Part:BBa_I714030

OriT-F (Origin of Transfer for the F-plasmid nic region)

OriT-F (Origin of Transfer for the F-type conjugative plasmid) is the nic region, where the relaxosome nicks the plasmid and conjugative transfer by F plasmid machinery begins via rolling circle replication.

Characterization of oriT-F: Transfer efficiency using a compatible conjugative F’-plasmid

(Characterized by SDU-Denmark 2019)

SDU 2019 has characterized the part oriT-F (BBa_I714030) by enabling transfer of the plasmid containing the oriT-F part via conjugation. The oriT-F part cannot promote the transfer of the plasmid on its own. Thus, we utilized an F'-plasmid containing the appropriate conjugative genes. These genes will enable the transfer of the plasmid containing oriT-F in combination with the conjugative plasmid's own transfer. The conjugation experiment was performed between E. coli Top10 cells, serving as both the donor and the recipient strain.

Experimental Procedure
The donor cells for conjugation were designed to contain two different plasmids; one plasmid containing the oriT-F part in a pSB1K3 backbone and the F’ plasmid. The recipient bacteria were designed to contain the pSB4C5 backbone only. Donor and recipient cells were grown to an OD₆₀₀ between 0.6-0.8 in LB broth. The donor and recipient samples were mixed in a 1:1 ratio to reach a total cell concentration of 10⁸ CFU per mL. The conjugating sample was incubated in 1.5 hours at 37℃. The sample was resuspended in LB broth by diluting 1:6. The conjugating sample was further incubated for 1.5 hours followed by plating on agar plates containing appropriate antibiotics. The CFU was counted by using the viable counting method. A control experiment was performed to confirm that the transfer of the plasmid containing the oriT-F is dependent on the presence of the conjugative plasmid. This was done by designing two new donors; a donor bacteria containing the conjugative plasmid and the pSB1K3 backbone without the oriT-F part incorporated, and another donor bearing the plasmid containing the oriT-F part. The control experiments were performed by following the same experimental procedure listed above. Since no colonies were to be observed on the plates selecting for recipient cells that have received the pSB1K3 backbone, it was concluded that the transfer of the plasmid (pSB1K3) is dependent on the presence of the conjugative plasmid and the oriT-F part.

The transconjugants were selected on agar plates containing antibiotics selecting for three different types of the former recipient cells. TN refers to the recipient cells having received the plasmid containing the oriT-F part only. TC refers to the recipient cells having received the conjugative plasmid only. TCN refers to the recipient cells having received both plasmids.

The experiments were carried out in the same way as the first experiment described for the oriT-R part (BBa_J01003).

Four replicates were carried out for this conjugation experiment to collect an appropriate amount of data points that were used to quantitatively distinguish the transfer efficiency. The results are shown in the following figure.

Figure 1 depicts the proportion of recipient cells having received either the plasmid containing the oriT-F (TN), the conjugative plasmid (TC) or both plasmids (TCN). The proportion of TN, TC and TCN were obtained by dividing the number of CFU to the total number of recipient cells. Data are represented as mean values with standard error of mean (SEM).

As the results indicate, the F’-plasmid seems to favor its own transfer rather than the plasmid containing the oriT-F. This contributes to a week dispersal of the plasmid containing the oriT-F part.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 278
  Illegal NheI site found at 324
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]