Part:BBa_K2996800
cas1-cas2 complex mutation
Using point mutation, the HindIII restriction site is changed.
Introduction CRISPR-Cas is a prokaryotic immune system in bacteria. The function of Cas1/2 complex is to recognize foreign DNA and insert a 33bp fragment into CRISPR locus of the bacteria’s genome as a new spacer. In the previous part(BBa_K2761009), there was a restrict digestion site of HindIII(AAGCTT) from 692-697 of Cas1 coding region. We used overlapping PCR to bring in a point mutation, changing it into AAGCAT so that HindIII could be used to assemble this part into other vectors.
Experiment and result
We designed primers ‘F’, ‘R’ on both end of Cas1/2 sequence and ‘Fm’ ‘Rm’ on restriction site Hind, and then ‘F’ and ‘Rm’ , ‘R’ and ‘Fm’ as two pairs of PCR primers respectively to produce two halves of the Cas1/2 mutant.
(PCR result image)
name as:
T--SJTU-BioX-Shanghai--partimp-1.jpg
Then overlap PCR is conducted with amplified products of the two PCR reactions above being template and ‘F’ and ‘R’ being primers.
(overlap PCR result image)
Name as:
T--SJTU-BioX-Shanghai--partimp-2.jpg
The mutant and original Cas1/2 was digested by HindIII. It is shown that the mutant was not cut while the original one was, which means our work has been successful.
(digest by HindIII image)
Name as:
T--SJTU-BioX-Shanghai--partimp-3.jpg
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 116
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 837
- 1000COMPATIBLE WITH RFC[1000]
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