BSLA: siRNA cassette

The main challenge of our project was to find a way to control Huanglongbing disease, which is caused by the bacteria Candidatus liberibacter asiaticus. After deep research, we found that this bacteria has not been cultured in vitro yet, so we concluded that it was the most convenient to target the vector instead of the bacteria, and the idea of using siRNA technology came to us.

For the design of our siRNA sequences we used several platforms, such as Dharmacon, InvivoGen, and Oligowalk in which we introduced our gene of interest and the platforms gave us many options of siRNAs. After this step, we compared the common results between platforms. CLUSTAL and BLAST analysis was run for each option to ensure that our siRNA will only silence our specie of interest. The siRNA stability, GC%, predicted thermodynamic ensemble and predicted hybridization were also taken into account. The final sequences of siRNAs were those which are completely specific for Diaphorina citri.

It is important to mention the method of siRNA production, Escherichia coli HT115 (DE3), an RNase III-deficient non-pathogenic strain that will be the one used since its genotype allows a better efficiency in siRNA production.

The design of our parts was based on the work by Xu et al. (2009), who use a device that works in mammal cells. We adapted this device to be used in E. coli. The PCR products were directly transformed into chemically competent E. coli HT115 and converted to functional vectors in vivo via homologous recombination. The devices were created as follows:

Each device includes a T7 promoter, a strong RBS, the coding region of a blue chromoprotein, the siRNA sense sequence, a 7-base loop, the siRNA antisense sequence and a T7 terminator, resulting in a plasmid for each of the selected siRNAs (Awd, WNT, SOD, RacI and a GFP control).

Figure 1. Device construction for RacI

The devices were named BSLA (Blue chromoprotein, sense, loop, antisense). After transcription, the sense and antisense siRNA sequences will hybridize, and as a RBS and a coding region from blue chromoprotein are present, a color will appear, reporting the production of the system. Then, after a treatment with RNAse A, the blue chromoprotein region will degrade (as it remains single-stranded), leaving only the assembled siRNA. The complete description of this device can be found here (link: http://parts.igem.org/Part:BBa_K2246000).