Part:BBa_K4344091
p19 pulldown gene and siRNA expression casette empty
We engineered a fragment containing the p19 gene of the tomato stunt virus with an added His-tag and a functional T7 promoter-driven expression cassette for a long dsRNA. The whole fragment was cloned into a pUC19 backbone. The engineering success of this construct was then validated by conduction of a western blot for p19 expression, NGS for sequence congruency and Native Page for siRNA presence. The functionality of the dsRNA expression cassette was validated by in vitro transcription using a commercial kit. To further enhance and regulate the production of p19, we added a Shine-Dalgarno and lacO sequence as addition to the tac-promoter-driven RNA transcription by primer extension PCR.
Methods For detailed methods please refer methods for siRNA production.
Engineering of pUC19-p19-siRNA UL19 We initially simulated the folding of the chosen loop element in the dsRNA expression cassette with UNAFold (N. R. Markham & M. Zuker, 2008) by selecting a sequence spanning from the +1 site of the T7 promoter to the 20th base of the T7 terminator. Two possible folding structures with ΔGs of -31.40 kcal/mol and -30.60 kcal/mol were identified.
We assessed the functionality of our plasmid in two different ways. At first we checked for expression of p19 by SDS-PAGE and Western-Blot. To determine the protein concentration a Bradford Assay was conducted (for results see Table 6, Fig 4A). 30 µg of Protein in the Wash Fraction 1C and a linear mass dilution from 3 µg to 3 ng of Ni-NTA bead bound p19-fraction 1 were analysed by SDS-PAGE. Ponceau staining of the Blot revealed in each fraction an accumulation of a protein with a mass around 28 kDa. This protein is most dominantly present in Wash fraction C together with other proteins of different sizes. In the linear mass gradient only a small band of a ~28kDa protein is visible with decreasing intensity at lower concentrations. In comparison to the loaded BSA masses (1 µg and 500 ng) only in the Wash fraction 1C contains comparable amounts of protein. The linear gradient shows high purity of the fraction with only one visible protein.
We optimized the sequence provided to us by Huang and Lieberman by addition of a T7-Terminator and a rrnB-T1 Terminator to more tightly regulate the gene expression. This allowed us to produce a single dsRNA with a size of 300 bp. In contrast to a size range which would have been obtained when no T7-Terminator is present. In casae of p19 expression we noticed that our initially design lacked a functional p19 expression casette. We therefore, altered our design and inserted the missing sequence for tac-mediated transcription as well as a lac operator for transcriptional control to redulate the gene expression more tightly since the tac promoter is a constitutive promoter with high expression levels. To enhance ribosom binding we included a Shine-Dalgarno sequnce downstream of the lac operator to ensure that the Shine-Dalgarno sequence is present in the transcribed mRNA. Whilst the T7 dsRNA expression casette is highly functional the p19 expression and purfification process can be enhanced. Purification can be eased and more yield can be achieved by usage of a different tag which binds more selectivly to it's counterpart therefore reducing unselective binding which occurs usually Ni-NTA beads (compare Westernblot wash fraction 1C). Tags like Haemaglotinin or GST may yield purer fractions. Overall, the proposed expression system for pro-siRNAs in E. coli is functional and the obtained pro-siRNAs are capable to faciliate knockdown in-vitro (see proof of concept, results cellculture assays).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 218
Illegal XbaI site found at 699
Illegal XbaI site found at 766 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 218
Illegal NotI site found at 807 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 218
Illegal XhoI site found at 760 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 218
Illegal XbaI site found at 699
Illegal XbaI site found at 766 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 218
Illegal XbaI site found at 699
Illegal XbaI site found at 766 - 1000COMPATIBLE WITH RFC[1000]
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