Difference between revisions of "Part:BBa K3017070"

Revision as of 06:44, 18 October 2019

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

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