Difference between revisions of "Team:Bielefeld-CeBiTec/siRNA"

Abstract

CRISPR/Cas is a widely used tool in Genome Engineering and strain development. Amongst other things it can be used for knock outs and insertion of new genes into the genome. However CRISPR/Cas has faced growing critics in the past, partly due to the large off target activity of CRISPR/Cas. The CRISPR/Cas system is a commercial product which contradicts the iGEM spirit and makes it difficult to legally share parts via the iGEM Registry. Here we want to propose RNA interference (RNAi) as an open source and free to use alternative to CRISPR/Cas for the iGEM community. We connected several known processes taking place in Escherichia coli to mimic the eukaryotic RNAi mechanisms. Additionally, we designed a two vector system to express and test siRNAs in E. coli, and developed a software tool to predict and rate possible siRNAs for a given target sequence.

Theorie

In Synthetic Biology it is often required to perform knockouts in the context of metabolic engineering. Currently knockouts are usually carried out by using the CRISPR/Cas9 system. One problem of these knockouts is that they are permanent and that the knockout of housekeeping genes can inhibit growth. In certain cases a knockout can lead to the death of the cell, making it impossible to knock a gene out for metabolic engineering. We want to propose an alternative: siRNAs and RNAi. The RNA interference system (RNAi) can be used to knock down genes by degradation of the corresponding mRNA, making the knockout inducible by using an inducible promoter. This enables us to let production cells grow until a desired cell density or production phase and lets us induce the knockdown when needed.
Protein biosynthesis is strongly regulated by different post- and pretranslational mechanisms. Post-translational regulation is realized by small interfering RNAs (siRNA)which block the ribosome binding site (RBS) and inhibit transcription by the RNA interference (RNAi) mechanism. Both mechanisms use the RNA chaperone Hfq to facilitate the hybridization of siRNA and mRNA (Na et al., 2013).
After transcription, RNAs are protected from degradation by a triphosphate at the 5’ terminus. Monophosphorylated ends are produced through maturation of the RNA by internal cleavage or by cleavage of pyrophosphate at the 5’ terminus (Foley et al., 2015).
Cleavage of the 5’ terminal phosphates is also the first step in RNA degradation. RNase E plays a major role in RNA degradation as well as in RNAi, as it does not only degrade monophosphorylated RNAs but can also accept a monophosphorylated 5’ terminus as an activator that triggers a conformational change by interaction with the 5’ sensing pocket. It has been shown that this allows the RNase E to cleave mRNA. Therefore, a complementary siRNA with unpaired bases at the 5’ terminus bound to an mRNA can induce cleavage of the mRNA. In a test four unpaired bases were sufficient and cleavage occurred 6 bp downstream of the homologue region. The required complementary sequence was rather short being only 9 bp long.
To enhance the degrading capability of siRNAs the 5’ terminus can be tailored to match the substrate specificity of the RNA pyrophosphohydrolase RppH by having a C at Position 2 from the 5’ terminus (Bandyra et al., 2012).
To find and design possible siRNAs for RNAi we designed and tested a program which also predicts secondary structures and the non-complementary 5’ terminus.
*Link here* Additionally we designed a two vector system to scan single siRNAs or siRNA libraries for the most effective RNAi silencing of a given target gene.

Mechanism

Two Vector System

To compare and scan multiple siRNAs or RNA libraries for RNAi we developed a two vector system comprised of an expression Vektor for the siRNAs and a Target Vektor for the production of a target sequence and the marker protein.
There are two versions of the Target Vektor. Version one is comprised of the PTet promoter, a lacZ-alpha cassette, a linker and the reporter protein AmilCP. To enable flourescence measurements a version containing the Blue Flourescent Protein (BFP) has been constructed as well. With higher silencing effectivity the absorption declines which can lead to false positive results in cases where the reporter protein does not work as expected. The second version has the lacZ-alpha cassette linked to the LacI protein. If transcribed correctly this inhibits the Lac promoter which in turn produces the reporter protein AmilCP if not inhibited.

This setup prevents false positive results as the absorption increases with higher silencing activity. Prior to use one of both biobricks needs to be cloned into another vector to achieve two different antibiotic resistances. The lacZ-alpha cassette can be replaced using the BbsI restriction enzyme. After the ligation of a target sequence into the Target vector it can be transformed. A library of possible siRNAs can be inserted into the expression vector followed by a transformation into competent cells containing the target vector. The emerging colonies then can be scanned for maximum silencing efficiency. Multiple colonies can be tested simultaneously in 96 well plates.

RNAi versus Knockouts

Using the siRNA Testing System

---

References ▾