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Team:UCSF/Project/Circuit/Data
From 2013.igem.org
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.
Altering Promoter 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 workin the literature characterizing the pLAC promoter, we chose to change both the number and orientation of repressor binding sites in pLAC. 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.
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 workin the literature characterizing the pLAC promoter, we chose to change both the number and orientation of repressor binding sites in pLAC. 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.
From Oehler et al., 2006.
Using a similar GFP reporter system as for the first promoter tests, we were able to show that our engineered pLAC promoters are now concentration sensors and can be used to create a circuit that responds differently to varying inducer levels. We plan to implement a similar strategy for the pTET promoter design to implement it in a parallel circuit.
PROMOTER DATA
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.
