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pUC19-p19-6xHis-siRNA UL19 (Tac-LacO-SD)

Plasmid used for production of siRNA in E. coli.

Sequence and Features

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 (Structure 1, Figure 1A) and -30.60 kcal/mol (Structure 2, Figure 1B) were identified.

Figure 1: Proposed folding of loop structure. Loop folding was simulated using the UNAFold algorithm. A: Folding structure 1 with a ΔG of -31.40 kcal/mol. B: Folding structure 1 with a ΔG of -30.60 kcal/mol.

pUC19-p19-siRNA empty was engineered by restriction based cloning of a PCR amplified backbone and an insert obtained by solid phase synthesis. Primers for pUC19 backbone amplification were analysed on a temperature gradient ranging from 64 °C to 71 °C. The annealing temperature proposed by <a href=”https://tmcalculator.neb.com/#!/main”>NEB T_m- Calculator</a> was 72 °C. For all temperatures we obtained amplicons with a size ranging from 1500 to 2000 bp as well as a second fragment between 600 and 700 bp (Figure 2A). An annealing temperature of 69 °C was chosen and the PCR was repeated (Figure 2B). Plasmids received by plasmid preparation were sequenced with p19-forward and AmpR reverse. Insert presence was proven as well as integrity of the loop structure.

siRNA target areas were amplified with SacI/XhoI or SalI/NotI sequence extension by PCR. We obtained amplicons with a size ranging from 200 to 300 bp (Figure 2C) which was congruent with the expected sizes of 266 bp for the SacI/XhoI Primer set and 268 bp for the NotI/SalI primer set. After performing a two-step cloning and transformation in E. coli, we analysed the obtained plasmids by restriction digest with SacI and NotI to confirm the presence of the insert in both restriction cassettes. Results were visualised on a 1.2 % agarose gel stained with ethidium bromide (Figure 2D).

Plasmids were sequenced with p19F and AmpR to confirm correct insertion and conservation of the loop structure as well as the integrity of p19 gene.

Subsequently we checked whether for the obtained sequence of the dsRNA expression cassette the formation of a loop structure is predicted. We chose a sequence from the +1 site of the T7 promoter to the 20th base of the T7 terminator to include the full expression cassette. Folding simulation resulted in one proposed structure with a ΔG of -605.90 kcal/mol.

We furthermore introduced a 7 bp long fragment between the tac promoter and the start codon of p19 by Primer Extension PCR.

After the successful cloning of pUC19-p19-siRNA UL19, we extend the insertion by a lac operator sequence and a Shine-Dalgarno sequence using the same principle. The lastly obtained plasmids were analysed by restriction digest with ClaI and HindIII on a 1.2 % agarose gel stained with ethidium bromide (Figure 2E).

Afterwards the Plasmid was sequenced with p19F and AmpR to confirm correct insertion of both inserts and conservation of loop structure as well as integrity of p19 gene. Furthermore the plasmid was sequenced with pUC19-pBR322ori-fwd to confirm complete presence of tac-lacO-Shine Dalgarno structural element. The proposed plasmid structure is displayed in Figure 2F.

Figure 2: Selection of results obtained during the engineering process of pUC19-p19-siRNA UL19. A: 1.2 % agarose gel of temperature gradient ranging from 64 °C to 71 °C for pUC19 backbone primer evaluation. B: 1.2 % Analytical gel of pUC19 backbone PCR at 69 °C. C: 1.2 % Analytical of UL19 siRNA target area PCR with primer sets SacI/XhoI and SalI/NotI. D: Analytical gel of pUC19-p19-siRNA UL19 after insertion of both siRNA target products. E: Analytical gel of pUC19-p19-siRNA UL19 after insertion tac-LacO-Shine Dalgarno extension. F: Proposed structure of the complete pUC19-p19-siRNA UL19 (tac-LacO-SD).

Evaluation of pUC19-p19-siRNA UL19 functionality

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 Figure 3A). 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 the Wash fraction 1C contains comparable amounts of protein. The linear gradient shows high purity of the fraction with only one protein visible (Figure 3C). Functionality of the T7 expression cassette was assessed by in-vitro transcription. Our self-designed and cloned plasmid was compared to a cassette derived by solid phase synthesis and a PCR full amplicon of this solid phase synthesis derived cassette. Both cassettes derived from the solid-phase synthesis did not yield any visible dsRNA, while our cassette produced a dsRNA at a size of ~300 bp (see Figure 3B).

Figure 3: Overview of functional analysis of pUC19-p19-siRNA UL19. A: Bradford standard curve used for calculation of protein of protein mass in the collected fractions. B: Analytical 2 % agarose gel stained with ethidium bromide for visualisation of dsRNA production self-designed and cloned plasmid was compared to a cassette derived by solid phase synthesis and a PCR full amplicon of this solid phase synthesis derived cassette. Both cassettes derived from the solid-phase synthesis did not yield any visible dsRNA, while our cassette produced a dsRNA at a size of ~300 bp. C: Western Blot of collected Protein in the Wash Fraction 1C and a linear mass dilution from 3 µg to 3 ng of Ni-NTA bead bound p19-fraction.

The originally designed plasmid for siRNA production showed no expression of p19 protein and therefore no pulldown of pro-siRNAs could be achieved. For this reason, we added a 7 bp consensus sequence of the tac promoter, a Lac Operator and a Shine-Dalgarno sequence. This solved our initial problem and added a regulatory element, which could be tuned with the same inductor as the expression of the genomic T7 RNA polymerase. The tac promoter is a synthetically generated constitutive promoter from the trp and lac operon and provides strong expression in E. coli (de Boer et al., 1983). The Shine-Dalgarno sequence in prokaryotes has the same function as the Kozak sequence in eukaryotes and is located near the start codon (Steitz & Jakes, 1975). To better control the expression of our plasmid, we added terminator regions to it. This kind of region wasn't included byHuang and Lieberman in their published sequence (Huang & Lieberman, 2013). Due to the improved expression control especially of the dsRNA cassette the desired amount of correctly folded and sequence matching dsRNA can be produced. Subsequently the yield of functional pro-siRNAs against the chosen target can be increased.