Part:BBa_K3017070
Combined CRISPRi and antisense RNA Toggle Switch - Model circuit
This construct is a proof of concept of a biological switch, novel to iGEM community, by our team. Our team has combined the CRISPRi system with RNA regulators to achieve a toggle switch. The switch utilizes the catalytically inactive form of Cas9 (dCas9) to achieve targeted and reversible repression of genes via specific single-guide RNAs (sgRNAs). Alternatively, the transcription of antisense RNA (asRNAs) reverses the effect of the dCas9 modulated repression on the desired genes. This method of regulation would allow for the ability to fine-tune and easily customize the execution of highly complex genetic circuits.
To bring our project to life, we have designed a circuit that drives the Escherichia coli (E.coli) strain DH5-alpha to alternately produce green fluorescent protein (GFP) and red fluorescent protein (RFP) depending on the inducer added to our system. The behavior of the construct, as a proof of concept design, relies much on the mathematical model based on data from characterizations of individual components, but the desired dynamics of the switch is illustrated below.
Circuit dynamics
| Inducer | Synthesis | Visual Effect | |
|---|---|---|---|
| State 0 | No inducer | dCas9, GFP, RFP | Orange |
| State 1 | Arabinose (Ara) | dCas9, sgRNA_RFP, asRNA_GFP, GFP | Green |
| State 2 | IPTG | dCas9, sgRNA_GFP, asRNA_RFP, RFP | Red |
State 0 -- initial state
dCas9, GFP, and RFP are constitutively produced
State 1 - Arabinose causes inhibition of RFP production
sgRNA_RFP (BBa_K3017002) is produced when arabinose is added. Then, the sgRNA forms a complex with the constitutively expressed dCas9 (which is always present in the system). This complex is guided by the sgRNA to specifically target the mrfp protein coding region, thereby suppressing the gene.
State 2 - IPTG reactivates RFP while suppressing GFP
Adding IPTG causes asRNA_RFP and sgRNA_GFP to be produced. As illustrated before, sgRNA will form a complex with dCas9. However, as sgRNA_GFP (BBa_K3017003) is produced in this state, the complex represses gfp instead of mrfp. Meanwhile, the asRNA_RFP produced will target the dCas9/sgRNA_RFP complex currently bound to the RFP gene. asRNA causes the dCas9 complex to dissociate by tightly binding to the sgRNA.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 852
Illegal PstI site found at 2274
Illegal PstI site found at 2478
Illegal PstI site found at 2508
Illegal PstI site found at 3720 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 5543
Illegal NheI site found at 6921
Illegal NheI site found at 7093
Illegal NheI site found at 7116
Illegal NheI site found at 8733
Illegal PstI site found at 852
Illegal PstI site found at 2274
Illegal PstI site found at 2478
Illegal PstI site found at 2508
Illegal PstI site found at 3720 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 313
Illegal BamHI site found at 5482
Illegal BamHI site found at 6860 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 852
Illegal PstI site found at 2274
Illegal PstI site found at 2478
Illegal PstI site found at 2508
Illegal PstI site found at 3720 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 852
Illegal PstI site found at 2274
Illegal PstI site found at 2478
Illegal PstI site found at 2508
Illegal PstI site found at 3720
Illegal NgoMIV site found at 1140
Illegal NgoMIV site found at 2244
Illegal NgoMIV site found at 2317
Illegal NgoMIV site found at 2802
Illegal NgoMIV site found at 3711
Illegal AgeI site found at 5317
Illegal AgeI site found at 6695
Illegal AgeI site found at 7702
Illegal AgeI site found at 7814
Illegal AgeI site found at 9342
Illegal AgeI site found at 9454 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 5299
Illegal SapI site found at 6677
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