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Indian Institute of Science Education and Research (IISER), Pune, India

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Project Description

In short, our project is meant to measure transcriptional noise and characterize the noise associated with different promoters in E. coli.

Transcription is the process of converting information stored in DNA to information stored in RNA. During this process, there is an amount of random variation in the rate of the conversion. We call this random variation ‘noise’. Transcriptional noise is largely influenced by sequences of DNA called promoters that help “tell” the cell to begin the process of transcription. Our project is to assess how much transcriptional noise is expressed by different commonly used promoters.

We are also working on a system of targeted repression of promoters using dCas9. Perhaps the most up and coming technique in synthetic biology is CRISPR/Cas9 genome editing. A CRISPR (clustered regularly interspersed short palindromic repeat) sequence is one which codes for a crisprRNA, an RNA which targets a particular DNA sequence (called the protospacer). In natural systems, the crisprRNA would bind to the protospacer and interact with another RNA sequence called a tracrRNA (which only serves to summon the Cas9 protein). The tracrRNA interacts with the Cas9 protein which recognizes a sequence in the DNA called the protospacer adjacent motif (PAM). The Cas9 protein would then cut the protospacer several base pairs upstream from the PAM. This opens the door for a synthetic biologist to insert an alternative gene or otherwise edit the genome. Additionally, in synthetic systems, the crisprRNA and tracrRNA sequences can be combined into a single guide RNA, or gRNA for short.

However, the CRISPR/ Cas9 system has many other applications. In our project, we are using a variation of the Cas9 which has been functionally deactivated (it no longer cuts the protospacer). It does, however, still clamp onto the protospacer and block the binding of other things. Therefore, if we want to inhibit the expression of a gene, we can do so by targeting the gRNA sequence to bind to the promoter region of the gene of interest. This will allow the functionally 'dead' Cas9 (dCas9) to bind to the promoter region of the gene, preventing the RNA polymerase which would have transcribed the sequence from binding to the promoter sequence because there is a giant protein in the way.

Within our project, we are referring to the idea of dCas9 repression as the Susan System, or Susan for short. We are working on a Suite of gRNA parts which will allow any iGEM team to easily repress genes expressed under any of the top ten most common iGEM promoters and then subsequently turn on expression of the gene on demand.

The importance of proper characterization of transcriptional noise in promoters cannot be overstated. The less transcriptional noise a cell exhibits, the more reliably and predictably a cell can manufacture proteins or functional RNA. When scientists use cells to manufacture tools, it is important that these tools be both accurate and precise. Reliable cells manufacture products with precision, and reliability requires low levels of transcriptional noise. Alternatively, a researcher could require cells with a high level of transcriptional noise. Therefore it is important to be able to identify which promoters result in which levels of transcriptional noise.

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Advice on writing your Project Description

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References

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Inspiration

See how other teams have described and presented their projects:

• Imperial
• UC Davis
• SYSU Software