Designed by: Yucheng Zhang   Group: iGEM23_Tongji-China   (2023-10-06)

trc promoter_SpdCas9

dCas9 controlled by a trc promoter. The trc promoter is a hybrid of the lac and trp promoters which is a stronger promoter in comparison to the lac promoter. This part is useful for producing dCas9 protein in bacteria.

Brief introduction

In the realm of genetic engineering and gene regulation, the trc promoter and dCas9 are key components of the CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats interference) system.

1. trc Promoter

The trc promoter, short for "Tetracycline Response Element (TRE)-regulated Cytomegalovirus (CMV) Promoter," is a synthetic promoter sequence commonly used in CRISPRi systems. It allows for precise control of gene expression in response to the presence or absence of tetracycline or its derivatives.

The trc promoter is widely employed to drive the expression of a modified version of the Cas9 protein called "dCas9" in CRISPRi systems. When tetracycline is added, it can suppress the activity of the trc promoter, thereby regulating the expression of dCas9 and, consequently, the level of gene silencing.

2. dCas9

dCas9 stands for "dead" or "catalytically inactive" Cas9. It is a variant of the Cas9 protein derived from the CRISPR/Cas9 system. Unlike the active Cas9 protein, which is known for its ability to cut DNA at specific target sites (CRISPR/Cas9 genome editing), dCas9 has been mutated to lose its DNA-cutting activity.

In CRISPRi systems, dCas9 is used as a programmable DNA-binding protein. It can be guided to specific gene sequences by a guide RNA (gRNA) molecule complementary to the target gene's sequence. Once dCas9 is bound to the target gene, it does not cleave the DNA but rather interferes with the transcriptional machinery, preventing the gene from being transcribed into RNA.

We designed specific gRNAs to direct dCas9 to accomplish the repression of Vibrio natriegens pilA promoter, thereby inhibiting the expression of the Vibrio natriegens' own pilus.


Fluorescence absorption

We produced a total of four plasmids, pdCas9-sg1-4 and then co-transformed with pGFPmod2 respectively. After IPTG induced dCas9 for 5 hours, the inhibition of GFP expression by dCas9 was shown by fluorescence values. Green fluorescence of the suspension was measured with microplate reader and the result was as follows.

From the measured results, it can be seen that all CRISPRi plasmids have some inhibition effect except for pdCas9-sg1, for which the result is not significant, and pdCas9-sg4 has the best inhibition effect. However, due to time constraints, we only used the initial pdCas9sg-1 plasmid for the construction of the bacterial hair expression strain.