Difference between revisions of "Part:BBa J01003"

Revision as of 18:48, 14 October 2019

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: the transfer efficiency using two compatible conjugative R-plasmids

(Characterized by SDU-Denmark 2019)

SDU 2019 used the part oriT-R (BBa_J01003) to enable the transfer of our designed plasmid via conjugation. However, the oriT-R part cannot promote the transfer of the plasmid on its own. Therefore, we utilized two different conjugative plasmids, pRK24 and RP4-8 (ordered from Addgene), 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.

Experimental procedure
The donor cells for conjugation was 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 was designed to contain the pSB4C5 backbone. Donor and recipient cells were grown to an OD_600 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 10^8 cfu per mL. The conjugating sample was incubated in 1.5 hours at 37 degrees Celsius. 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; a 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 listed above. Since no colonies were to be observed on the plates selecting for recipient cells that have been receiving 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 either containing ampicillin, kanamycin, and chloramphenicol (TN), ampicillin and chloramphenicol (TC) or kanamycin and chloramphenicol (TNC). TN refers to the recipient cells that have received the plasmid containing the oriT-R part only. TC refers to the recipient cells that have received the conjugative plasmid only. TNC refers to the recipient cells that have 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 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 seemed 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 differences between the two mobilizing 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 OD_600 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 10^8 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 was performed to collect an appropriate amount of data points that was 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 making linear regression on the data points the rate of the formation of either the TN, TC or TNC can be estimated. (Data coming up).

Sequence and Features