OriT-R (Origin of transfer for the R-plasmid nic region)
OriTR, the R plasmid nic region, is where the relaxosome nicks the plasmid and conjugative transfer by R plasmid machinery begins.
This oriT was characterized by Heidelberg iGEM2008 team (see experience). In this characterization the conjugation rate, conjugation time, conjugation efficiency between different strains, at different times and temperatures was measured. Furthermore the influence of donor : recipient ratio was analysed.
Characterization of oriT-R: Transfer efficiency using two compatible conjugative R-plasmids
(Characterized by SDU-Denmark 2019)
SDU 2019 used the part oriT-R (BBa_J01003) to enable transfer of our designed plasmid via conjugation. However, the oriT-R part cannot promote transfer of the plasmid on its own. Therefore, we utilized two different conjugative plasmids, pRK24 and RP4-8 (ordered from Addgene, no. 51950 and no. 79813, respectively), in order to be able to characterize the transfer efficiency of the plasmid containing the oriT part. The conjugation experiments were performed between E. coli Top10 cells, serving as both the donor and the recipient strain.
The donor cells for conjugation were designed to contain two different plasmids; one plasmid containing the oriT-R part in a pSB1K3 backbone and either of the two conjugative plasmids (pRK24 or RP4-8). The recipient bacteria were designed to contain the pSB4C5 backbone. Donor and recipient cells were grown to an OD600 between 0.6-0.8 in LB broth.
For the experimental procedure on conjugation, inspiration was found in the following article and modified for our project .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 for 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-R is dependent on the presence of either of the conjugative plasmids. This was done by designing two new donors; donor bacteria containing either of the conjugative plasmids and the pSB1K3 backbone without the oriT-F part incorporated, and another donor bearing the plasmid containing the oriT-R part. The control experiments were performed by following the same experimental procedure as listed above. Since no colonies were to be observed on the plates selecting for recipient cells having received the pSB1K3 backbone, it was concluded that the transfer of our plasmid (pSB1K3) is dependent on the coexistence of either of the conjugative plasmids and the oriT-R part.
The transconjugants were selected on agar plates containing antibiotics selecting for three different types of the former recipient cells. TN refers to recipient cells having received the plasmid containing the oriT-R part only. TC refers to recipient cells having received the conjugative plasmid only. TCN refers to recipient cells having received both plasmids.
Four replicates were carried out for the conjugation including the pRK24 plasmid to collect an appropriate amount of data points that was used to quantitatively distinguish the transfer efficiency. Eight replicates were carried out for the conjugation including the RP4-8 plasmid also to collect an appropriate amount of data points that was used to quantitatively distinguish the transfer efficiency. The results are shown in the following figure.
As the results indicates, the RP4-8 plasmid ensures an overall uniform transfer of both the plasmid containing the oriT-R, the conjugative plasmid and both of the plasmids at once. The pRK24 plasmid seem to favor its own transfer rather than the plasmid containing the oriT-R, contributing to a less uniform dispersal of the different plasmids to recipient cells. However, no significant difference between the two conjugative plasmids was observed based on the two-way ANOVA with Tukey’s multiple comparisons test.
A second experiment was performed in order to follow the transmission of both the plasmid containing the oriT-R and the conjugative plasmid over time. Again, the 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 mixed cells were resuspended in LB broth by diluting 1:5 and subsequently allocated into six separate tubes, for plating at appropriate time points. The conjugating samples were incubated at 37 degrees Celsius under slow rotation to ensure mixing of cells.
Four replicates of the conjugation experiment over time were performed to collect an appropriate amount of data points that were used to quantitatively distinguish the rate of dispersal of the two plasmids. The results are shown in the following figure.
The results display a linear correlation between the formation of either of the three transconjugants over time. By fitting a linear regression on the data points the rate of the formation of either the TN, TC or TCN can be estimated. The slope for TN is 0.003639 (± 0.000454), for TC the slope is 0.0.003487 (± 0.000594) and the slope for TCN is 0.003049 (± 0.000357).
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 117
Illegal NgoMIV site found at 127
- 1000Illegal SapI site found at 19