Overview

This year we made the components of CRISPRi system into parts and submitted to the iGEM Registry. With continuously increasing interest in work relevant to CRISPRi system, we believe these 2 parts will provide lots of convenience to users. Since in our project we hope that light could be the only parameter that we directly adjust, we did some improvements to the pre-existing parts: inserting them into a constant expressing operon.

All of our parts can be divided into 2 categories, the CRISPRi system and the light sensing system.

CRISPRi

Original Components: dCas9 protein(BBa_K1026001) and sgRNA(BBa_K1026003)

Constitutive Expressing Components: dCas9 protein(BBa_K1026000) and sgRNA(BBa_K1026002)

We have tested CRISPRi system though the following experiment: After successful transformation, the control group expressed dCas9 and mRFP constitutively while the case group expressed dCas9, sgRNA and mRFP. The color of these two groups should be different due to the knock down effect of CRISPRi system.

The result is in consistent with our expectation:

Additionally, the base-pairing region can be changed according to different people's needs. Inverted PCR will provide an easy way to replace this region and make it a universal and convenient regulating tool.

We also built the part that combines a promoter regulated by light sensor with sgRNA(BBa_K1026004), if used together with light sensing system, the red light will negatively control the expression amount of sgRNA, thus affecting the reporter genes or functional genes.

Light Sensor

After careful discussion and selection, we chose three light-controlled gene expression systems induced by red, green, and blue respectively. Main parts of these 3 sensing systems all have been used by 2011_Uppsala Team.

From the perspective of convenience, we improved several of these parts:

Constitutively Expressed Red Light Sensor Operon(BBa_K1026005)

Constitutively Expressed Blue Light Sensor Operon(BBa_K1026011)

Constitutively Expressed pcyA Operon(BBa_K1026014)

When we use green light sensing system we find that green light upregulated genes downstream the promoter, and we would like to construct 3 downregulated systems. After studying the mechanism and structure characteristics of this system, we designed another promoter. The regulatory factor will bind to the G-box thus blocking the way of RNA Polymerase. As a result, we get the wanted promoter part: A rationally designed promoter negative regulated by CcaR (BBa_K1026009)

After combining 2 systems together, we get the following parts that have direct relationship of light-controlled CRISPRi system:

A gRNA targeting mRFP, negatively regulated by Red Light Sensor(BBa_K1026004)

A gRNA targeting mRFP, negatively regulated by Green Light Sensor(BBa_K1026007)

A gRNA targeting mRFP, negatively regulated by Blue Light Sensor(BBa_K1026010)

With these parts we can use this light-controlled CRISPRi system as a universal gene regulating tools and it is very easy to control.

Correct Combination makes Suitable Solution. We believe this system will bring about plenty of new applications.

<groupparts>iGEM013 SJTU-BioX-Shanghai</groupparts>