Part:BBa_K4643003

incP origin of transfer

The IncP (Incompatibility group P) origin of transfer is a DNA sequence found in certain bacterial plasmids of the IncP group. These plasmids are typically associated with soil bacteria, especially pseudomonads and Burkholderia species. IncP plasmids are known for their ability to transfer their genetic material between different bacterial strains, including distinct bacterial species, making them important for the dissemination of antibiotic resistance genes and other biotechnologically relevant genes.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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Experience of 2024 team Heidelberg

The IncP origin of transfer (oriT) is a conserved DNA sequence that is essential for conjugation-mediated DNA transfer between bacteria. The oriT sequence contains a nic site that is recognized by the TraI relaxase, which generates a nick and initiates the conjugation process (Virolle et al., 2020). We tested the functionality of this oriT sequence by performing conjugation assays on both solid and liquid media. As part of the PICasSO toolbox,the IncP oriT will facilitate transfer of mobilizable plasmids in inter-kingdom conjugation assays for delivery of large genetic payloads to mammalian cells in the future.

Results from inter-bacterial conjugation assay

We carried out conjugation assays using the RP4 system (IncP) in the trans configuration: a dual-plasmid-system where the RP4 conjugation machinery (encoded by pHelper_RP4) and the oriT sequence (present in pmob_b) are present on separate plasmids and thus, only the plasmid carring the oriT (the mobilizable plasmid) is transferred between bacteria. All plasmids possessed distinct antibiotic resistance genes and so, conjugation was implied from the survival of recipient bacteria on agar plates selecting for bacteria with mobilizable plasmid. Additionally, all recipients carried a plasmid conferring a common antibiotic resistance to enable selection against donors. Appropriate experimental controls were included (refer Table 1).

Table 1. Donors and recipients in the different controls and in the test group used for the bacteria-bacteria conjugation assay.

Sample	Donor	Recipient
Negative Control 1	E. coli 10-beta with pHelper_RP4	E. coli BL21(DE3)
Negative Control 2	E. coli 10-beta with pmob_b	E. coli BL21(DE3)
Positive Control	-	E. coli BL21(DE3) transformed with pmob_b
Test Group	E. coli 10-beta with both pHelper_RP4 and pmob_b	E. coli BL21(DE3)

The conjugation assay was performed as described by Silbert et al., (2021) on both solid and liquid media (without shaking) for 18 hours at 37 °C. After conjugation, the OD₆₀₀ of the bacteria was adjusted to 2.4 and serial diltutions (ranging from 10^-2 to 10^-9) were plated on two types of LB agar plates (one selecting for transconjugants and another selecting for recipients). Finally, the conjugation efficiency was calculated by dividing the number of transconjugant colonies by the number of recipient colonies at a particular dilution.

It was observed that the conjugation efficiencies (calculated as transconjugants per recipient) differed depending on the OD₆₀₀ of the donors and recipients used at the start of conjugation. Notably, a 1000-fold (from 1.8 x 10^-2 to 6.7 x 10^-5) increase in conjugation efficiency was observed between the test groups upon increasing the OD₆₀₀ from 1.2 to 10 (Figure 1). This provides a clear indication that cell density and therefore, enhanced cell-cell contact is an important parameter for conjugation on solid media. Interestingly, at an OD₆₀₀ of 10, we also observe some natural transformation happening in case of the negative control 2 samples, which could be a consequence of high cell density resulting in increased chances of natural transformation. In liquid media however, we could not show any conjugation happening, which confirms the inefficiency of the wild-type RP4 system to mediate conjugation in liquid media (Robledo et al., 2022). Moreover, the positive controls that were chemically transformed grew on selective agar plates, suggesting that the apparent lack of conjugation in liquid media is not due to the experimental conditions but rather the inefficiency of the conjugative system itself, under liquid conditions.

Figure 1: Efficiency of conjugation on solid media. Bar charts depicting the conjugation efficiency (reported as transconjugants/recipient) of the various experimental groups. Three technical replicates were used for the OD₆₀₀ 10 test group.

References

Robledo, M., Álvarez, B., Cuevas, A., González, S., Ruano-Gallego, D., Fernández, L. Á., & De La Cruz, F. (2022). Targeted bacterial conjugation mediated by synthetic cell-to-cell adhesions. Nucleic Acids Research, 50(22). https://doi.org/10.1093/nar/gkac1164

Silbert, J., Lorenzo, V. De, & Aparicio, T. (2021). Refactoring the Conjugation Machinery of Promiscuous Plasmid RP4 into a Device for Conversion of Gram-Negative Isolates to Hfr Strains. ACS Synthetic Biology, 10(4). https://doi.org/10.1021/acssynbio.0c00611

Virolle, C., Goldlust, K., Djermoun, S., Bigot, S., & Lesterlin, C. (2020). Plasmid transfer by conjugation in gram-negative bacteria: From the cellular to the community level. In Genes (Vol. 11, Issue 11). https://doi.org/10.3390/genes11111239