Team:UCSF/Project/Circuit/Data

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CRISPRi Decision-Making Circuit: Promoter Design and Data

Summary: We have designed a CRISPRi circuit that can choose between two outcomes based on the level of input This circuit is also scalable (i.e. many parallel circuits can be deployed in the same cell) due to the use of CRISPRi gRNA's

Before we could create promoters that respond to a high and low amount of inducer, we first had to select promoters with two favorable characteristics we could exploit to make them concentration-dependent which would be a low basal level and a wide dynamic range. We chose four well-known promoters as possible candidates for our circuit: pLAC, pTet, pBAD, and PprpB. We constructed separate plasmids containing each of the promoters and characterized these promoters by measuring the GFP expression over time. We determined the basal level, highest induction level, and inducer range. We selected pLAC & pTET as the promoters for the synthetic circuit, because of their low basal expression and the varying responsiveness at different inducer concentrations.

Dose-response curve for pLAC promoter induced with different amount of inducers (0, 0.1, 1, 10, 20, 40, 60, 80, 120 uM IPTG). Cells were grow to mid-log phase and then start induction. OD600 value and GFP fluorescence level of each sample were measured by plate reader after saturation. GFP fluorescence were corrected for OD600 value. The red line indicated Hill function fit of the dose-response curve and error bars indicate standard deviation calculated on the basis of technical replicates.

Dose-response curve for pTET promoter induced with different amount of inducers (0, 0.004, 0.01, 0.04, 0.06, 0.11 uM aTc). Cells were grow to mid-log phase and then start induction. OD600 value and GFP fluorescence level of each sample were measured by plate reader after saturation. GFP fluorescence were corrected for OD600 value. The red line indicated Hill function fit of the dose-response curve and error bars indicate standard deviation calculated on the basis of technical replicates.

Engineering pLAC Sensitivity

Our next goal after our promoter assays was to create engineered versions of our promoters responsive to high and low levels of inducer, either by modifying activation or repression of the specific promoter. Based on previous work in the literature characterizing the pLAC promoter, we chose to change both the number and orientation of repressor binding sites in pLAC.

From Oehler et al., 2006.

To create our "low" inducer promoter, we shifted the O1 operator site further downstream of our promoter sequence and removed another operator site from pLAC to relieve repression. To create our "high" promoter, we took the "low" promoter and added the O3 operator site further upstream near the start of the promoter sequence. The O3 operator site will then form a loop with our O1 operator site, thus creating a physical barrier to prevent transcription, reducing the promoter activity at low levels of inducer.

On this diagram (A) denotes the very original pLAC which is found in the parts registry. (B) is our re-engineered pLAC that has an added O3 binding site. We found in literature that by adding an O3 binding site, a higher inducer level is required to start transcription for a loop to form between the 03 and 01 binding sites. We expect we would need more inducer to produce similar levels of GFP.

When we engineered the pLAC promoter, the dynamic range is insignificantly different. This graph shows that the green fluorescent protein is expressing at the same inducer concentration for both of the promoters. So we went back to the drawing board and redesigned the pLAC promoter.

Improved Designs for High & Low Sensitivity Promoters

In the diagram below, (C) is our low promoter and (D) is our high promoter. To create our low inducer promoter, we shifted the O1 operator site further downstream of our promoter sequence and removed another operator site from pLAC to relieve repression. To create our high promoter, we took the low promoter and added the O3 operator site further upstream near the start of the promoter sequence. The O3 operator site will then form a loop with our O1 operator site, thus creating a physical barrier to prevent transcription, we require higher levels of inducer for expression.

The sequence of four different pLAC promoters constructed for the purpose of high and low sensor. (A) was the original pLAC promoter, (B) was the engineered one that have reduced expression level, (C) and (D) was the expected low and high sensor respectively. Yellow region of each promoter indicated its -35 and -10 core region for RNA polymerase binding, the red characters of each promoter indicates +1 start point of transcription, and the blue square indicated different LacI binding sites (LacO1 and LacO3).

Our second round of promoter engineering shows that the outputs are close to our desired outputs. However, the sensitivity of the promoters did not change. Though we did not get our desired high and low promoters, we have created new promoters that will be available at the parts registry and have been fully characterized.

We also created a mathematical model of our system and were able to identify important variables and parameters that would help produce our decision making circuit.

CRISPRi Decision-Making Circuit: Circuit Output

The essential benefit of using the CRISPR system for the circuit design is that it has a very specific target -- a guideRNA contains nucleotides which are complementary to a unique DNA sequence. As a result, we can use a vast number of individual gRNAs to repress several genes, giving us the ability to design circuits to make numerous and more complex decisions.

Our proof-of-concept design is, at a low level of inducer, to express GFP simultaneously with a gRNA to repress RFP. When the inducer level increases, the "high" sensing promoter turns on expression of RFP and a gRNA to repress GFP, making a switch-like decision based on the input. We were able to construct an early design of the circuit in the pCDF plasmid that contains both fluorescent proteins and gRNAs. We initially put both components of the circuit under individual promoters, pLAC and pTET, as a control to test whether or not we could obtain expression of both products and repression from the gRNAs, both under individual induction and after switching inducers. Since this circuit relies on CRISPR repression, we had to also construct a plasmid (pACYC) containing the catalytically dead dCas9 protein as well as the Csy4 protein to cleave the RNA products (see Design page or Parts Registry for more information).

We are in the final stages of cloning the dCas9 plasmid and we hope to test the circuit soon.

@@ Line 24: / Line 24: @@
-<div id="description" style = "width:950px; height:300px" align="justify">
+<div id="description" style = "width:950px; height:30px" align="justify">
-<font face="arial" size = "5"><b><center>Decision-Making Circuit: Data</font></b> </center> <br>
+<font face="calibri" size = "5"><b><center>CRISPRi Decision-Making Circuit: Promoter Design and Data</font></b> </center> <br> </div>
-<font face="arial" size = "4">
+<div id="description" style = "width:950px; height:80px" align="justify">
-Building upon our CRISPR conjugation project, we began to think about what types of alterations we could confer to cells using the CRISPRi system. The unique capabilities of the CRISPR system allow for the design of a circuit that can achieve decision-making ability. Many synthetic circuits have been created using multiple repressors as their switch. In our circuit design <FONT COLOR="#008000"><b>we utilize guideRNAs (gRNAs) in lieu of repressors, which will allow for a highly scalable design. </b></FONT COLOR="#008000"><br>
+<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>
+<font face="calibri" size = "5">
-</div>
+<b><FONT COLOR="#008000">Summary: We have designed a CRISPRi circuit that can choose between two outcomes based on the level of input This circuit is also scalable (i.e. many parallel circuits can be deployed in the same cell) due to the use of CRISPRi gRNA's </FONT COLOR="#008000"></font></b>
+</font></div>
+<div id="description" style = "width:950px; height:140px" align="justify">
+<font face="calibri" size = "4">
+Before we could create promoters that respond to a high and low amount of inducer, we first had to select promoters with two favorable characteristics we could exploit to make them concentration-dependent which would be a low basal level and a wide dynamic range. We chose four well-known promoters as possible candidates for our circuit: pLAC, pTet, pBAD, and PprpB. We constructed separate plasmids containing each of the promoters and characterized these promoters by measuring the GFP expression over time. We determined the basal level, highest induction level, and inducer range. We selected pLAC & pTET as the promoters for the synthetic circuit, because of their low basal expression and the varying responsiveness at different inducer concentrations. <br></div>
 <div id="photos">
-<center><img style="margin-top:10px"; padding:0;"
+<center>
-src="https://static.igem.org/mediawiki/2013/f/fe/Circuit.jpg"> </center><br></div>
+<img style= "width:425px; height:355px; margin-top:10px; "
+src="https://static.igem.org/mediawiki/igem.org/0/04/PLAC_Induction.jpg">
+ </center><br></div>
-<div id="description" align="justify" style = "width:950px; height:280px">
+<div id="description" style = "width:700px; height:100px; margin-left:120px; margin-bottom:20px" align="justify">
-<font face="arial" size = "4">
+<font face="calibri" size = "2">Dose-response curve for pLAC promoter induced with different amount of inducers (0, 0.1, 1, 10, 20, 40, 60, 80, 120 uM IPTG). Cells were grow to mid-log phase and then start induction. OD600 value and GFP fluorescence level of each sample were measured by plate reader after saturation. GFP fluorescence were corrected for OD600 value. The red line indicated Hill function fit of the dose-response curve and error bars indicate standard deviation calculated on the basis of technical replicates.</font>
-<br><FONT COLOR="#008000"><u>Design Requirements: </FONT COLOR="#008000"></u><br>In order for this circuit to properly function, we had to address two main challenges: <br><br><center><b><FONT COLOR="#008000"> 1) </font></b> identifying or constructing promoters that were differentially responsive to both <b>high</b> and <b>low</b> levels of an inducer</center>
+</div>
-<br><center><b><FONT COLOR="#008000">2)  </font></b> ensuring that the fluorescent proteins and gRNAs were produced in the same amount under the same promoter.</center>
-<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><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>.
+<div id="photos">
+<center>
+<img style= "width:425px; height:355px; margin-top:10px; "
+src="https://static.igem.org/mediawiki/igem.org/6/63/PTET_Induction.jpg">
+ </center><br></div>
+<div id="description" style = "width:700px; height:100px; margin-left:120px" align="justify">
+<font face="calibri" size = "2">Dose-response curve for pTET promoter induced with different amount of inducers (0, 0.004, 0.01, 0.04, 0.06, 0.11 uM aTc). Cells were grow to mid-log phase and then start induction. OD600 value and GFP fluorescence level of each sample were measured by plate reader after saturation. GFP fluorescence were corrected for OD600 value. The red line indicated Hill function fit of the dose-response curve and error bars indicate standard deviation calculated on the basis of technical replicates.</font>
 </div>
+<div id="description" style = "width:950px; height:100px" align="justify">
+<font face="calibri" size = "4">
+<FONT COLOR="#008000"><u>Engineering pLAC Sensitivity </FONT COLOR="#008000"></u><br>
+<br>Our next goal after our promoter assays was to create engineered versions of our promoters responsive to high and low levels of inducer, either by modifying activation or repression of the specific promoter. Based on <a href="http://www.ncbi.nlm.nih.gov/pubmed/16432263">previous work</a> in the literature characterizing the pLAC promoter, we chose to change both the number and orientation of repressor binding sites in pLAC.
+</div>
 <div id="photos">
 <center><img style="margin-top:20px"; padding:0;"
-src="https://static.igem.org/mediawiki/2013/5/59/Scalable.jpg"> </center><br></div>
+src="https://static.igem.org/mediawiki/2013/e/ef/Promoter_Affinity.jpg">
+<br><font face="calibri" size = "2">From <a href="http://www.ncbi.nlm.nih.gov/pubmed/16432263">Oehler <i>et al.</i>, 2006.</a> </center><br></div>
-</center>
+<div id="description" style = "width:950px; height:100px" align="justify">
+<font face="calibri" size = "4">
+To create our "low" inducer promoter, we shifted the O1 operator site further downstream of our promoter sequence and removed another operator site from pLAC to relieve repression. To create our "high" promoter, we took the "low" promoter and added the O3 operator site further upstream near the start of the promoter sequence. The O3 operator site will then form a loop with our O1 operator site, thus creating a physical barrier to prevent transcription, reducing the promoter activity at low levels of inducer. </font></div>
+<div id="photos">
+<center><img style="height:150px; margin-top:20px"; padding:0;"
+src="https://static.igem.org/mediawiki/2013/4/41/PLAC_Prom_Eng_UCSF1.png">
+<div id="description" style = "width:700px; height:70px; margin-left:120px" align="justify">
+<font face="calibri" size = "2">On this diagram (A) denotes the very original pLAC which is found in the parts registry. (B) is our re-engineered pLAC that has an added O3 binding site. We found in literature that by adding an O3 binding site, a higher inducer level is required to start transcription for a loop to form between the 03 and 01 binding sites. We expect we would need more inducer to produce similar levels of GFP.
+</font>
 </div>
-<div id="description" align="justify" style = "width:950px; height:160px">
+<div id="description" align="justify" style = "width:950px; height:50px">
-<font face="arial" size = "4">
+<font face="calibri" size = "4"> When we engineered the pLAC promoter, the dynamic range is insignificantly different. This graph shows that the green fluorescent protein is expressing at the same inducer concentration for both of the promoters. So we went back to the drawing board and redesigned the pLAC promoter.
-<br><FONT COLOR="#008000"><u>Using CRISPR to Create Scalable Circuits: </u></FONT COLOR="#008000"></b>texttexttexttext.
 </div>
+<div id="photos">
+<center><img style="height:340px; margin-top:20px"; padding:0;"
+src="https://static.igem.org/mediawiki/2013/7/7d/RedesignedpLAC_1_UCSF.png">
+<div id="description" style = "width:950px; height:120px" align="justify">
+<font face="calibri" size = "4">
+<FONT COLOR="#008000"><u>
+Improved Designs for High & Low Sensitivity Promoters</FONT COLOR="#008000"></u><br>
+<br>In the diagram below, (C) is our low promoter and (D) is our high promoter. To create our low inducer promoter, we shifted the O1 operator site further downstream of our promoter sequence and removed another operator site from pLAC to relieve repression. To create our high promoter, we took the low promoter and added the O3 operator site further upstream near the start of the promoter sequence. The O3 operator site will then form a loop with our O1 operator site, thus creating a physical barrier to prevent transcription, we require higher levels of inducer for expression.
+</div>
+<div id="photos">
+<center><img style="height:300px; margin-top:20px"; padding:0;"
+src="https://static.igem.org/mediawiki/2013/2/22/RedesignedpLAC_2_UCSF.png">
+<div id="description" style = "width:700px; height:70px; margin-left:120px; margin-bottom:25px" align="justify">
+<font face="calibri" size = "2">The sequence of four different pLAC promoters constructed for the purpose of high and low sensor. (A) was the original pLAC promoter, (B) was the engineered one that have reduced expression level, (C) and (D) was the expected low and high sensor respectively. Yellow region of each promoter indicated its -35 and -10 core region for RNA polymerase binding, the red characters of each promoter indicates +1 start point of transcription, and the blue square indicated different LacI binding sites (LacO1 and LacO3).
+</font>
+</div>
+<div id="photos">
+<center><img style="height:300px; margin-top:20px"; padding:0;"
+src="https://static.igem.org/mediawiki/2013/d/d1/PLAC_Prom_Eng_UCSF2.png">
+<div id="description" style = "width:950px; height:120px" align="justify">
+<font face="calibri" size = "4">Our second round of promoter engineering shows that the outputs are close to our desired outputs. However, the sensitivity of the promoters did not change. Though we did not get our desired high and low promoters, we have created new promoters that will be available at the parts registry and have been fully characterized. <br> <br>
+We also created a <a href="https://2013.igem.org/Team:UCSF/Modeling" >
+mathematical model</a><span>
+ of our system and were able to identify important variables and parameters that would help produce our decision making circuit.
+</div>
+<div id="description" align="justify" style = "width:950px; height:350px">
+<br><font face="calibri" size = "5"><b>CRISPRi Decision-Making Circuit: Circuit Output</font></b> <br>
+<font face="calibri" size = "4">
+<br>The essential benefit of using the CRISPR system for the circuit design is that it has a very specific target -- a guideRNA contains nucleotides which are complementary to a unique DNA sequence. As a result, we can use a vast number of individual gRNAs to repress several genes, giving us the ability to design circuits to make numerous and more complex decisions.
+<br><br>Our proof-of-concept design is, at a low level of inducer, to express GFP simultaneously with a gRNA to repress RFP. When the inducer level increases, the "high" sensing promoter turns on expression of RFP and a gRNA to repress GFP, making a switch-like decision based on the input. We were able to construct an early design of the circuit in the pCDF plasmid that contains both fluorescent proteins and gRNAs. We initially put both components of the circuit under individual promoters, pLAC and pTET, as a control to test whether or not we could obtain expression of both products and repression from the gRNAs, both under individual induction and after switching inducers. Since this circuit relies on CRISPR repression, we had to also construct a plasmid (pACYC) containing the catalytically dead dCas9 protein as well as the Csy4 protein to cleave the RNA products (see Design page or Parts Registry for more information).
+<br><br>We are in the final stages of cloning the dCas9 plasmid and we hope to test the circuit soon.
+</div>
+<div id="photos">
+<center><img style="height:300px; margin-top:20px"; padding:0;"
+src="https://static.igem.org/mediawiki/2013/9/9d/UCSF_Circuit_TestSystem.png"></div>
+</html>