Team:Tec-Monterrey/Improve
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iGEM Team UIUC 2015 designed Part BBa_K1681001 based on the work of Lu and Farzadfard, the SCRIBE system, which integrates a specific DNA sequence into a determined part of a bacterium's genome by action of a Beta Recombinase. The target DNA sequence is produced by a retron and a reverse transcriptase.
SCRIBE(BsaI)
Part BBa_K1681001 holds a single SCRIBE cassette induced by a lac operon, holding an editable retron sequence and the expression of a reverse transcriptase and a beta recombinase for DNA recombination into the bacteria's genomic DNA. iGEM Team UIUC did not include results regarding this specific part, however, they did obtain successful recombination of target DNA into the genome with a variant of this part with the same principle.From their work, as well as that from Farzadfard, a different design for this construct is proposed, where is it further adapted for the implementation of Cas1 and Cas2 proteins, instead of a recombinase, in the integration of target DNA to the bacteria's genome.
Improvement
In the integration of a DNA spacer into bacteria's CRISPR loci by means of Cas1-Cas2 integrase activity, the target DNA sequence to be deliberately inserted must be produced. This sequence is generated following the same basis as the SCRIBE system: a reverse transcriptase that retrotranscribes a retron sequence. Improved Part BBa_K2761010 contains two cassettes responsible for the production of this DNA message.
Inducible message generator (RT/msr-msd)
To adapt this DNA sequence generation for the integration with Cas1-Cas2 proteins, the target DNA in the msd region of the retron must first contain a PAM sequence, recognizable by the Cas proteins. Along with the PAM sequence, the rest of the msd must be a specific sequence to be associated with the specific inductor that triggers the system.Additionally, the reverse transcriptase is set to be expressed in a separate cassette as the retron, both under a same promoter, so as to avoid false positives resulting from leaking.
Characterization
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