Part:BBa_K4807027

Rluc8 insert

pOSIP is the vector mediating the insertion of Rluc8 in the bacterial chromosomal DNA as outlined in the clonetegration protocol.

Among the various plasmids within the OSIP family capable of performing this task, we chose pOSIP-TT (P21) (BBa_J435116) due to its tetracycline resistance, which was useful for simultaneous transformation with other plasmids.

This vector features a ColE1/pMB1/pBR322/pUC ori as well as a gamma replication origin from E. coli plasmid R6K and it is tetracycline resistant.

pOSIP-TT (P21) lacks a T7 promoter, an RBS and a T7 terminator. To ensure the expression of our Rluc8 gene after its integration into the chromosomal DNA, it's essential to incorporate these regulatory elements (BBa_K4807001, BBa_K4807002) into our insert.

The vector was linearized through digestion with two different restriction enzymes, XhoI and EcorI. These enzymes were responsible for removing the ccdB toxin and ColE1/pMB1/pBR322/pUC origin of replication in order to make the detection of the cloned vectors easier. Within this region, the Rluc8 gene (BBa_K4875014) was inserted, complete with its regulatory elements.

It's unnecessary to incorporate a tag into our insert to verify the proper expression of the Rluc8 gene because Rluc8 is an enzyme that interacts with its substrate, yielding a visible outcome.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 667