Part:BBa_K2643012
T7p expressing sgRNA targeting the EPO coding sequence
T7p-gRNA_EPO
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
BBa_K2643012 carries a DNA template to produce single guide RNA (sgRNA) targeting the second exon-exon junction of EPO cDNA. This template was used to produce active sgRNA by in vitro T7 RNA transcription. The sgRNA was proven to be bounded to dxCas9 and guided it to the target EPO. The sgRNA with this same sequence was used to test the functionality of the dxCas9 [http://part.igem.org/Part:BBa_K2643000 dxCas9-Tn5] fusion protein.
Characterization
Biobrick construction
sgRNA template was designed [1] and ordered as gBlock from IDT . Fw primer gaattcgcggccgcttctagagaagcTAATACGACTCACTATAGG and rv primer ctgcagcggccgctactagtaGCACCGACTCGGTGCCAC were used in a PCR to introduce biobrick suffix and prefix.
Figure 1, lane 1 to 4 confirms the amplification of sgRNA (162bp) with biobrick suffix and prefix. PCR products isolated, restricted with EcoRI and PstI restriction enzyme and ligated to pSB1C3.
Figure 1. PCR amplification of gBlock to introduce biobrick prefix and suffix (expected size:162bp). The ladder represents the size of DNA in bp.
In vitro transcription
The T7 in vitro transcription was done following protocol found on our [http://2018.igem.org/Team:TUDelft/Experiments#gRNAtranscription-scroll wiki]. The transcripts were cleaned and visualized on 2% TBE agarose gel shown on figure 2. In this figure, lane 1 and 2 confirms the success of sgRNA transcription with this template.
Figure 2. Visualization of sgRNA (expected size 96bp) on 2% TBE agarose gel stained with ethidium bromide. The ladder represents the size of DNA in bps.
sgRNA loading assay
The same sgRNA sequence was produced by ArborBio (namely J2.1 sgRNA) and its functionality was tested by means of trypsin resistance assay (dxCas9), DNA mobility assay (dxCas9 ), and integration assay ([http://part.igem.org/Part:BBa_K2643000 dxCas9-Tn5]).
Source
gBlocks. Template design was based on HiScribe™ Quick T7 High Yield RNA Synthesis Kit [1].
Safety
This part can be used in ML1 level biosafety laboratory.
References
- ↑ 1.0 1.1 Biolabs, N. E. (n.d.). SgRNA Synthesis Using the HiScribe™ Quick T7 High Yield RNA Synthesis Kit (NEB #E2050). Retrieved from https://international.neb.com/protocols/2015/11/24/sgrna-synthesis-using-the-hiscribe-quick-t7-high-yield-rna-synthesis-kit-neb-e2050
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