Part:BBa_K3001015
Lsh crRNA targeting GFP with T7 promoter
This CRISPR RNA (crRNA) is designed to interact with Cas13a isolated from Leptotrichia shahii (Lsh). It is designed to target a segment of GFP mRNA transcript. It is designed to be inducibly expressed under a T7 promoter and was used for in vivo experiments in E. coli BL21(DE3). Experiments were conducted in the plasmid pUC19.
wetlab data
We completed a dual plasmid transformation. To confirm that we had both plasmids present we completed a digest test because PCR amplification from the colony was not working. This should separate our construct from the plasmid confirming that both parts are present. In figure 1, only linearized cuts are seen. Due to both plasmids being approximately the same size it is difficult to see. However, there are two bands present. In figure 2, only linearized cuts are seen near the 3000 bp mark. Due to both plasmids being approximately the same size it is difficult to see. However, there are two bands present. The second band at approximately 2000 bp is plasmid backbone, while the 1000 bp mark is the GFP insert. The crRNA insert (expected size is ~260 bp) is likely not seen in the gel due to having to run the gel longer to get better resolution for the upper bands.
Figure 2. 1% agarose of restriction digests using PstI and EcoRI from Escherichia coli BL21(DE3) cells containing plasmids for GFP and Lbu crRNA, Lwa crRNA, Lba crRNA, or Lsh crRNA. Left to right: lane 1: Lwa crRNA colony 1; lane 2: Lwa crRNA colony 2; lane 3: Lwa crRNA colony 3; lane 4: Lwa crRNA colony 4; lane 5: Lsh crRNA colony 2; lane 6: Lba crRNA colony 1; lane 7: Lba crRNA colony 2; lane 8: Lba crRNA colony 3; lane 9: Lbu crRNA colony 1; lane 10: Lbu crRNA colony 2; lane 11: Lbu crRNA colony 3; lane 12: Lbu crRNA colony 4; lane 13: 1 kb ladder.
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]
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