Part:BBa_K4344090
p19 pulldown gene and UL19 siRNA target expression casette
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.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 220
Illegal XbaI site found at 701
Illegal XbaI site found at 1015 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 220
Illegal NotI site found at 1303 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 220
Illegal XhoI site found at 1009 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 220
Illegal XbaI site found at 701
Illegal XbaI site found at 1015 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 220
Illegal XbaI site found at 701
Illegal XbaI site found at 1015
Illegal NgoMIV site found at 978
Illegal NgoMIV site found at 1078 - 1000COMPATIBLE WITH RFC[1000]
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