Part:BBa_K2657002

Rp4 Origin of Transfer PhytoBrick

BBa_K2657002 is a PhytoBrick (MoClo standard) part plasmid using the Universal Acceptor (modified pSB1C3) as its backbone. The Origin of Transfer was cloned from the RP4 origin with primers that added Phytobrick functional elements and BsmBI and BsaI restriction sites for use in Golden Gate Assembly. The origin of transfer was then assembled into a Phytobrick via a BsmBI Golden Gate reaction which inserted the OriT into the Universal Acceptor, BBa_P10500.

Functionality

The functionality of this origin of transfer was demonstrated by a successful conjugation into Serratia marcescens. The OriT was used in a BsaI golden gate assembly reaction to create a self-replicating plasmid containing a p15a origin of replication, and genes encoding GFP and resistance to kanamycin. The assembly was transformed into a promiscuous donor strain of E. Coli , Mu Free Donor. The Mu Free Donor was then conjugated with Serratia marcescens . The conjugation was deemed successful by the organism's ability to express GFP and grow on a kanamycin plate.

Fig 1. Conjugation of Serratia marcescens . The plates on the left show the red bacteria under visible light while the plates on the right demonstrate the organism's expression of GFP when viewed under blue light.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4