Difference between revisions of "Part:BBa K2996007"
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Our pRec plasmid has pTet upstream of cas1-cas2, addition of tetracycline will activate the expression of cas1-cas2 complex, thus accomplishing information storage. | 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. | (1) Inoculate 1% overnight bacterial culture in Erlenmeyer flask. | ||
(2) Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3). | (2) Incubate for 2h to reach early exponential stage (OD600 is 0.2-0.3). | ||
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(4) Incubate overnight at 20℃. | (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. | 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. | Its biological function, which is to insert protospacer into RSRL array, is confirmed by observable florescence, microplate reader and fluorescence microscope. | ||
Revision as of 05:00, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 170
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 891
- 1000COMPATIBLE WITH RFC[1000]