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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 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.
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:
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.
