Part:BBa_J01003

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 No part name specified with partinfo tag. to enable the transfer of a donor plasmid containing other genes apart from oriT-R. The conjugation was performed from e. coli to e. coli (K12, DH10B) containing a streptomycin resistance gene [1, 2]. To identify the most efficient transfer of the donor plasmid containing the oriT-R part, conjugation experiments using two different conjugative plasmids (pRK24 and RP4-8) was carried out. The plasmids were ordered from addgene and each of them has ampicillin resistance [3, 4]. The oriT-R part was inserted into a kanamycin backbone (pSB1K3) and was transformed into the e. coli strain. To make the e. coli cells able to conjugate either the pRK24 or the RP4-8 mobilizing plasmid was conjugated into the cells.

Experimental procedure
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. 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 the appropriate antibiotics. The cfu was counted by using the viable counting method. To ensure that conjugation only takes place when donor and recipient samples are mixed, the donor and recipient bacteria were incubated separately and treated in the same way as the conjugating sample. Since no colonies on the control plates were observed (data not shown) we concluded that conjugation only takes place when donor and recipient strains are mixed.

The transconjugants were selected on agar plates either containing ampicillin, kanamycin, and chloramphenicol (TMD), ampicillin and chloramphenicol (TM) or kanamycin and chloramphenicol (TD). Four replicates were carried out for the conjugation with 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 with the pRK24 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.

https://parts.igem.org/File:T--SDU-Denmark--oriT_optimization.png

Figure 1 depicts the proportion of recipient cells that have been receiving either the donor plasmid (TD), the mobilizing plasmid (TM), or both plasmids (TMD). The proportion of TD, TM and TMD 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). A two-way ANOVA with Tukey’s multiple comparisons test revealed no significant differences between groups (N=4-8).

As the figure indicates, the RP4-8 ensures an overall uniform transfer of both the donor plasmid, the mobilizing plasmid and both of the plasmids at once. The pRK24 mobilizing plasmid seemed to favor the transfer of the mobilizing plasmid rather than the donor plasmid, contributing to a less uniform transfer of the different plasmids. However, no significant differences between the two mobilizing plasmids was observed.

[1],[2],[3],[4].

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 NgoMIV site found at 117
  Illegal NgoMIV site found at 127
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 19