iGEM 2022 — Team heidelberg


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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# Engineering Success

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

![](https://static.igem.wiki/teams/4344/wiki/wetlab/engineering/loop-folding-unafold.png)

**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 NEB Tm- Calculator (https://tmcalculator.neb.com/#!/main) 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 (Fig 2A). An annealing temperature of 69 °C was chosen and the PCR was repeated (Fig 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.

Here sequence alignments can be found for [pUC19-*p19*-empty p19F](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/sequencing-files/puc19-p19-sirna-empty-p19f.pdf) and [pUC19-*p19*-empty AmpR](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/sequencing-files/puc19-p19-sirna-empty-ampr.pdf).

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 (Fig 2D). We compared the resulting fragment sizes to the theoretical sizes obtained by digest in NEB Cutter v3 (Tab. 1).

**Table 1: Comparison of theoretical fragment sizes and obtained sizes.** Plasmids were isolated with QIAGEN Spin Mini Kit and fragmented with EcoRI & HindIII followed by analysis on a 1,2 % Agarose gel. 500 ng of NEB 1kb+ served as a size marker. Plasmid sequence was digested using NEB Cutter v3 to obtain theoretical fragment sizes.

| Plasmid | Theoretical No. of fragments | Theoretical size of obtained fragments [bp] | Obtained No. of fragments | Obtained fragment sizes [bp] |
| --- | --- | --- | --- | --- |
| pUC19-*p19*-siRNA *UL19* | 2 | 2758 + 547 | 2 | ~2500 + ~550 |

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.

Here sequence alignments can be found for [pUC19-p19-siRNA UL19 p19F](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/sequencing-files/puc19-p19-ul19-sirna-p19f.pdf) and [pUC19-p19-siRNA UL19 AmpR](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/sequencing-files/puc19-p19-ul19-sirna-ampr.pdf).

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 (UL 19 dsRNA.pdf ).

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

Sequence Alignments for pUC19-*p19*-siRNA tac extension can be found: [here](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/sequencing-files/puc19-p19-ul19-sirna-tac-extension.pdf).

After the successful cloning of pUC19-*p19*-siRNA *UL19* in, 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 (Fig 2E). We compared the obtained fragment sizes to the theoretical sizes acquired by digest in NEB Cutter v3 (Tab. 2).

**Table 2: Comparison of theoretical fragment sizes and obtained sizes.** Plasmids were isolated with QIAGEN Spin Mini Kit and fragmented with ClaI & HindIII followed by analysis on a 1,2 % Agarose gel. 500 ng of NEB 1kb+ served as a size marker. Plasmid sequence was digested using NEB Cutter v3 to obtain theoretical fragment sizes.

| Plasmid | Theoretical No. of fragments | Theoretical size of obtained fragments [bp] | Obtained No. of fragments | Obtained fragment sizes [bp] |
| --- | --- | --- | --- | --- |
| pUC19-*p19*-siRNA *UL19 tacO-LacO* | 2 | 3005 + 341 | 2 | ~3000 + ~350 |

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**.

Sequence Alignments for pUC19-*p19F*-UL19 siRNA Tac-LacO-SD pBR322 fwd can be found: [here](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/sequencing-files/taco-laco-pbr322-fwd.pdf).

![](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/fig-2.png)

**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 70,5 °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-p*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 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 (Fig 4C).

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 4B**).

![](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/fig-3.png)

**Figure 3: Bradford and gelelectrophoresis**

**Table 6: Protein amounts of different fractions.**Protein amounts were calculated using Bradford standard curve (compare **Figure 3A**) using OD obtained at 610 nm in duplicates. Samples 1C and p19-1 were loaded on SDS PAGE and analysed by Western Blot.

| Sample | OD1 1:10 [610 nm] | OD2 1:10 [610 nm] | mean | standard deviation | c (diluted) [µg/mL] | c (undiluted) [µg/mL] |
| --- | --- | --- | --- | --- | --- | --- |
| 1a | 0.109 | 0.099 | 0.104 | 0.007 | 74.3 | 742.9 |
| 1b | 0.033 | 0.034 | 0.010 | 0.001 | 23.9 | 239.3 |
| 1c | 0.363 | 0.335 | 0.363 | 0.020 | 249.3 | 2492.9 |
| P19 1 | 0.336 | 0.355 | 0.336 | 0.001 | 239.6 | 2396.4 |
| 2a | 0.123 | 0.127 | 0.125 | 0.003 | 89.3 | 892.9 |
| 2b | 0.024 | 0.023 | 0.024 | 0.001 | 16.8 | 167.9 |
| 2c | 0.365 | 0.360 | 0.363 | 0.004 | 258.9 | 2589.3 |
| P19 2 | 0.368 | 0.369 | 0.369 | 0.008 | 263.2 | 2632.1 |

## Production of pro-siRNA in *E. coli* *T7* Express *lysY/Iq*

Production of pro-siRNA was analysed by native PAGE. This resulted in a methylene blue stained gel (Fig 4). For each fraction of 500 ng two different fragment patterns can be observed.: oOne above the positive control (siRNA 21 nts) and one slightly below the positive control. Fragments running roughly at the same height as the positive control are therefore between 18 and 21 nts in size. These were cut from the gel for further processing.

![](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/fig-4.png)

**Figure 4: Purified pro-siRNAs on a native TBE-PAGE.** 500 ng of eluted RNAs (Input samples p19 Isolation) were loaded on a 15 % native TBE-PAGE and stained with methylene blue. A distinct fragment pattern is visible with the fragments with roughly the same height as the positive control deemed with size between 18 and 21 nts.

## Discussion

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).

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Experiments siRNA

## Production and Analysis of siRNA loaded liposomes for treatment of HSV infection

We adapted a method for production of recombinant siRNAs in *E. coli* published by Huang *et al.* in 2013
(Huang *et al.*, 2013; Huang & Lieberman, 2013).

To evaluate our chosen method in a simple and efficient way, the *EGFP* gene was chosen as a proof-of-concept target
for our liposomal siRNA delivery system. We used the p*EGFP*-C1 plasmid (Addgene No. 2487) containing the *EGFP*
gene published by Yang *et al.* (Yang *et al.*, 1998). We then selected the gene
*UL19* encoding for HSV-1 major capsid protein for evaluation of potential HSV treatment methods using liposomal formulations.

## Plasmid design

The sequence of *UL19* encoding for HSV-1 major capsid protein was extracted from the complete
[human herpesvirus 1 genome](https://www.ncbi.nlm.nih.gov/nuccore/KF498959.1). The sequence was human codon optimised
using [Benchling](https://www.benchling.com/).

The sequence was screened for published antibody binding sites and functional structure elements using the
[UniProt database](https://www.uniprot.org/). 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.

## Production of pRK5-HSV *UL19* AA816:1148-6xHis

100 ng of pEX-A258-HSV-*UL19*-6xHis and p*EGFP*-C1 were transformed into competent *E. coli* DH5α
(New England BioLabs) according to manufacturer’s protocol. pEX-A258-HSV-*UL19*-6xHis transformants were plated onto
LB-Amp plates (100 µg/mL Ampicillin) and incubated at 37 °C overnight. p*EGFP*-C1 transformants were plated onto LB-Kan plates
(75 µg/mL Kanamycin) and incubated at 37 °C overnight. Subsequently they were inoculated into 5 mL LB-Amp (100 µg/mL Ampicillin) or
LB-Kan (75 µg/mL Kanamycin) media and incubated at 37 °C at 180 rpm overnight. The plasmids were isolated from 4 mL of overnight culture
with the QIAPREP Spin Miniprep Kit. For quality control, 500 ng of each plasmid were digested with 10 units of EcoRI-HF
(New England BioLabs) in a total volume of 30 µL and analysed on a 1.2 % TAE-Agarose gel stained with ethidium bromide.

100 ng of pRK5 were transformed into competent *E. coli* NEB10-beta (NEB). The pRK5 transformants were plated onto LB-Amp plates
(100 µg/mL Ampicillin) and incubated at 37 °C overnight before subsequently being inoculated into 5 mL LB-Amp (100 µg/mL Ampicillin)
and incubated at 37 °C at 180 rpm overnight. Plasmids were then isolated from 4 mL of the overnight culture with the
QIAPREP Spin Miniprep Kit. For quality control 500 ng of each plasmid were digested with 10 units of EcoRI-HF (New England BioLabs)
in a total volume of 30 µL and analysed on a 1.2 % TAE-Agarose gel stained with ethidium bromide.

The congruence of the sequence was confirmed by NGS for pRK5 and p*EGFP*-C1 using CMV-Forward and SV40A-Reverse.

## Amplification of *UL19* AA816:1148-6xHis by PCR and Insertion of the Kozak-Sequence

A missing Kozak-sequence was inserted to enhance the translation of HSV *UL19* AA816:1148-6xHis via PCR.
The HSV *UL19* fragment was amplified with PCR using Phusion 2x Mastermix with HF-Buffer (NEB), 0.5 µM pRK5-Kozak-fwd.,
HindIII-HSV *UL19*-rev. and roughly 1 ng of pEX-A258-HSV-*UL19*-6xHis, the total reaction volume being 20 µL,
for this purpose. The success of the PCR was evaluated on a 1.2 % TAE-Agarose gel stained with ethidium bromide.
Successful PCR products were pooled and purified using the Qiagen PCR Clean-Up Kit. The concentration was measured using Nanodrop 2000.

**Table 1: Primers used for production of *UL19* AA816:1148-6xHis insert.** Primers were ordered at IDT. Tm describes the calculated melting temperature by IDT. TA describes the proposed annealing temperature, calculated with NEB Tm Calculator. TPCR describes the finally used annealing temperature.

| Primer | Sequence | Tm [°C] | TPCR [°C] |
| --- | --- | --- | --- |
| pRK5-Kozak-fwd. | 5’-CCAACTGCACCTCGGTTCTATCGATTGAATTCGCCACCATGGTGCCTGCGTTCAGTCGAG-3’ | 72.1 | 68 |
| HindIII-HSV *UL19*-rev. | 5’-ATCCAAGCTTTTAGTGATGGTGATGGTGATGTCGT-3’ | 63.5 | 68 |

## Restriction Digest of *UL19* AA816:1148-6xHis and pRK5 backbone

500 ng of the obtained PCR product and pRK5 were each digested with 10 units of EcoRI-HF and 10 units of HindIII-HF (NEB) for 1 h at
37 °C separately. The restriction digests were then purified using the Qiagen PCR Clean-Up Kit. The concentration was measured using
Nanodrop 2000.

## Ligation and Transformation

The fragments were ligated at a 3:1 (insert:vector) molar ratio. The 65.22 ng of *UL19* insert were thus ligated with 100 ng of
pRK5 vector using T4 ligase (Jena Biosciences) in a total reaction volume of 20 µL (supplied with 2 µL 10x Standard Ligation Buffer)
at 16 °C for 30 min. After that, the ligase was inactivated at 65 °C for 15 min and chilled on ice afterwards for 5 min. 5 µL of the
ligation mix were transformed into NEB 5α competent *E. coli* (NEB) according to manufacturer’s protocol, plated onto LB-Amp plates
(100 µg/mL Ampicillin) and incubated at 37 °C overnight. 12 clones were picked,inoculated into 5 mL LB-Amp (100 µg/mL Ampicillin) and
incubated at 37 °C at 180 rpm overnight. The plasmids were isolated from 4 mL of the overnight culture with the QIAPREP Spin Miniprep Kit
according to the manufacturer's protocol. For quality control 500 ng of each plasmid were digested with 10 units of EcoRI-HF
(New England BioLabs) and 10 units of HindIII-HF (NEB) in a total volume of 30 µL and analysed on a 1 % TAE-Agarose gel stained with
ethidium bromide.
The congruence of the sequence was confirmed by NGS for pRK5-HSV *UL19* AA816:1148-6xHis using CMV-Forward and SV40A-reverse primer.

## 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’- TCTAGAGCGCACGTA*tac*ACGCGCTGATCAGC-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](http://parts.igem.org/Part%3ABBa_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.

**Table 2: Primers used for production of *UL19* AA816:1148-6xHis insert.** Primers were ordered at IDT. Tm describes the calculated melting temperature by IDT. TA describes the annealing temperature used in the PCR, calculated with NEB Tm Calculator.

| Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| --- | --- | --- | --- | --- |
| pUC19-BamHI-fwd. | 5’-TCCGGATCCGAAACGCGCGAGACGAAAGGG-3’ | 68.9 | 72.0 | 69.0 |
| pUC19-ClaI-rev. | 5’-ATCCATCGATTCAGGGGATAACGCAGGAAAGAACA-3’ | 64.4 | 72.0 | 69.0 |
| *p19*-BamHI-rev. | 5’-GGATGGATCCGCAAAAGGCCAG-3’ | 60.9 | 69.0 | 68.0 |
| *p19*-ClaI-fwd. | 5’-ATTATCGATAACGCCAGCAACGCG-3 | 59.9 | 69.0 | 68.0 |

## 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.

**Table 3: Primers used for production of siRNA target areas of *UL19* AA816:1148-6xHis insert.** Primers were ordered at IDT. Tm describes the calculated melting temperature by IDT. TA describes annealing temperature used in the PCR, calculated with NEB Tm Calculator.

| Part-Nr. | Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| --- | --- | --- | --- | --- | --- |
| BBa\_K4344055 | SacI-HSV-*UL19*-6xHis-fwd. | 5’-ATGAGCTCGTCGTACCTGAGATTGCTCCAG-3’ | 64.0 | 72.0 | 65.0 |
| BBa\_K4344075 | XhoI-HSV-*UL19*-6xHis-rev. | 5’-ATCTCGAGTGTTAGTGGTAGAGGCGGTGTTG-3’ | 63.7 | 72.0 | 65.0 |
| BBa\_K4344033 | NotI-HSV-*UL19*-6xHis-fwd. | 5’-ATGCGGCCGCGTCGTACCTGAGATTGCTCCAG-3’ | 70.0 | 72.0 | 65.0 |
| BBa\_K4344059 | SalI-HSV-*UL19*-6xHis-rev. | 5’-ATGTCGACTGTTAGTGGTAGAGGCGGTGTTG-3’ | 64.0 | 72.0 | 65.0 |

## 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.

**Table 4: Primers used *tac*-LacO-SD-Extension PCR.** Primers were ordered at IDT. Tm describes the calculated melting temperature by IDT. TA describes the annealing temperature used in the PCR, calculated with NEB Tm Calculator.

| Part-Nr. | Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| --- | --- | --- | --- | --- | --- |
| BBa\_K4344052 | pUC19-pBR322-ori-fwd | 5’-GGGAAACGCCTGGTATCTTT-3’ | 55,1 | 63.0 | 63.0 |
| BBa\_K4344071 | *Tac-Tac* Extension-rev. | 5’-TTTCCTTGTATAGCTCGTTCCATCACCACACCATTATACGAGCCGATGATTAATTGTCAA-3’ | 67.5 | 63.0 | 63.0 |
| BBa\_K4344039 | *p19-Tac* Extension-fwd. | 5’-TTGACAATTAATCATCGGCTCGTATAATGGTGTGGTGATGGAACGAGCTATACAAGGAAA-3’ | 67.5 | 65.0 | 63.0 |
| BBa\_K4344037 | *p19*-HindIII-rev | 5’-AGTGAAGCTTCCGTCCTGTC-3’ | 56.9 | 65.0 | 63.0 |
| BBa\_K4344052 | pUC19-pBR322-ori-fwd. | 5’-GGGAAACGCCTGGTATCTTT-3’ | 55.1 | 63.0 | 63.0 |
| BBa\_K4344037 | *p19*-HindIII-rev. | 5’-AGTGAAGCTTCCGTCCTGTC-3’ | 56.9 | 63.0 | 63.0 |

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/Iq* 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.

# Production of pro-siRNA in *E. coli* *T7* Express *lysY/Iq*

## Bacterial culture

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

One positive clone was selected and inoculated into 100 mL LB-Amp (100 µg/mL Ampicillin) and incubated at 37 °C, 180 rpm.
OD600 was measured every hour with a conventional photometer. After OD600 reached 0.4 to 0.6 production was
induced by addition of 100 µL of 500 mM IPTG (0.5 mM final concentration). The cells were incubated at 20°C overnight.

## Biomass harvest and lysis

Biomass was harvested by centrifuging at 3200 g, 4°C for 45 min. The resulting 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. Lysate was sonicated with an OmniRuptor Pulse for two minutes,
at 30 % Power with ten second intervals on ice. 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.

## Recovery of *p19* bound pro-siRNA

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.

Ni-NTA beads were resuspended in 500 µL of SDS buffer (50 mM phosphate (pH 7.5), 100 mM NaCl and 0.5 % (wt/vol) SDS)
and incubated at 700 rpm on an Eppendorf Mixer for 10 minutes. Beads were pelleted by centrifugation at 500 g for 1 min and
supernatant was transferred in a fresh 1.5 mL tube. This procedure was repeated once and the obtained supernatants were combined.
Total amount per production 1 mL.

100 µL of cold 2 M KCl was added and the mixture was incubated on ice for 10 min. The precipitate was pelleted by centrifugation for
5 min at 16000 g, 4 °C. The supernatant was split into two samples containing around 500 µL and 50 µL of 3 M sodium acetate. 550 µL of
isopropanol and 10 µg glycogen were added to precipitate pro-siRNAs. Samples were incubated at -20 °C for 2 h.

pro-siRNAs were pelleted through centrifugation at 16000 g, 4°C for 30 min. Supernatant was discarded and the pellet was washed with 1 mL
of ice-cold 75 % (vol./vol.) ethanol. Supernatant was carefully removed. Pellets were air-dried for 5 min and resuspended in 100 µL of
nuclease-free water. Concentration was measured with IMPLEN Nanophotometer.

## Isolation of pro-siRNA

15 % (wt/vol) TBE-PAGE was used for separation of pro-siRNA. 500 ng of pro-siRNAs were mixed with 1 volume of gel loading buffer.
Gel was run at 150 V until bromophenol blue line was 1 cm from the bottom of the gel.

The gel was stained with methylene blue staining solution (0.2 % methylene blue in 0.4 M Sodium acetate : 0.4 M acetic acid (1:1)) for 15 min.
Background staining was removed by incubation in distilled water. The pro-siRNA bands (~21-25 nts) were identified and cut from the gel.

Gel fragments were cut into pieces, transferred into a 1.5 mL tube and incubated in >500 µL 0.3 M NaCl at 4 °C on a rocking incubator
overnight. 500 µL of isopropanol (500 µL isopropanol per 500 µL 0.3 M NaCl) and 10 µg glycogen were added to precipitate pro-siRNAs.
The samples were incubated at -20 °C for 2 h. pro-siRNAs were pelleted through centrifugation at 16000 g, 4°C for 30 min.
The supernatant was discarded and the pellet was washed with 1 mL of ice-cold 75 % (vol./vol.) ethanol.
Any remaining supernatant was carefully removed. The pellets were air-dried for 5 min and resuspended in 100 µL of nuclease-free water.
Concentration was measured with Nanodrop 2000.

# Liposome production

## Lipid stocks

The lipids and cholesterol were weighed in a 25 mL volumetric flask. The flask was filled up with CHCl3:MeOH (9:1)
and shaken until the lipid was dissolved. 4 mL aliquots were stored at -20 °C until usage. For cholesterol the end concentration was
set to 100 mM, for mPEG2000 10 mM, for DOTAP 46 mM and for DOPE 42 mM.

## Liposome production method: Two step dual centrifugation and three step dual centrifugation

### Lipid film production

The lipid stocks were thawed to room temperature and volumes pipetted together in ratios set to the desired composition
(mPEG:cholesterol:DOTAP:DOPE, 0.3:2:1:1), which can be seen in **Table 5**. For the 1 mM and 0.185 mM lipid films 56 µL and 91 µL
methanol was added to the lipid solution. After mixing the lipid stocks, they were heated in a heating block to 50 °C and steamed with
N2 gas for 15 to 45 minutes until the organic solvent had evaporated. After that step, the vial was dried under vacuum for
1 hour. The final lipid films were stored at -20 °C with the lid taped shut with parafilm.

**Table 5: Pipetting scheme for 100 mM, 1 mM and 0.185 mM lipid films.** Volumes needed for differently concentrated lipid films for mPEG, cholesterol, DOTAP and DOPE are shown.

| Final liposome concentration | 100 mM | 1 mM | 0.185 mM |
| --- | --- | --- | --- |
| mPEG2000 (10 mM stock) | 139.4 µL | 14 µL | 3 µL |
| Cholesterol (100 mM stock) | 93.0 µL | 9 µL | 2 µL |
| DOTAP (46 mM stock) | 101.1µL | 10 µL | 2 µL |
| DOPE (42 mM stock) | 110.8 µL | 11 µL | 2 µL |

### Liposome production by dual centrifugation

Dual centrifugation is used in combination with SiLibeads to rehydrate and break the lipid film into liposomes.
We compared two protocols: two step centrifugation and three step centrifugation. For each protocol the lipid film had to be thawed to
room temperature.

Two step dual centrifugation (2-fold DC):
SiLibeads (type SL) were added to the lipid film together with the first volume of PBS and centrifuged for 15 min at 2500 rpm
in the Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9. The amount of PBS volume was calculated with the following
formula. If the formula gives a volume below 2 µL, 2 µL PBS should be used instead.

The vial was removed from the centrifuge and the second volume of PBS was added. It was centrifuged for 5 min at 2500 rpm in the
Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9. The PBS volume was calculated with the following formula.
If the formula gives a volume below 2 µL, 2 µL PBS should be used instead.

Three step dual centrifugation (3-fold DC):
This protocol was only used for DNA and RNA loaded liposomes. 2 µL of DNA or siRNA (containing 3 µg of oligonucleotides)
in PBS were added to the lipid film and the vial was centrifuged for 15 min at 2500 rpm in the Zentrimix 380 R at 20 °C with
acceleration of 5 and deceleration of 9.

The vial was removed from the centrifuge and SiLibeads (type SL) were added. It was centrifuged again for 15 min at 2500 rpm in the
Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9.

The vial was removed from the centrifuge and 2 µL PBS were added. It was centrifuged for a third time for 5 min at 2500 rpm in the
Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9. The vial was removed and 96 µL PBS were added.
The vial was vortexed.

## Liposome production method: VPG method

### Preparation of Dry Lipid Mixture

In the protocol published by Hirsch *et al.* in 2008, dry lipids were dissolved in ethanol. Since we had already solved our
lipids in CHCl3:MeOH (9:1), we pursued two different ways we could implement their procedure.

For testing whether alternative solvents made a difference in liposome production, lipids solved in CHCl3:MeOH and
lipids solved in ethanol were used. To extract the lipids for the ethanol experiment, the CHCl3:MeOH dissolved lipids
were dried under N2 and solved in 400 µL ethanol. Lipid solutions with a total lipid mass of 24 mg were created with both lipids
solved in ethanol and lipids solved in CHCl3:MeOH (59.5 µL mPEG, 288.7 µL cholesterol, 173.7 µL DOTAP and 178.6 µL DOPE).
After pipetting the volumes in the respective tubes, the lipid solutions were stripped of the solvent under N2 and finally put under
vacuum for 90 min at 35 mbar. The respective dry lipid mixtures can be stored in the fridge.

### Liposome production by dual centrifugation

Dry lipid mixtures were thawed and 36 µL of PBS added to reach a final batch weight of 60 mg. Beads were added in a 1:1 ratio
to the mass (60 mg beads). The samples were incubated at room temperature for 10 min and afterwards centrifuged for 30 min at
2500 rpm in the Zentrimix 380 R at 20 °C with acceleration of 5 and deceleration of 9. The vial was removed from the centrifuge
and 120 µL PBS (200 % PBS in relation to VPG) were added. It was centrifuged for 30 s at 2500 rpm in the Zentrimix 380 R at 20 °C
with acceleration of 5 and deceleration of 9. Samples were stored at 4 °C.

## Size and PDI measurement

For measuring the size (hydrodynamic diameter, dH) of liposomes and their polydispersity index (PDI) the
Malvern Zetasizer Nano series Nano-ZS was used. The sample was directly prepared in a polystyrol/polystyrene cuvette from Sarstedt.
1 to 10 µL of sample (depending on sample concentration) was added to PBS for a total volume of 1000 µL and measured in the zetasizer
with a size measuring SOP.

## Zeta potential measurement

For measuring the zeta potential of liposomes the Malvern Zetasizer Nano series Nano-ZS was used. The sample preparation was performed in
a reaction tube. 1 to 10 µL of sample (depending on sample concentration) was added to 10 % PBS for a total volume of 1000 µL.
The sample was applied to a folded capillary zeta cell and the sample measured with a zeta potential measuring SOP.

## Concentrate decoy DNA and siRNA in vacuum centrifuge

For concentrating nucleotides we evaporated the solvent in a SpeedVac SC100. For the decoy DNA primers we used medium heat with a
vacuum of below 30 bar for 1 hour or longer until the desired concentration was achieved. The sample was measured with a NanoDrop 2000.
For siRNA the vacuum centrifuge was set on low temperature at under 30 bar for 1 hour or longer.

## NanoDrop measurement of nucleic acids

For different experiments knowing the concentration of DNA primers and siRNA was elemental. We used a NanoDrop 2000 for this task.
1 µL of water or 1 µL of the buffer that the nucleic acid was dissolved in was used as blank. The next step was to measure 1 µL of the
sample. The measuring device was cleaned before and after each measurement with a non-linting cloth.

## Agarose gel for encapsulation efficiency

In order to evaluate the encapsulation efficiency (EE), we adapted a protocol published by Li & Al-Jamal in 2021.
First, 20 µL of the final liposome solution were loaded with an appropriate amount of loading buffer (in our case 7 µL of
6x Loading Buffer, to ensure that the solution completely drops down and stays in the pocket) onto an 1.8 % Agarose gel stained with
1:10000 ethidium bromide. It was run for 20 min at 85 V in 1x TAE, so that free DNA or siRNA was separated from the liposomes that
had encapsulated DNA or siRNA. Since Liposomes are larger than free nucleotide strands, they stayed within the pocket or moved
significantly slower.
To effectively analyse the amount of nucleotides that was not encapsulated by the liposomes, DNA or siRNA was pipetted into one
pocket as reference. By comparing band intensities with this standard the amount of unencapsulated nucleotides could be measured.
By subtracting and then dividing the obtained value from the initial amount of DNA or siRNA in the given liposome solution, one could
calculate the EE of the liposomes with the following equation.

## Nuclease affinity testing

To test if nucleotides bind to SiLibeads, we incubated a solution of DNA primers in PBS with and without SiLibeads for one day
(100 µL of 0.78 mg/mL DNA with 142 mg SiLibeads). We measured the DNA concentration after 0 min, 5 min, 10 min, 30 min, 1 h, 2 h and
1 d with the NanoDrop 2000 and compared the measurements with a sample of PBS.

## Chitosan coating

In order to coat our loaded liposomes with chitosan, we adapted a protocol published by Mady *et al.* in 2009.
0.3 g chitosan was dissolved in 1 % (w/v) acetic acid. Then, the desired amount of the chitosan solution was added to 10 µL
of the liposome solution in a small Eppendorf tube. The mixture was first incubated at room temperature and 400 rpm in a heat block for 2 h.
Then, it was incubated overnight at -4°C.

## Ion detection reactions

***Fe3+***: A small crystal of NH4SCN was added into 50 µL of the sample to be tested.
Positive control was conducted with a small spatula tip of iron(III)chloride. If the solution contains Fe3+,
it will turn dark red (compare with pos. control), and if not, the solution will retain its original colour.

***Fe2+***: 50 µL of the sample to be tested was first acidified with 10 µL 1M HCl, then a small crystal of
K3[Fe(CN)6] was added to the solution. Positive control was conducted with a small spatula tip of iron(II)chloride.
If the solution contains Fe2+, it will turn blue (or greenish if there is only a small amount of Fe2+,
always compared with pos. control), and if not, the solution will be slightly yellowish due to the colour of
K3[Fe(CN)6] itself in an aqueous solution.

# Cell culture

## Maintenance of HeLa cells

The HeLa cells were grown to 80 to 90 % confluency in DMEM supplied with 10 % FCS and 1 % Pen-Strep (Standard DMEM).
The cells were trypsinized with 0.025 % trypsin-EDTA and seeded at a density of 2.1 \* 10^6 cells in a T75 flask.

## Assessment of expression capabilities of our promoter construct

The cells were seeded at a density of 3 \* 10^5 cells per well in a 6 well-plate in triplicates and incubated for 4 to 6 h at
37 °C in 2 mL standard DMEM. Medium was then replaced with 1.5 mL of Pen-Strep free medium and the cells were then transfected with
6 µg of p*EGFP*-C1 in 10 µL PEI-max in a total volume of 0.6 mL for 12 h at 37 °C, 5 % CO2. Transfection medium was replaced
with 1.5 mL standard DMEM medium before transferring the plate to the IncuCyte. Pictures were then taken every 2 h for 3½ consecutive days.
These were then analysed for determination of *EGFP* expression pattern using R-Studio.

## Transfection with p*EGFP*-C1 and pRK5-HSV *UL19* AA816:1148-6xHis for testing of liposomal formulation

HeLa cells were grown to a confluency of 80 to 90 %, were then harvested with Trypsin-EDTA and seeded at a density of
1.5 \* 10^5 cells in 1 mL of standard DMEM. Cells were left 4 to 6 hours to adhere and were then transfected with either 3 µg of
p*EGFP*-C1 or pRK5-HSV *UL19* AA816:1148-6xHis using PEI-Max. Transfection media was removed after 12 h and replaced with
standard DMEM. After 24 h cells were treated with the liposomal formulations according to **Figure 1**.

![Transfection and Treatment scheme for treatment](https://static.igem.wiki/teams/4344/wiki/wetlab/methods/method-12-treatment.png)

**Figure 1: Transfection and Treatment scheme for treatment.** (A): Transfection setup for each well. *EGFP*-C1 = Transfection with *EGFP*-C1, pRK5-*UL19* = Transfection with pRK5-HSV *UL19* AA816:1148-6xHis, HeLa = no transfection. (B): Treatment of cells after 24h.

Cells were incubated in the respective treatment solution for transfection with

* PEI + pos cntrl: synthetic siRNA against *EGFP* with PEI as transfection reagent
* Lipo + pos cntrl: synthetic siRNA against *EGFP* encapsulated in the liposomal formulation as described above
* PEI + pro-siRNA: produced siRNA against *UL19* with PEI as transfection reagent
* Lipo + pro-siRNA: produced siRNA against *UL19* encapsulated in the liposomal formulation as described above
* Empty lipo: non-loaded liposomal formulation as described above
* PEI: just PEI for qualitative comparison of cytotoxicity
* dsRNA: direct transfection with dsRNA produced by *in-vitro* transcription using pUC19-*p19*-*UL19* siRNA as template using PEI as transfection reagent

after 4 ½ hours. The treatment solution was then removed and replaced with 1 mL standard DMEM. Cells were incubated for 2 ½ days to
wait for knockdown induction. The experiment was then stopped by Trypsin-EDTA treatment an1 mL of standard DMEM was added to stop the
trypsin treatment. The detached cells were transferred into 1.5 mL eppis per well on ice.

# RNA isolation

A 750 µL aliquot was taken from each sample. This aliquot underwent RNA isolation using the QIAGEN RNeasy Plus Mini Kit.
RNA concentration was measured using Nanodrop 2000 and the quality of the isolated RNA was assessed by 2 % agarose-gel electrophoresis.
Each sample was used as a triplicate for bulk-RNAseq with each triplicate containing 350 ng total RNA. Bulk-RNAseq was carried out at
GeneCore Facility (EMBL Heidelberg).

# qPCR for Primer evaluation

Three different primer sets were designed using Primer3 for detection of *EGFP*-RNA and *UL19*-RNA.
Primers were evaluated for proposed annealing temperatures by T-Gradient PCR using 2x Taq Master-Mix (NEB) in a total volume of 25 µL.
Plasmid titration was conducted with each primer set to evaluate primer efficiency and Cut-Off values of the
Luna Universal One-Step RT-qPCR Kit (NEB) using p*EGFP*-C1 and pRK5-HSV *UL19* AA816:1148-6xHis as template
(for plate and reagent setup see **Figure 2**) in a Roche LightCycler480. Reaction setup was carried out according to the
manufacturer's protocol. Standard ranged from 1.31 \* 10^10 to 1.31 \* 10^2 copies/µL and 5.,37 \* 10^9 to 5.37 \* 10^1 copies/µL for
p*EGFP*-C1 and pRK5-HSV *UL19* AA816:1148-6xHis respectively. To validate selective binding High-Resolution-Melt (HRM)
analysis was performed.

![Plate setup for plasmid titration](https://static.igem.wiki/teams/4344/wiki/wetlab/methods/method-12-treatment.png)

**Figure 2: Plate setup for plasmid titration.** (A): Plate setup for p*EGFP*-C1 titration with primer sets 1 to 3. Standards ranging from 1.31 \* 10^10 to 1.31 \* 10^2 copies/µL. (B): Reaction mixture volume scheme. (C): Plate setup for p*EGFP*-C1 titration with primer sets 1 to 3. Standards ranging from 5.37 \* 10^9 to 5.37 \* 10^1 copies/µL. (D): RT-qPCR program for plasmid titration.

**Table 6: Assessed qPCR Primers.** Primers were ordered at IDT. Tm describes the calculated melting temperature by IDT. TA describes the annealing temperature used in the PCR, calculated with NEB Tm Calculator.

| Part-Nr. | Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| --- | --- | --- | --- | --- | --- |
| BBa\_K4344023 | HSV-*UL19*-fwd-1 | 5’-ACCCAATTTTCCACCAGCAC-3’ | 56.1 | 64.0 | 60.0 |
| BBa\_K4344026 | HSV-*UL19*-rev.-1 | 5’-GCTGATGATGTAAGGCCACG-3’ | 56.1 | 64 | 60 |
| BBa\_K4344021 | HSV-*UL19*-fwd-2 | 5’-AGCATTAGACAGGTGGT-3 | 56.3 | 58.0 | 60.0 |
| BBa\_K4344024 | HSV-*UL19*-rev-2 | 5’-CAAATCTGTCTTGGCGCACT-3’ | 56.0 | 58.0 | 60.0 |
| BBa\_K4344022 | HSV-*UL19*-fwd-3 | 5’-TCAGGTTCGATCGGGTGTAC-3’ | 56.4 | 64.0 | 60.0 |
| BBa\_K4344025 | HSV-*UL19*-rev-3 | 5’-TGTGCGAGAACCTGAAGAGT-3’ | 56.1 | 64.0 | 60.0 |
| BBa\_K4344009 | EGFP-fwd-1 | 5’-GGTGAACTTCAAGATCCGCC-3’ | 56.1 | 64.0 | 60.0 |
| BBa\_K4344012 | EGFP-rev-1 | 5’-CTTGTACAGCTCGTCCATGC-3’ | 56.0 | 64.0 | 60.0 |
| BBa\_K4344010 | EGFP-fwd-2 | 5’-GACGACGGCAACTACAAGAC-3’ | 55.8 | 61.0 | 60.0 |
| BBa\_K4344013 | EGFP-rev-2 | 5’-GGCGGATCTTGAAGTTCAC-3’ | 53.6 | 61.0 | 60.0 |
| BBa\_K4344011 | EGFP-fwd-3 | 5’-TAAACGGCCACAAGTTCAGC-3’ | 55.9 | 64.0 | 60.0 |
| BBa\_K4344014 | EGFP-rev-3 | 5’-AAGTCGTGCTGCTTCATGTG-3’ | 55.9 | 64.0 | 60.0 |

# RT-qPCR for knockdown efficiency evaluation

Knockdown efficiency was evaluated by RT-qPCR using the Luna Universal One-Step RT-qPCR Kit (NEB).
In case of *UL19* primer set 1 was used. *GAPDH* served as a housekeeping gene reference. Samples used for bulk RNA-Seq
were pooled per condition and treatment and purified with Monarch RNA Extraction Kit. RT-qPCR was carried out as described before
(compare **Figure 2 B &D**). A total of 50 ng RNA was used per reaction. Samples were analysed in duplicates. **Figure 3**
visualises the pipetting scheme.

![Pipetting scheme for RT-qPCR](https://static.igem.wiki/teams/4344/wiki/wetlab/methods/method-12-treatment.png)

**Figure 3: Pipetting scheme for RT-qPCR.** Samples were analysed in duplicates for target RNA expression (orange) and *GAPDH* expression (yellow).

**Table 7: *GAPDH* primer sequences.** Primers were ordered at IDT. Tm describes the calculated melting temperature by IDT. TA describes the annealing temperature used in the PCR, calculated with NEB Tm Calculator.

| Part-Nr. | Primer | Sequence | Tm [°C] | Ta [°C] | TPCR [°C] |
| --- | --- | --- | --- | --- | --- |
| BBa\_K4344016 | *GAPDH*-fwd. | 5’-GTCTCCTCTGACTTCAACAGCG-3’ | 57.4 | 65.0 | 60.0 |
| BBa\_K4344017 | *GAPDH*-rev. | 5’-ACCACCCTGTTGCTGTAGCCAA-3’ | 61.3 | 65.0 | 60.0 |

# Qualitative Western Blot analysis of Knockdown efficiency

The remaining 750 µL of the triplicates were pooled and the cells were pelleted at 16000 rpm at 4°C for 5 min.
Supernatant was removed and replaced by a protein lysis buffer. The mixture was well-vortexed.
Samples were then kept on ice to precipitate excess SDS. The precipitate was spun down at 16000 rpm at 4°C for 15 min.
The supernatant was transferred to a 1.5 mL eppi. For measurement of protein concentration a Bradford Assay was conducted as
described earlier. 40 µg of total protein for each condition were loaded onto a SDS-Page. 5 µL prestained protein ladder was
loaded as a size standard. The gel was run at 110 V for 1½ h on ice. The proteins were extracted from the gel by semi dry Western Blotting
on a nitrocellulose membrane at 15 V for 60 min. The blot membrane was blocked with 5 % milk powder in TBS-T for 1 h.
Afterwards blot was incubated with primary antibody Anti-His-tag mouse monoclonal antibody (1:10000, Proteintech) in case of
pRK5-HSV *UL19* AA816:1148-6xHis or anti-GFP-tag mouse monoclonal antibody (1:20000) in blocking solution supplied with
10 mM sodium azide at 4°C overnight. Secondary antibody goat anti-mouse HRP conjugated antibody(1:5000, Proteintech) in
blocking solution supplied with 10 mM sodium azide at 4°C for 2 h. The membrane was washed five times for 3 min to remove
unselective bound antibodies. After that the membrane was stained with DAB to detect *UL19* fragments or *EGFP*.
α-tubulin served as a housekeeping gene for relative quantification, anti-tubulin mouse monoclonal antibody (1:20000, Proteintech) was used.
The images were analysed using a ChemiDoc XRS+ (BioRad Laboratories).

## REFERENCES

* Han, M., Napier, C. E., Frölich, S., Teber, E., Wong, T., Noble, J. R., Choi, E. H. Y., Everett, R. D., Cesare, A. J., & Reddel, R. R. (2019). Synthetic lethality of cytolytic HSV-1 in cancer cells with ATRX and PML deficiency. *Journal of Cell Science, 132(5)*, jcs222349. <https://doi.org/10.1242/jcs.222349>.
* Hirsch, M., Ziroli, V., Helm, M., & Massing, U. (2009). Preparation of small amounts of sterile siRNA-liposomes with high entrapping efficiency by dual asymmetric centrifugation (DAC). *Journal of controlled release: official journal of the Controlled Release Society*, 135 (1), 80–88.
* Huang, L., Jin, J., Deighan, P., Kiner, E., McReynolds, L., & Lieberman, J. (2013). Efficient and specific gene knockdown by small interfering RNAs produced in bacteria. *Nature Biotechnology, 31(4)*, 350-356. <https://doi.org/10.1038/nbt.2537>.
* Huang, L., & Lieberman, J. (2013). Production of highly potent recombinant siRNAs in Escherichia coli. *Nature Protocols, 8(12)*, 2325-2336. <https://doi.org/10.1038/nprot.2013.149>.
* Li, D., Al-Jamal, K.T. (2021). siRNA Design and Delivery Based on Carbon Nanotubes. In: Ditzel, H.J., Tuttolomondo, M., Kauppinen, S. (eds) Design and Delivery of SiRNA Therapeutics. *Methods in Molecular Biology, vol 2282*. Humana, New York, NY.
* Mady, M.M., Darwish, M.M., Khalil, S. *et al.* (2009) Biophysical studies on chitosan-coated liposomes. *Eur Biophys J 38*, 1127–1133.
* Metwally, A. A., Blagbrough, I. S., & Mantell, J. M. (2012). Quantitative Silencing of *EGFP* Reporter Gene by Self-Assembled siRNA Lipoplexes of LinOS and Cholesterol. *Molecular Pharmaceutics, 9(11)*, 3384-3395. <https://doi.org/10.1021/mp300435x>.
* Mulligan, M. E., Brosius, J., & McClure, W. R. (1985). Characterization in vitro of the effect of spacer length on the activity of Escherichia coli RNA polymerase at the *tac* promoter. *Journal of Biological Chemistry, 260(6)*, 3529-3538. [https://doi.org/https://doi.org/10.1016/S0021-9258(19)83654-2](https://doi.org/https%3A//doi.org/10.1016/S0021-9258%2819%2983654-2).
* Yang, T.-T., Sinai, P., Green, G., Kitts, P. A., Chen, Y.-T., Lybarger, L., Chervenak, R., Patterson, G. H., Piston, D. W., & Kain, S. R. (1998). Improved Fluorescence and Dual Color Detection with Enhanced Blue and Green Variants of the Green Fluorescent Protein. *Journal of Biological Chemistry, 273(14)*, 8212-8216. <https://doi.org/10.1074/jbc.273.14.8212>.

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{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# Results

### Our results can be found on the corresponding sub-pages:

[![](https://static.igem.wiki/teams/4344/wiki/wetlab/sirna/banner-sirna.jpg)

siRNA](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results-siRNA%27%29%20%7D%7D)
[![](https://static.igem.wiki/teams/4344/wiki/wetlab/lnps/bannerbild-lnp.png)

Liposomes](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results-liposomes%27%29%20%7D%7D)
[![](https://static.igem.wiki/teams/4344/wiki/wetlab/cellculture/banner-cellculture.jpg)

Cell-Culture](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results-cell-culture%27%29%20%7D%7D)

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