You are interested in whether the protein you are studying for your dissertation, ZyxR, is capable of binding to and regulating expression of the ZyxABC operon. ZyxR is a 24kDa protein with a slightly negative overall charge and no known inherent tags. ZyxR is known to bind to the zyxY gene to regulate expression. yopA (100bp) is not regulated by ZyxR. The first step in this process is to determine whether ZyxR is capable of binding to the regulatory regions surrounding the zyxABC operon. 1. Give one potential strategy that you could use to purify ZyxR. Your strategy should be a multistep process beginning with cell lysate (containing millions of proteins) that you will take to purify the protein, should address potential shortcomings of each step, and should attempt to rectify those shortcomings. If your strategy requires any special conditions or changes to ZyxR, you should include those in the details. Drawings of your strategy/steps are highly encouraged!
2. You decide to conduct an EMSA to determine whether ZyxR binds zyxABC-adjacent sequences. You have successfully purified ZyxR and amplified the appropriate sequences (termed zyxABC). In setting up the EMSA, you are unsure about what samples/reagents should be present in each well. Fill in the table below with +/- to describe the contents of each well (there should be either a + or - in each empty cell, some have been completed for you). (4pts, 0.33/pt per correct cell) Contents Negative control (probe only) Lane purpose Negative control Positive control (non-specific probe) (known binding) Experimental sample + ZyxR ZYXABC zxY vopA