmid [ref], which was used in all of our constructions. So, we designed the sRNA to target the sequence of the sfGFP mRNA that goes from the RBS to the start codon to ensure its specificity of to its target and an efficient hybridization.
To design the sRNA, we used the software RiboMaker [ref], which gave us the sequence, hybridization energy values and the possible secondary structure of the sRNA. Analyzing the data, we selected the best two results and then performed a new analysis using the software NUPACK [ref]. By this new analysis we were able to choose the best sRNA and build our final construction. To learn more, see our modeling section [link].
Our final construction was then composed by:
the gal operon promoter, which contains the cre site and has already been well studied [3]** explicar melhor??
the designed sRNA
the terminator T1 from E. coli rrnb [BB code] and its optimized version for B. subtilis [BB code]
Figure 2 shows our construction and its mechanism of action.
Figure 2.
References
[1] Zomer et. al. Time-Resolved Determination of the CcpA Regulon of Lactococcus lactis subsp. cremoris MG1363. Journal of Bacteriology. vol. 189, p. 1366-1381. American Society for Microbiology, 2007.
[2] Görke, B.; Stülke, J. Carbon catabolite repression in bacteria: many ways to make the mos out of nutrients. Nature Reviews: Microbiology. vol. 6, p. 613-624. Macmillan Publishers Ltd, 2008.
[3] Luesink et. al. Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Molecular Microbiology. vol. 30, n. 4, p. 789-798. Blackwell Science Ltd, 1998.
