Contribution

Introduction

With this part future iGEM teams are able to design their own siRNAs and express a dsRNA specific for the specific siRNA target area in E. coli. The tightly regulated expression of the p19 protein and the dsRNA allows for a precise induction of siRNA production and an easy regulation by IPTG. The production method is easily scalable to large volumes and the cloning steps can be carried out in every laboratory. With the provided documentation and protocol every team with access to basic reagents used in molecular biology and biochemistry can insert their own target area for siRNAs and test the generated siRNAs after purification for their needs. No need for expensive solid phase synthesis.

Methods

Plasmid design

The sequence of UL19 encoding for HSV-1 major capsid protein was extracted from the complete human herpesvirus 1 genome. The sequence was human codon optimised using Benchling.

The sequence was screened for published antibody binding sites and functional structure elements using the UniProt database. We identified the amino acids 862 to 880 in UL19 as a published antibody binding site (Han et al., 2019). Since it is a published antibody site, we anticipate that the corresponding DNA sequence is highly conserved in the HSV genome. Therefore, we chose a sequence for cloning and expression including this structure element. For UL19 bases 2446 to 3444 were selected resulting in a fragment of 999 bp in size. The fragment was further modified to mask unwanted restriction sites and ease primer design while keeping the amino acid sequence unchanged. UL19 was modified at the 16-18TCC>AGT and 21G>A. A His-tag (5’-CATCACCATCACCATCAC-3’) and a TAA stop codon were added at the 3’ sequence end. The modified sequence was synthesised via Eurofins Genomics gene synthesis service and delivered cloned in a pEX-A258 vector.

Selection of siRNA target area

Huang et al. recommend that the siRNA target area’s size should range between 250 and 500 bp (Huang et al., 2013). We selected a 249 bp area for UL19 (Sequence: 2527 – 2775; Fragment: 82 - 330). The siRNA target area includes the previously described structural motif. For EGFP knockdown a previously published siRNA sequence (sense: 5’-GCAAGCUGACCCUGAAGUUCAUTT-3’; (Metwally, A. A., Blagbrough, I. S., & Mantell, J. M., 2012) was chosen.

The analysis of our target areas with siRNA Design Tool from IDT revealed 13 potential siRNA candidates for UL19. 2 of these 13 candidates were indicated as cross-reacting with human gene transcripts.

Design of pUC19-p19-siRNA empty vector

We designed a construct containing two expression cassettes. One for p19-6x His expression and one for expression of prä-pro-siRNA. The p19 sequence was a gift from Linfeng Huang and Judy Lieberman at the Boston Children’s Hospital and is congruent with the sequence of Addgene (cat. no. 46306).

The p19 gene is flanked by a tac-promotor and a rrnBT1 transcription terminator. The empty siRNA expression cassettes are flanked by a T7-promotor and a T7 transcription terminator. The first siRNA cassette is located between the SacI and XhoI restriction site. The second is between the SalI and NotI restriction sites. Both cassettes are connected by a 32 bp long linker sequence facilitating the folding of ss-pre-pro-siRNA to ds-pre-pro-siRNA (5’- TCTAGAGCGCACGTAtacACGCGCTGATCAGC-3’) after transcription. The whole construct is flanked by non-coding backbone sequences of pUC19 containing ClaI restriction site at 5’-end and BamHI restriction site at 3’-end. The sequence was ordered at Twist Bioscience. During the evaluation of the full plasmid we noticed missing p19 expression and therefore, redesigned the tac-promoter site using primer extension PCR. We increased the spacer length as published by Mulligan, M. E., Brosius, J., & McClure, W. R. (Mulligan, M. E., Brosius, J., & McClure, W. R., 1985), inserted a lacO element and added a Shine-Dalgarno sequence. The registry part BBa_K2172009 served as the design template.

Production of pUC19-p19-siRNA empty vector

pUC19-p19 6xHis-siRNA empty was produced by restriction based cloning of PCR amplified pUC19 Backbone and p19-siRNA empty construct. PCR was performed using Phusion 2x Master Mix with HF Buffer (New England BioLabs) with 0.5 µM forward primer and 0.5 µM reverse primer and around 1 ng template in a total reaction volume of 20 µL. Success of PCR was validated on 1.2 % agarose gel stained with ethidium bromide.

Recovery of PCR products and restriction digest

The PCR products were pooled, purified with QIAGEN PCR Clean Up Kit and concentration was measured with Nanodrop 2000. 500 ng of each PCR was digested with 10 units BamHI and 10 units ClaI (NEB) for 1 h at 37 °C in 50 µL. Samples were recovered with QIAGEN PCR Clean Up Kit and concentration was measured with a Nanodrop 2000.

Ligation and Transformation of pUC19-p19-siRNA empty

The fragments were ligated at a 3:1 (Insert:Vector) molar ratio, 100 ng vector, with T4 DNA Ligase (Jena Bioscience) at 23 °C for 30 min in a total volume of 20 µL in duplicates. 5 µL of each ligation was transformed into NEB5α competent E. coli (NEB) according to manufacturer's protocol, plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight.

Of each transformation four positive clones were picked and inoculated into 5 mL LB-Amp media (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm overnight. The plasmids were isolated from 4 mL of each overnight culture with QIAGEN Plasmid Mini Kit according to manufacturer's protocol and eluted into 30 µL of the provided elution buffer. Concentration was measured with Nanodrop 2000.

Quality control

For quality control, restriction digestion was performed. 500 ng plasmid was digested with EcoRI-HF (NEB) to assure correct size and insert presence. It was analysed on 1.2 % TAE-Agarose gel stained with ethidium bromide.

The construct was sequenced with p19-Seq.-fwd. and pUC19-AmpR-rev.

Amplification of siRNA-target area

Designated siRNA target area was amplified by PCR using two different primer sets. pEX-A258-UL19-6xHis served as template. PCR was performed using Phusion 2x Master Mix with HF Buffer (New England BioLabs) with 0.5 µM forward primer and 0.5 µM reverse primer and ~1 ng template in a total reaction volume of 20 µL. The success of the PCR was validated on 1.2 % agarose gel stained with ethidium bromide. PCR products were pooled, purified with QIAGEN PCR Clean Up Kit and their concentration was measured with Nanodrop 2000.

Restriction digest of PCR products and pUC19-p19-siRNA empty with SacI-HF and XhoI

500 ng of pUC19-p19 6xHis-siRNA empty and 1 µg UL19 siRNA target PCR Product obtained by primer set 1 were digested with 20 units of SacI-HF and XhoI in CutSmart buffer in a total volume of 50 µL at 37 °C for 2 h. Products were recovered with the QIAGEN PCR Clean Up Kit and concentrations were measured with Nanodrop 2000.

Ligation and Transformation of pUC19-p19-siRNA 1/2

Fragments were ligated at a 5:1 (Insert:Vector) molar ratio, 100 ng vector, with T4 DNA Ligase (Jena Bioscience) at 16 °C for 30 min in a total volume of 20 µL. 5 µL of the ligation was transformed into NEB5α competent E. coli (NEB) according to manufacturer's protocol, plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight. Of each transformation four positive clones were picked, inoculated into 5 mL LB-Amp media (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm overnight. Plasmids were isolated from 4 mL of each overnight culture with the QIAGEN Plasmid Mini Kit according to manufacturer's protocol and eluted into 30 µL of the provided elution buffer. Concentration was measured with Nanodrop 2000.

Restriction digest of PCR products and pUC19-p19-UL19 siRNA 1/2 with SalI-HF and NotI-HF

500 ng of a positive clone and 1 µg of UL19 siRNA target PCR product obtained by primer set 2 were digested with 20 units of NotI-HF and SalI-HF in CutSmart buffer in a total volume of 50 µL at 37 °C for 2 h. Products were recovered with the QIAGEN PCR Clean Up Kit and concentrations were measured with Nanodrop 2000.

Ligation and Transformation of pUC19-p19-UL19 siRNA

Fragments were ligated at a 5:1 (Insert:Vector) molar ratio, 100 ng vector, with T4 DNA Ligase (NEB) at 4 °C overnight in a total volume of 20 µL. 5 µL of each ligation was transformed into NEB5α competent E. coli (NEB) according to manufacturer's protocol, plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight.

Of each transformation six positive clones were picked, inoculated into 5 mL LB-Amp media (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm overnight. Plasmids were isolated from 4 mL of each overnight culture with the QIAGEN Plasmid Mini Kit according to manufacturer's protocol and eluted using 30 µL of the provided elution buffer. Concentration was measured with Nanodrop 2000.

Quality control

For quality control 500 ng of plasmid was digested with 20 units SacI and 20 Units XhoI, 20 units NotI-HF and 20 units SalI-HpF, 20 units SacI-Hf and NotI-HF to assure correct size and presence of both inserts. Results were analysed on 1.2 % TAE-Agarose gel. Positive clones were sequenced for presence of loop structure with p19-fwd. and pUC19-AmpR-rev.

Insertion of tac-LacO-SD-Extension by PCR

We inserted a 7 bp long fragment by primer extension PCR between the tac promoter and the p19 starting codon using the primers sets pUC19-ori-fwd./tac-tac Extension-rev. and p19-tac Extension-fwd/p19-HindIII-rev in two different PCR reactions as described earlier with pUC19-p19-UL19 siRNA serving as template. Success of PCR was validated on 1.2 % TAE-Agarose gel stained with ethidium bromide. All successful PCR products were pooled and recovered with the QIAGEN PCR Clean Up Kit and concentration was measured with Nanodrop 2000. Both products were used as a template at equimolar ratios for ligation of both fragments by PCR using the primers pUC19-ori-fwd. and p19-HindIII-rev. Results were validated on 1.2 % TAE-Agarose gel. The PCR product with the correct size (502 bp) was extracted from the gel and recovered with the Qiagen Gel Extraction Kit.

500 ng of the obtained PCR product and pUC19-p19-UL19 siRNA were each digested with 10 units ClaI and 10 units of EcoRI (NEB) for 1 h at 37 °C separately. The restriction digests were purified using the Qiagen Gel Extraction Kit. The concentration was measured using Nanodrop 2000.

The fragments were ligated at a 3:1 (Insert:Vector) molar ratio, 100 ng vector, with T4 DNA Ligase (Jena Bioscience) at 16 °C for 30 min in a total volume of 20 µL. 5 µL of the ligation was transformed into NEB5α competent E. coli (NEB) according to manufacturer's protocol, plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight. Of each transformation four positive clones were picked, inoculated into 5 mL LB-Amp media (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm overnight. Plasmids were isolated from 4 mL of each overnight culture with the QIAGEN Plasmid Mini Kit according to manufacturer's protocol and eluted into 30 µL of the provided elution buffer. Concentration was measured with Nanodrop 2000.

Process was repeated for insertion of LacO and Shine-Dalgarno Sequence using primer sets pUC19-ori-fwd./tac-LacO-SD-rev. and tac-LacO-SD-p19-fwd./HindIII-p19-rev with the previously produced plasmid served as template. For ligation of both fragments pUC19-ori-fwd. and p19-HindIII-rev. were used.

The quality control was conducted by restriction digest of 500 ng plasmid with HindIII and ClaI and subsequent analysis on a 1.2 % Agarose gel. Sequence congruence was assessed by NGS with pUc19-ori-fwd., p19-fwd. and AmpR-rev.

Evaluation of p19 expression and functionality of RNA expression cassette

Assessment of protein expression

100 ng of pUC19-p19 6xHis-UL19 siRNA was transformed into NEB high efficiency T7 Express lysY/I^q competent E. coli (NEB C3016I) according to manufacturer’s protocol. Transformants were plated onto LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight.

One positive clone was selected, inoculated into 300 mL LB-Amp (100 µg/mL Ampicillin) and incubated at 37 °C and 180 rpm. OD₆₀₀ was measured every hour with a conventional photometer. After OD₆₀₀ reached 0.4 to 0.6 protein expression was induced by addition of 300 µL of 500 mM IPTG (0.5 mM final concentration). The cells were incubated at 37 °C for 2 h. Biomass was harvested by centrifuging at 3200 g, 4 °C for 45 min. The produced pellets were resuspended in 10 mL cold lysis buffer (50 mM phosphate buffer (pH 7.5), 300 mM NaCl, 10 mM Imidazole, 1 % (vol/vol) Triton X-100, 1 mg/mL lysozyme), transferred into a 50 mL Falcon tube and incubated at 4 °C on a rocking incubator for 1 h. Afterwards, the lysate was sonicated with an OmniRuptor Pulse for two minutes at 30 % power with ten second intervals on ice. The lysate was centrifuged at 4000 g, 4°C for 45 minutes. Supernatant was filtered clear using a 0.45 µm followed by a 0.22 µm syringe filter into a 15 mL falcon tube. 1 mL of Ni-NTA beads were added to the supernatant and the mixture was incubated at 4°C on a rocking incubator for 12 h. Ni-NTA beads were washed by centrifuging at 500g, 4°C for 10 minutes and removing the supernatant. 5 mL of fresh lysis buffer (without lysozyme) was added and beads were incubated at 4 °C on a rocking incubator for 10 minutes. This was repeated three times.

The purified p19 protein was separated from the beads by addition of 500 µL of lysis buffer (1 % SDS, 1 mM EDTA, 250 mM Imidazole in PBS pH 7.4) and centrifugation at 500g for 5 min at room temperature. The purified protein was then incubated on ice for 10 min to precipitate excess SDS present in the lysis buffer. The solution was centrifuged clear at 16000 rpm at a temperature of 4 °C for 15 min.

The protein amount in the purified p19 protein fraction was assessed by a modified Bradford-Assay published by Grinztalis et al.. A BSA standard curve ranging from 10 µg to 100 µg was established. The lysates were diluted to a total protein amount of 50 µg in 10 µL and prepared for the SDS-PAGE. 10 µL of a 2x Laemmli-buffer were added to each sample and heated at 95 °C for 10 min. The samples were then allowed to cool down at room temperature. All samples were loaded onto a 12 % Tris-SDS-PAGE. A loading control of 5 µL prestained protein ladder was loaded as a size standard. The gel was run at 110 V for 1 ½ h on ice. The proteins were extracted from the gel by semi dry Western Blotting on a nitrocellulose membrane at 15 V for 30 min. The blot membrane was blocked with 5 % milk powder in TBS-T for 1 h. Afterwards the blot was incubated with primary antibody Anti-His-tag mouse monoclonal antibody (1:10000, Proteintech) in a blocking solution, supplied with 10 mM sodium azide at 4 °C overnight. Secondary antibody solution (1:5000, Proteintech) in blocking solution supplied with 10 mM sodium azide at 4°C for two hours. The membrane was washed five times for 3 min to remove unselective bound antibodies. After that, the membrane was stained with DAB to detect the p19 protein. The images were analysed using a ChemiDoc XRS+ (BioRad Laboratories).

In vitro transcription of cloned UL19 RNA-sequences

To assess the functionality of our T7-driven expression cassette HiScribe-T7 transcription kit from NEB was used. 100 ng of linearized pUC19-p19-UL19 siRNA plasmid were in a reaction according to manufacturer’s protocol in a total volume of 20 µL and incubated at 37 °C overnight. An aliquot was taken from the crude sample. The DNA template was removed by a DNase I digest at 37 °C for 10 min. This was followed by the clean-up with Monarch RNA clean-up kit (NEB). An aliquot was taken from the purified sample for comparison.

Discussion

We added a 7 bp consensus sequence of the tac promoter, a Lac Operator and a Shine-Dalgarno sequence. This 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 has the same function as the Kozak sequence in eukaryotes but in prokaryotes 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 is something that Huang and Lieberman didn’t include in their published sequence (Huang & Lieberman, 2013). Due to this more precise expression control especially of the dsRNA cassette the 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. For overnight expression our findings suggest a different type of protein-tag which is more unique and has a very high affinity for its ligand such as a GST-Tag or FLAG-Tag should be used (Lichty et al., 2005). His-Tag is very much suitable for short induction periods for example one to two hours as proposed by Huang and Lieberman (Huang & Lieberman, 2013). The proposed expression system for pro-siRNAs is a simple workflow which can be achieved with basic techniques of molecular biology and is therefore a simple and efficient way once established to test different sequences for presence of possible siRNA sequences or to facilitate a knockdown of genes with a high amount of polymorphism or which are prone to mutate likely, such as viral genomes. We are confident that the obtained pro-siRNAs are capable of initiating the knock-down of our target gene.