Part:BBa_K1062000

Guide RNA (gRNA) target for RFP

This part is a guide RNA (gRNA) that targets the RFP gene, when bound to Cas9. It also contains a cleavage site recognized by the Csy4 enzyme which allows it to be cleaved from neighboring DNA/RNA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

In both of our projects, we used gRNAs and dCas9 as:

1) To specifically target and repress the "virulence gene" (we used flourescent proteins as a proof of concept) in our conjugation project

2) Improve past circuits with better decision making by using dCas9 instead of traditional repressors.

Conjugation Project

In the Conjugation Project, our target contained a RFP cassette that is knocked into the chromosome of the strain. Due to the fact that the target strain is essential to proving the Conjugation Project, this gRNA is essential in order to prove that repression of a specific target can be possible.

Synthetic Circuit

In the Synthetic Circuit, the target is the RFP gene that would be located in the High Concentration Pathway. The purpose of the gRNA is to prevent any possibility of the RFP gene expressing, due to a possible leaky promoter, when the Low Concentration Pathway is being induced.