Part:BBa_K5465999

ONERING-TetO

The ONERING-TetO plasmid consists of a pRPF144 backbone which contains RepA, RepB, OriV, ColE1, CatP, OriT and TraJ.

RepA and RepB are replication proteins required to activate OriV which allows for stable propagation of plasmid in Gram Positive bacteria. OriV is a high copy number origin of replication.

ColE1 is the high copy number origin of replication required for stable propagation in Gram negative bacteria.

CatP is the gene sequence coding for Chloramphenicol resistance. It produces the enzyme Chloramphenicol acetyltransferase which is the main effector for inactivation of the antibiotic - chloramphenicol

OriT and TraJ are components required for conjugation of plasmid from donor into recipient. OriT is the sequence which targets the plasmid for transfer into the recipient via conjugation. TraJ encodes for the protein required for efficient conjugation of the plasmid. OriT is only compatible with T4SS secretion systems.

The ONERING-TetO plasmid is a part used in the TetR Fluorescent Repressor Operator System (FROS) which is based on the Tetracycline repressor protein (TetR) and Tetracycline operator sequence (TetO).

The function of this part is it allows quantification of conjugation efficiency between a donor strain and a recipient strain through imaging. This is done by exploiting the interaction between TetR protein and the TetO sequences. The donor contains a plasmid with TetO sequences (ONERING-TetO), which upon conjugation will be transferred to the recipient, which contains a genetically encoded TetR protein fused to mNEONGreen (MnG) fluorescent protein. TetR will bind to the TetO sequences on the plasmid, and the fused MnG will produce foci which can be imaged by fluorescence microscopy. These foci will show exact locations in the recipient where the plasmid is present.

Requirements for this part is an adequate donor strain constitutively expressing a fluorescence protein emitting in a single channel. For our project, we produced a CA434::BFP strain. We did this by transforming CA434 with pGRG25-pMax-sfBFP plasmid. The BFP gene is inserted into the CA434 genome through a Tn7-mediated integration mechanism.

For your recipient, you need to produce a strain that contains TetR:MnG integrated into its genome. For our project, we used the strain MG1655::TetR:MnG. This is important, as the TetR:MnG fusion protein will be primarily used to image and detect foci showing successful conjugation. The fluorescent protein can be customised, but ensure that it does not overlap with fluorescence assigned to the donor.

The last requirement is the ONERING-TetO plasmid which contains the TetO array. For our project, we inserted a TetO array into the pRP4144 plasmid with the array containing around 20 TetO sequences which allowed us to adequately estimate conjugation efficiency.

The data will have to be analysed using napari-bacseg and conj-assay, which are both open-source softwares. Napari is used to produce segmentations of bacteria from the brightfield images and localisations of the foci from fluorescence images.

Conj-assay uses this data to identify donors, recipients and transconjugants and then calculate conjugation efficiency.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4843
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4463
  Illegal NgoMIV site found at 4589
  Illegal NgoMIV site found at 4599
  Illegal AgeI site found at 4850
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4853
  Illegal BsaI.rc site found at 3280
  Illegal BsaI.rc site found at 3309
  Illegal BsaI.rc site found at 3652
  Illegal SapI site found at 3260
  Illegal SapI site found at 4491
  Illegal SapI site found at 5887