The overall goal of this mating assay is to assess the effects of mutations within the conjugative transfer gene on its ability to affect plasmid transfer within the context of a functional type four secretion complex. This method allows for asking protein specific questions, such as identifying regions of protein-protein interactions that occur between transfer proteins as they assemble a functional type four secretion system in bacterial conjugation. In this technique, genes on large conjugative plasmids are knocked out by homologous recombination.

Gene function is assessed by providing the target gene to the knock-outs on a smaller plasmid. Generally, individuals new to this method will struggle because organization and set-up are key to successfully conduct this procedure. Proper experimental preparation is necessary to get interpretable results.

Begin this protocol with preparation of the digested pBAD33 plasmid as described in the text protocol. Run each digest on an agarose gel. Using a UV cabinet and a sterile razor, cut the 2.8 kilo baseband that cotresponds to the cath sequence out of the gel.

Work quickly to minimize the UV exposure of the DNA. Extract the DNA from the cut out gel slice using a gel extraction kit as per the manufacturer's protocol. To amplify the extracted cat cassette by polymerase chain reaction or PCR.

Prepare the reaction mixture in a sterile PCR tube. Use primers that contain overhangs that are homologous to the target gene sequence for homologous recombination. Set-up a negative control that bears the same components, except replace the template DNA with double distilled water.

Also, set-up a positive control using template DNA and primers that have been proven to work in a PCR reaction. Mix all the reaction contents gently by pipetting. Then, amplify the extracted cat cassette using the PCR parameters listed in the text protocol.

Use only five microliters of each reaction to confirm the correct size of amplification via agarose gel electrophoresis. Purify the PCR amplicon using a PCR purification kit with the manufacturer's protocol. Add 300 nanograms of the amplified cat cassette into 50 microliters of electrocompetent DY330R cells, harboring the pOX38-Tc plasmid on ice.

Gently pipette up and down to mix. Repeat this step using unmodified pBAD33 plasmid as a positive control. Then, transfer the cells to a pre-cooled one millimeter electroporation cuvette.

Electroporate the cells at 1.8 kilovolts with a time constant of 5.5 milliseconds using an electroporator. Immediately, after applying the pulse, dilute the cells with one milliliter of SOC media and transfer to a fresh microfuge tube. Incubate the cells at 32 degrees Celsius for two hours.

Next, aliquot 100 microliters of each sample unto agar plates containing 10 micrograms per milliliter tetracycline and 20 micrograms per milliliter chloramphenicol. Spread the aliquot over the plate using a sterile spreader. Incubate the plate upside down at 32 degrees celsius overnight.

The next day, select for the successful recombinants. The cat cassette introduced into the cell will undergo homologous recombination with the gene of interest and create the pOX38-Tc chloramphenicol knock-out clone. Separate the electroporate 300 nanograms of pK184 gene or pK184 gene mutant plasmid into 50 microliters of electrocompotent DY330R-cells harboring the pOX38-Tc chloramphenicol knock-out plasmid as before.

To generate the XK1200 donor cells perform conjugative mating followed by electroporation to generate XK1200 cells harporing both the chloramphenicol knock-out and the PK184 plasmid as described in the text protocol. Preparing overnight culture of these XK1200 donor cells in 20 milliliters of sterile LB with chloramphenicol and kanamycin. Also prepare MC4100 recipients cells in 50 milliliters of LB with 50 micrograms per milliliter of streptomycin using cells from a glycerol stock or from a single colony on ang agar plate.

Grow the cultures at 37 degrees celsius, shaking at 200 rpm. Add glucose to a final concentration of 100 millimolar to all donor cells. The next day, make 1 in 70 dilutions from each overnight culture separately in two milliliters of sterile LB with the same antibiotics.

Grow the cells to mid log phase at 37 degrees celsius with shaking at 200 rpm. Centrifuge the cell culture to pellet the cells and discard the supernatant. Then, wash the cell pellet once with cold sterile LB to remove the antibiotics.

After centrifuging a second time as before we suspend the cells in two milliliters of cold sterile LB.In duplicate, aliquot 100 microliters of donor cells and 100 microliters of recipient cells to 800 microliters of sterile LB media for one milliliter total volume. Allow the cells to mate at 37 degrees celsius for one hour without shaking. After an hour, vortex the cells for 30 seconds to disrupt the mating pairs.

Then, place the cells on ice for 10 minutes to prevent further mating. Using the mid log cultures and fresh sterile LB, prepare six serial dilutions of the donor and recipient cells from 1 and 100 to 1 and 10 million. On each of two halves of an agar plate containing naladixic acid, chloramphenicol, and kanamycin, spot 10 microliter aliquots of each dilution of XK1200 donor cells.

Repeat spotting for the dilutions of the recipient MC4100 cells on agar plates containing streptomycin. Then, incubate the plates overnight at 37 degrees celsius. Next, using the vortex mixture and fresh sterile LB, prepare six dilutions of the transconjugants.

Select for the transconjugant MC4100 cells that harbor the pOX38-Tc chloramphenicol knock-out bu spotting ten microliter aliquots of each dilution on each half of the agar plates containing streptomycin and chloramphenicol. Repeat for both duplicate mixtures. Then, incubate the plates overnight at 37 degrees celsius.

Calculate the mating efficiency by counting the number of colonies from the same dilution spotting for each donor, recipient and transconjugants cells on their respective plates. Also, count the recipient colonies to test for any bias resulting from a larger number of transconjugants than recipients at that given dilution. Calculate the mating efficiency as the number of transconjugant colonies divided by the number of donor colonies, multiplied by 100 to obtain the the efficiency value per 100 donor cells.

When the type four secretion system gene-tra-F is knocked out of the pOX38-Tc plasmid, it results in a loss of conjugative transfer of the pOX38-Tc traF chloramphenicol knock out plasmid from donor to recipient cells. However, when the traF gene is provided in trans by the PK184 traF plasmid an the donor cells, a recovery of conjugative transfer of the pOX38-Tc traF chloramphenicol knock-out plasmid is observed. When provided with dilution mutants of traF and the PK184 plasmid in the donor cells, loss of conjugative transfer of the pOX38-Tc traF chloramphenicol knock-out plasmid can be observed.

Loss of conjugative transfer indicates the region of the protein important for protein-protein interactions within the conjugative type four secretion system. This region can then be probed in more detail by point mutations that affect the efficiency for transfer. Once mastered, this technique can be performed in a single work day with expected growth times.

This protocol can be done on cells other than the ones demonstrated here. The critical aspect, at this point, is that, donor and recipient cells don't harbor the plasmid of interest. So that when you add the knock-out plasmid, you don't get conflicting results.

While attempting this procedure it is important to be very organized as there are different growth conditions and antibiotics for donor, recipient, and transconjugate cells. Following this procedure, other methods such as crystallography, NMR, or mass spectrometry can be performed in order to obtain a detailed structural and functional picture of the protein of interest.