Team:UCSF/Project/Circuit/Data

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<font face="arial" size = "5"><b><center>Decision-Making Circuit: Data</font></b> </center> <br>
 
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<FONT COLOR="#008000"><u>Promoter Selection and Testing: </FONT COLOR="#008000"></u><br>
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<FONT COLOR="#008000"><u>Altering Promoter Sensitivity: </FONT COLOR="#008000"></u><br>
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<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>
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<br>Our next goal after our promoter assays was to create high and low affinity versions of our promoters. We only changed the affinity of pLAC. To create our low affinity promoter we shifted the 01 operater site further downstream of our promoter sequence. We also removed another operator site from pLAC. To create our high affinity promoter we took our low affinity promoter and added the 03 operator site further upstream near the start of the promoter sequence. The 03 operator site will then form a loop with our o1 operator site thus creating our high affinity promoter.</font>
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<br>A large portion of our project was extensively <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Data">testing</a> and <a href="https://2013.igem.org/Team:UCSF/Modeling">modeling</a> promoter activity for use in the circuit, as well as designing a new <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Data">lactose-reponsive</a> promoter to sense changing levels of inducer.  
<br>A large portion of our project was extensively <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Data">testing</a> and <a href="https://2013.igem.org/Team:UCSF/Modeling">modeling</a> promoter activity for use in the circuit, as well as designing a new <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Data">lactose-reponsive</a> promoter to sense changing levels of inducer.  
<br><br>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 <a href="https://2013.igem.org/Team:UCSF/Parts">registry</a>.
<br><br>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 <a href="https://2013.igem.org/Team:UCSF/Parts">registry</a>.
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Revision as of 07:00, 27 September 2013

Decision-Making Circuit: Data

Promoter Selection and Testing:
In order to create synthetic circuit sensitive to differing levels of inducer, we first had to select promoters with 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 separate plasmids containing each of the promoters individually driving the expression of GFP, and using this system we characterized these promoters by measuring the GFP expression over time as a function of different inducer levels. We determined the basal level, highest induction level, and inducer range. Because of the low basal expression and the varying responsiveness at different inducer concentraions, we selected pLAC & pTET as the promoters for the synthetic circuit.
pLAC Driven GFP Expression

Altering Promoter Sensitivity:

Our next goal after our promoter assays was to create high and low affinity versions of our promoters. We only changed the affinity of pLAC. To create our low affinity promoter we shifted the 01 operater site further downstream of our promoter sequence. We also removed another operator site from pLAC. To create our high affinity promoter we took our low affinity promoter and added the 03 operator site further upstream near the start of the promoter sequence. The 03 operator site will then form a loop with our o1 operator site thus creating our high affinity promoter.


Design Requirements:
In order for this circuit to properly function, we had to address two main challenges:

1) identifying or constructing promoters that were differentially responsive to both high and low levels of an inducer

2) ensuring that the fluorescent proteins and gRNAs were produced in the same amount under the same promoter.

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.

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