Research Proposal

Part 3: conjugation

Selecting the Conjugation system

There are many different conjugation systems, with F and IncP being among the most used by previous iGEM teams. The IncP incompatibility group is further divided into two groups IncP-alpha aka Birmingham aka RP4 plasmid and the IncP-beta R751 plasmid. There are several reasons why the IncP system would be better suited for us:

• Smaller plasmid size (R751 is 53423 [1]) (RP4 is 60099 [2]) (F is 99159 [3])
• F has a self-imposed fertility inhibition system (new donors fertile for only about 6 generations), IncP systems have no self-imposed fertility inhibition system [4]
• F has two surface exclusion proteins (TraT, TraS), IncP has only one (trbK) [4]

Selecting genes to knockout

Entry exclusion
The goal of this part of the project is to allow a message plasmid to be transmitted between cells containing conjugation helper plasmids (modified R751). In nature this occurs but at very low levels, due to the presence of entry exclusion proteins [5]. These membrane-bound proteins block incoming transfers from cells containing the conjugative plasmid. Note that in order for the transfer to be blocked their presence is required only in the recipient. These entry exclusion proteins are present for two primary reasons: i) to prevent redundant transfer of the conjugative plasmid among a population of cells and ii) to prevent lethal zygosis [6]. In R751 the gene trbK encodes for the entry exclusion protein [7] in R751 and RP4. Its presence is not required for conjugation to occur [8]. Some papers have reported that knocking out the trbK genes in other plasmids did not affect the conjugation frequency: “transfer of the trbK mutant occurred at near-wild-type frequencies.” [9]. Given this information we propose to knockout the trbK gene.
Lethal Zygosis
What is lethal zygosis: “We find that diaminopimelic acid in the recipient membrane is released into the medium during bacterial matings, indicating that membrane damage was inflicted on the recipient by the donor, probably for forming a channel for DNA transfer. When the damage is extensive, as in matings with an excess of Hfr bacteria, the F- bacteria are killed (lethal zygosis). The transfer of a large amount of DNA in Hfr matings appears to enhance the killing.” [6] By knocking out the trbK gene we may be exposing our cells to lethal zygosis. If this is indeed the case than knocking out one of the critical mating pair formation genes (trbB, trbC, trbD, trbE, trbF, trbG, trbH, trbI, trbJ, trbL) from the trb operon would prevent this. The gene trbC was selected for this due to its small size and position on the operon. trbC is known to encode for the pilin subunits needed for pilus formation. Given this information we propose to knockout the trbC gene as well.

Experimental Procedures

Section 1: Helper Plasmid
Part 1A:

• Acquire R751 and/or RP4 plasmid
• Electoporate plasmid into cells and confirm conjugation. See [http://openwetware.org/wiki/Conjugation conjugation protocol]
• Characterize conjugation efficiency

Part 1B: oriT knockout

• Knockout oriT using either [http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement lambda red], [http://openwetware.org/wiki/IGEM:Peking/2007/Count:Knockout knockout protocol used by Peking '07] or [http://openwetware.org/wiki/Berk2006-ConjugationTeam knockout protocol used by Berkeley '06]
• Electroporate into cells
• Verify that conjugation stopped
• Electroporate PlasmidG as well and characterize conjugation efficiency
• If works send R751 oriT knockout plasmid to registry

Part 1C: trbK knockout

• Knockout oriT + trbK
• Electroporate into cells
• Verify that conjugation takes place among R+ cells using PlasmidG (see below)
• Characterize conjugation efficiency
• If works send R751 oriT + trbK knockout plasmid to registry

Part 1D: trbC knockout

• Knockout oriT + trbK + trbC
• Electroporate into cells and create culture for communication (cultureCom)
• Verify that no conjugation takes place in presence of PlasmidG
• If works send R751 oriT + trbK + trbC knockout plasmid to registry

Section 2: Message Plasmid
Part 2A: BioBrick Assembly

• Order DNA synthesis for [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175000 BBa_K175000] (trbC) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175001 BBa_K175001] (trbK)
• Amplify BioBricks needed [http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840 BBa_E0840] (GFP generator), [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0034 BBa_B0034] (strong rbs), [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] (constitutive promoter), [http://partsregistry.org/wiki/index.php?title=Part:BBa_I714031 BBa_I714031] (oriT-R).
• Assemble: [oriT][promoter] and keep this for later
• Assemble PlasmidG: [oriT][promoter][GFP generator] + plasmid backbone with different resistance than helper plasmid and low copy number.
• Assemble [oriT][promoter][rbs][trbC][rbs][trbK] and keep this intermediate assembly product for possible use in integration stage
• Assemble [oriT][promoter][rbs][trbC][rbs][trbK][GFP generator] and keep this intermediate assembly product for possible use in integration stage
• Assemble PlasmidCKG: [oriT][promoter][rbs][trbC][rbs][trbK][GFP generator] + plasmid backbone with different resistance than helper plasmid and low copy number.

Part 2B: Full Communication testing

• Electroporate PlasmidCKG into some cells from cultureCom creating initiatorCells
• Select for presence of both message and helper plasmid
• Mix initiatorCells with cells not containing any plasmids and characterize conjugation efficiency
• Add initiatorCells to cultureCom and observe signal propagation, characterize rate of signal propagation.
• If signal propagation observed, do victory dance.

References

[1] Thorsted, P. B., D. P. Macartney, P. Akhtar, A. S. Haines, N. Ali, P. Davidson, T. Stafford, M. J. Pocklington, W. Pansegrau, B. M. Wilkins, E. Lanka, and C. M. Thomas. 1998. [http://www.ncbi.nlm.nih.gov/nuccore/113911681?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Complete sequence of the IncPbeta plasmid R751: implications for evolution and organisation of the IncP backbone]. J. Mol. Biol. 282:969-990.

[2] Pansegrau, W., E. Lanka, P. T. Barth, D. H. Figurski, D. G. Guiney, D. Haas, D. R. Helinski, H. Schwab, V. A. Stanisich, and C. M. Thomas. 1995. [http://www.ncbi.nlm.nih.gov/nuccore/508311?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Complete nucleotide sequence of Birmingham IncP alpha plasmids]. Compilation and comparative analysis. J. Mol. Biol. 239:623-663.

[3] Frost, L., K. Ippen-Ihler, and M. Skurray. 1995. [http://www.ncbi.nlm.nih.gov/nuccore/9507713?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Analysis of the sequence and gene products of the transfer region of the F sex factor]. Microbiol. Rev. 58:162-210.

[4] Plasmid Biology. Barbara E. Funnell and Gregory J. Phillips, eds. American Society for Microbiology Press, Washington, DC, 2004. Chapter 9.

[5] Garcillán-Barcia MP, de la Cruz F. Why is entry exclusion an essential feature of conjugative plasmids? Plasmid. 2008;60:1–18. doi: 10.1016/j.plasmid.2008.03.002.

[6] Ou, J. T. 1980. Role of surface exclusion genes in lethal zygosis inEscherichia coli K12 mating. Molecular and General Genetics178:573–581.

[7] Haase, J., Kalkum, M. & Lanka, E. TrbK, a small cytoplasmic membrane lipoprotein, functions in entry exclusion of the IncP alpha plasmid RP4. J. Bacteriol. 178, 6720–6729 (1996).

[8] Haase J, Lurz R, Grahn A M, Bamford D H, Lanka E. Bacterial conjugation mediated by plasmid RP4: RSF1010 mobilization, donor-specific phage propagation, and pilus production require the same Tra2 core components of a proposed DNA transport complex. J Bacteriol. 1995;177:4779–4791.

[9] Li, P. L., I. Hwang, H. Miyagi, H. True, and S. K. Farrand. 1999. Essential components of the Ti plasmid trb system, a type IV macromolecular transporter. J. Bacteriol. 181:5033-5041.

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