Simple biochemical principles and methods are often applied to complex problems in cell and molecular biology. This assignment has elements of both. In addition, sometimes research projects take an odd turn, and we need to go back and check our work.
We are studying the enzyme lactase, which cleaves the disaccharide lactose into galactose and glucose. A DNA sequence of the lactase gene was recombined with a plasmid. We intended to introduce the plasmid into E. coli cells and then follow the expression of lactase from the plasmid gene from the bacteria. The lactase plasmid was produced by a company for us, but it does not seem to be expressing a functional protein when we isolate it from bacterial cultures.
1. We want to ensure the correct gene was actually cloned and sent to us. What method could you use to show that the entire specific and complete DNA sequence we require is present in the product we were sent? This method also has to verify that the base sequence is free of errors and mutations.
2. The result from your method above shows that the authentic lactase gene is present in the plasmid. You now want to test whether the E. coli cells express lactase mRNA. Which single method do you suggest to use to accomplish this? Use a different method than the one used for Q1 and you may use a method that was not described in class.
3. You read a report suggesting that E. coli expresses a protein repressor of the lactase gene. The repressor binds directly to the DNA and could be responsible for the absence of lactase mRNA. A recent report contains the DNA base sequence the repressor binds. Given this information, name the specific feature on the B-form of DNA where would you expect the protein regulator to bind.
4. Using only methods described in MCDB 310 and all the information above, devise a method to purify the DNA binding protein from E. coli lysate (i.e. broken up bacteria in a buffer solution). You may only use two chromatography methods (not just two steps) for purification from the lysate, and you need the purest sample of the protein possible. Because this protein is newly discovered, there is no antibody available. Again, you already have lysed cells. The question becomes: what two methods will get rid of most of the protein you don't want and which one is really specific for the protein you do want?