Part:BBa_K2996002
minimal CRISPR array-A
The cassette consists of 69 bp of the respective leader, including the sequence elements essential for adaptation, and one spacer flanked by two repeats. Wildtype E.coli has two types of CRISPRER array, with their repeat sequence differ in one base pair. We applied A-type repeat sequence to our design, due to a higher acquisition ratio demonstrated by previously published papers.
- Usage and Biology
In our design, this integration cassette RSRL in pRead consists of a leader and two repeats interspaced by a spacer fused downstream, to the complete coding sequence of an out-of-frame EGFP gene (EGFP +2).
Originally, there is a stop codon within the RSRL sequence and the fluorescent protein gene was not in the correct reading frame, so there would be no fluorescent protein expressed. After induction, spacer acquisition carried out by cas1/2 would cause an addition of 61 base pairs, a protospacer and another repeat sequence, thus resulting in frame shifting. In that case, the stop codon would disappear, and EGFP enter the correct reading frame.
When you want to read the information, inducer should be added and expression of fluorescent protein would be activated.
- result
图
PCR Microplate reader Sequencing
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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