I am working on a bacterial hypothetical protein, which belongs to a drug transporter family but other than that nothing is know about this protein. I want to characterise it, I have made a knockout already, can anyone help with suggesting some transporter assays or drug transport assays ?
In addition to making a knockout, you should also overexpress the protein. The wild-type, knockout and overexpression strains should be isogenic otherwise. Then you can test for the effect of the protein on the resistance of the bacteria to antibacterial drugs, using broth microdilution or agar plates to measure the minimal inhibitory concentration of the drugs. This will provide evidence of whether or not the protein confers drug resistance, but it will not directly show that the protein is a drug transporter.
Measuring drug uptake in efflux in bacteria is difficult, but it has been done. The easiest way is to use a radioactive drug, allowing you to measure the radioactivity of the cells. Fluorescent molecules can also be used in some cases, such as ethidium bromide, if they are substrates for the transporter. It is a very challenging measurement with drugs that are neither fluorescent nor radioactive.