Difference between revisions of "Part:BBa K2996007"

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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.

Mechanism and Usage

Cas1-Cas2 is a heterohexamer containing a Cas2 dimer sandwiched by two Cas1 dimers, where Cas1 is the catalytic subunit and Cas2 substantially increases integration activity.
The optimal substrate for E. coli Cas1-Cas2 is a 33-bp prespacer, which contains a central 23-bp double-stranded duplex with 5-nt overhangs on each side splayed into single-stranded DNA ends by tyrosine wedges in Cas1.The length of the prespacer is determined by the distance between two Cas1 integrase active sites. The 23-bp duplex sits on the flat surface of Cas1-Cas2, which positions the 3′ends into the catalytic site of each Cas1. In the E. coli type I-E system, Cas1-Cas2 complex which has high binding affinity for prespacer substrates with a canonical PAM (5′-CTT-3′). Once loaded with the prespacer,Cas1-Cas2 recognizesthe CRISPR array and catalyzes spacer integration. 3 ′-OH group of the prespacer attacks the backbone at the leader-proximal end of the first repeat with in the CRISPR array,forming a half-site intermediate.The end of the prespacer containing the cytosine from the PAM attacks at the leader-distal end of the opposite strand of the repeat, forming a full-site intermediate.The single-stranded repeats on either end of the integrated spacer are filled by DNA polymeraseI,and the nicks are ligated to create an intact CRISPR array.
In the end, the CRISPR will have an addition of 61 base pairs.
The constructed pRec plasmid can express Cas1 and Cas2 upon addition of anhydrotetracycline (aTc), which results in spacer acquisition, as the information input.
Similar to pLux-cas plasmid, addition of AHL molecules will lead to the expression of Cas1-Cas2 complex.

Protocol

Induction
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℃.

Results

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, see <a href="https://parts.igem.org/Part:BBa_K2996011"></a>.

Sequence and Features