Difference between revisions of "Part:BBa K2996800"
 
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Using point mutation, the HindIII restriction site is changed.
 
Using point mutation, the HindIII restriction site is changed.
  
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Introduction  
+
<br/>Introduction  
 +
<br/>
 
<br/>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.
 
<br/>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.
 
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
+
<br/>Experiment and result
 +
<br/>
 
<br/>We designed primers ‘F’, ‘R’ on both end of <i>Cas1/2</i> 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 <i>Cas1/2</i> mutant.
 
<br/>We designed primers ‘F’, ‘R’ on both end of <i>Cas1/2</i> 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 <i>Cas1/2</i> mutant.
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(PCR result image)
 
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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.
 
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
 
−
<br/>https://static.igem.org/mediawiki/parts/e/e3/T--SJTU-BioX-Shanghai--partimp-2.png
 
−
<br/>The mutant and original <i>Cas1/2</i> 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
 
 
<br/>https://static.igem.org/mediawiki/parts/d/d8/T--SJTU-BioX-Shanghai--partimp-3.jpg
 
<br/>https://static.igem.org/mediawiki/parts/d/d8/T--SJTU-BioX-Shanghai--partimp-3.jpg
 +
<br/>The mutant and original <i>Cas1/2</i> 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.
 +
<br/>https://static.igem.org/mediawiki/parts/e/e3/T--SJTU-BioX-Shanghai--partimp-2.png
  
  

Latest revision as of 03:41, 22 October 2019


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. Then overlap PCR is conducted with amplified products of the two PCR reactions above being template and ‘F’ and ‘R’ being primers.
T--SJTU-BioX-Shanghai--partimp-3.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.
T--SJTU-BioX-Shanghai--partimp-2.png


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 116
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 837
  • 1000
    COMPATIBLE WITH RFC[1000]