"
Team:UCSF/Project/Circuit/Data
From 2013.igem.org
(Difference between revisions)
| Line 28: | Line 28: | ||
<font face="arial" size = "4"> | <font face="arial" size = "4"> | ||
| - | + | <FONT COLOR="#008000"><u>Promoter Selection and Testing: </FONT COLOR="#008000"></u><br> | |
| + | In order to create synthetic circuit sensitive to differing levels of inducer, we first had to select promoters had favorable characteristics we could exploit to make them concentration-dependent, namely a low basal level and a wide dynamic range. We chose four well-known promoters as possible candidates for our circuit: pLAC (lactose), pTet (tetracycline), pBAD (arabinose), and PprpB (proprionate). We constructed plasmids containing each of the promoters individually driving the expression of GFP, and using this system we characterized these promoters by measuring their expression over time with different inducer levels. We determined the basal level, highest induction level, and inducer range. Based on our promoter assays, we found that pLAC & pTET were the best promoters to use in our synthetic circuit.<br> | ||
<br>Our synthetic circuit has been engineered to give cells a decision making ability between differential outputs and will utilize CRISPRi as a switching mechanism between these outputs. Depending on a high or low amount of chemical signal, our cells can produce either RFP or GFP. If there was a low amount of chemical signal, GFP and a gRNA to RFP will be produced. The gRNA will then combine with dCas9, which has been incorporated on a separate plasmid. The dCas9 will then repress the RFP. The inverse follows the same principle; if suddenly the chemical signal increases, RFP and gRNA to GFP will be produced. The gRNA will combine with dCas9 and repress GFP expression.</font> | <br>Our synthetic circuit has been engineered to give cells a decision making ability between differential outputs and will utilize CRISPRi as a switching mechanism between these outputs. Depending on a high or low amount of chemical signal, our cells can produce either RFP or GFP. If there was a low amount of chemical signal, GFP and a gRNA to RFP will be produced. The gRNA will then combine with dCas9, which has been incorporated on a separate plasmid. The dCas9 will then repress the RFP. The inverse follows the same principle; if suddenly the chemical signal increases, RFP and gRNA to GFP will be produced. The gRNA will combine with dCas9 and repress GFP expression.</font> | ||
</div> | </div> | ||
Revision as of 06:50, 27 September 2013
Promoter Selection and Testing:
In order to create synthetic circuit sensitive to differing levels of inducer, we first had to select promoters had favorable characteristics we could exploit to make them concentration-dependent, namely a low basal level and a wide dynamic range. We chose four well-known promoters as possible candidates for our circuit: pLAC (lactose), pTet (tetracycline), pBAD (arabinose), and PprpB (proprionate). We constructed plasmids containing each of the promoters individually driving the expression of GFP, and using this system we characterized these promoters by measuring their expression over time with different inducer levels. We determined the basal level, highest induction level, and inducer range. Based on our promoter assays, we found that pLAC & pTET were the best promoters to use in our synthetic circuit.
Our synthetic circuit has been engineered to give cells a decision making ability between differential outputs and will utilize CRISPRi as a switching mechanism between these outputs. Depending on a high or low amount of chemical signal, our cells can produce either RFP or GFP. If there was a low amount of chemical signal, GFP and a gRNA to RFP will be produced. The gRNA will then combine with dCas9, which has been incorporated on a separate plasmid. The dCas9 will then repress the RFP. The inverse follows the same principle; if suddenly the chemical signal increases, RFP and gRNA to GFP will be produced. The gRNA will combine with dCas9 and repress GFP expression.
Design Requirements:
In order for this circuit to properly function, we had to address two main challenges:
A large portion of our project was extensively testing and modeling promoter activity for use in the circuit, as well as designing a new lactose-reponsive promoter to sense changing levels of inducer.
To address the second issue, we made strategic design choices and utilized an RNA-cutting enzyme called Csy4 in order to equivalently express both the fluorescent protein and guideRNA for each part of the circuit. Both the protein and gRNA are behind the same promoter and linked together with a sequence coding a Csy4 cut site. After transcription, the RNA product is cleaved to make both mRNA for the fluorescent protein and the gRNA. To see more of our design strategies for the guideRNAs and using Csy4, please refer to our parts submitted to the registry.
Using CRISPR to Create Scalable Circuits: texttexttexttext.
