Parts

Overview

The aim of our project was to inhibit the infection of sugar beets by degrading the Beet Yellows Virus (BYV) genome with the help of the RNA interference (RNAi) mechanism. To do so, we wanted to produce RNAi precursors with genetically engineered bacteria. We aim to protect these precursors by encapsulating them with the coat protein of the Tobacco Mosaic Virus (TMV). We also wanted to produce these proteins with the help of genetically engineered bacteria. Therefore, our project was composed of two major components to inhibit the infection of sugar beets by the BYV: the RNAi technology and the capsid protein.

RNAi technology

For RNAi therapy, we selected two targets for our RNAi precursors:

• The RNA-dependent RNA polymerase (RdRp) of the BYV, the enzyme that allows the BYV to replicate its genome and colonize new cells and plants.

• The protein p21, which acts as an RNAi silencing suppressor of the virus, binds to siRNAs, preventing them from being incorporated in the RISC complex.

We designed one RNAi precursor for each gene, one for both genes at the same time, and one against the phytoene desaturase (PDS) of the sugar beet. The last RNAi precursor serves as a control to make sure that the plant machinery well processes our RNAi precursors to produce specific siRNAs. The PDS is an enzyme of the b-carotenoid synthesis that, when inhibited, makes the plant leaves appear white instead of the usual green.

To be able to synthesize our RNAi precursors, we had to divide their sequences into two parts: one corresponding to the sense sequence surrounded by NdeI and BamHI restriction sites, and one corresponding to the antisense sequence surrounded by NdeI and BlpI restriction sites. In addition, at the end of the antisense sequence, we added a sequence called the Origin of Assembly Sequence (OAS). This specific sequence of nucleotide is the one that is recognized by the TMV coat proteins, leading to the formation of the viral capsid around the sequence containing the OAS.

To clone our RNAi precursors, we used the pET21a(+) as a backbone since it already contained the promoter and terminator of our interest: the T7 promoter and terminator (Figure 1).

Figure 1: pET-21a(+)

In addition to the lhRNA, we decided to design basic parts encoding small hairpin RNAs (shRNAs). We chose to design these parts to be able to compare the effects of shRNAs to the ones of lhRNAs. This way, we could show if lhRNAs are more, less or equally efficient than shRNAs.

The table below shows the different parts for the RNAi technology section:

The figures below illustrate the three new composite parts that we designed for the RNAi technology.

Figure 2: NdeI_PDS_lhRNA

Figure 3: NdeI_p21_lhRNA

Figure 4: NdeI_poly_lhRNA

Coat protein production

To produce the Coat Proteins (CPs) of the Tobacco Mosaic Virus (TMV) that will encapsulate the RNAi precursors, we constructed a plasmid encoding this protein using Golden Gate assembly. To do so, we used as a backbone the BBa_J435500 (Figure 5).

Figure 5: AE_lacZ_pDest

Here is the list of the parts we used and created for this section of our project: