Difference between revisions of "Part:BBa K2996007"
Line 3: Line 3:
 
<partinfo>BBa_K2996007 short</partinfo>
 
<partinfo>BBa_K2996007 short</partinfo>
  
−
Expression of the CRISPR adaptation proteins Cas1 and Cas2 promotes unidirectional integration of 33–base pair DNA spacers into our designed RSRL array.
+
Expression of the CRISPR adaptation complex cas1-cas2 promotes unidirectional integration of 33–base pair DNA spacers into our designed RSRL array.
 +
Our pRec plasmid has pTet upstream of cas1-cas2, addition of tetracycline will activate the expression of cas1-cas2 complex, thus accomplishing information storage.
 +
 
 +
=protocol
 +
(1) Inoculate 1% overnight bacterial culture in Erlenmeyer flask.
 +
(2) Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3).
 +
(3) Add tetracycline to final concentration of 100 ng/μL.
 +
(4) Incubate overnight at 20℃.
 +
 
 +
=result
 +
Since this plasmid was a gift from Professor. Wang from Columbia University, we were confident of the expression of cas1-cas2 complex from pRec.
 +
Its biological function, which is to insert protospacer into RSRL array, is confirmed by observable florescence, microplate reader and fluorescence microscope.
 +
For more detailed results, click BBa_K2996011.
 +
 
  
−
Addition of tetracycline will activate the expression of cas1-cas2 complex, thus accomplishing information storage.
 
  
  

Revision as of 04:59, 20 October 2019


pTet upstream of cas1/2

Expression of the CRISPR adaptation complex cas1-cas2 promotes unidirectional integration of 33–base pair DNA spacers into our designed RSRL array. Our pRec plasmid has pTet upstream of cas1-cas2, addition of tetracycline will activate the expression of cas1-cas2 complex, thus accomplishing information storage.

=protocol (1) Inoculate 1% overnight bacterial culture in Erlenmeyer flask. (2) Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3). (3) Add tetracycline to final concentration of 100 ng/μL. (4) Incubate overnight at 20℃.

=result Since this plasmid was a gift from Professor. Wang from Columbia University, we were confident of the expression of cas1-cas2 complex from pRec. Its biological function, which is to insert protospacer into RSRL array, is confirmed by observable florescence, microplate reader and fluorescence microscope. For more detailed results, click BBa_K2996011.



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 170
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 891
  • 1000
    COMPATIBLE WITH RFC[1000]