Introducing a foundation advancement into iGEM

Combined CRISPRi and antisense RNA toggle switch is a new biological switch system and is novel to the iGEM community. It has the potential to replace switches that rely on repressor protein - operon pairs. To bring the switch to life we have:

Introduced new basic parts into the iGEM part registry

Custom-designed sgRNA for this switch: BBa_K3017001, BBaK3017002
Custom-designed asRNA for this switch: BBa_K3017003, BBaK3017004

Created a model circuit using the above parts, on target proteins GFP and RFP: BBa_3017070

Here's our part collection!

Demonstration of the switch with a mathematical model

Our switch is brought to life by the model we built. It is a descriptive model that characterizes the expression of the output proteins of our system, to evaluate the switch’s effectiveness and the elasticity of variables. Our model shows that the system will take around 4 hours to reach that state, which is shorter than the Collins switch. Check out our model here!

Characterization of RNAs

We characterised our new composite parts BBa_K3017061 and BBa_K3017060 for their transcription and their interaction as a pair.

T7-BBa_K3017061 and T7-BBaK3017060 were synthesised by PCR
T7-BBa_K3017061 and T7-BBaK3017060 were transcribed in vitro
Transcripts of T7-BBa_K3017061 and T7-BBaK3017060 were hybridized. Results show that the custom-designed sgRNA and asRNA pair hybridize as expected. We expect even higher hybridization rate in vivo.

Click to see the details of our experiment!

Characterization of dCas9

We obtained our Sp.dCas9 gene, new to iGEM, from a professor in our university. Since the plasmid was not compatible with standard assembly, we had to make extra efforts to achieve the assembly of our circuit:

SalI cut site, 6XHis tag and terminator were added to the dCas9 gene by PCR
Transformed plasmid was verified by colony PCR and restriction digestion check
Expression of dCas9 was verified by SDS-PAGE, Coomassie Blue staining and Western Blot

Here is how we got dCas9!

Characterization of Old Part BBa_R0010

LacI-regulated promoter BBa_R0010 is used in our model circuit. The relative expression levels of GFP under BBa_R0010 at different pH values were implied. This characterization helps us assess how different environmental conditions affect promoter activity. This, we believe, would aid in future application development:

A graph of fluorescence/OD against pH (pH 5-9) was plotted from the plate reader measurements
We found out:

pLac on its own shows leak expression while that with added glucose shows repressed activity
The overall trend of GFP expression decreases with an increase in pH

This is how we characterized BBa_R0010!

Characterization of competition between constitutive promoters

Professor Fei SUN’s postdoctoral fellows pointed out that the three constitutive promoters in our proposed circuit may display competitive behaviour among each other, thus leading to reduced protein expression by our circuit. We conducted an experiment to assess the level of interference between different numbers of promoters:

Plasmids containing 1, 2 and 3 constitutive promoter-RBS-repeats (BBa_J23104-B0034) are constructed to control a mrfp gene BBa_E1010
A plot of Fluorescence/OD shows no notable correlation between the number of promoters present in the plasmid and the level of fluorescence protein expression

Click to see our promoter competition assay!

//After iGEM//

Future Updates

Unfortunately, we did not manage to test out our full circuit before the wiki-freeze deadline :(
However, some of our team members are planning to continue doing experiments to confirm that our Biscuit does work! We'll update this wiki once we've progressed.