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Lecture 2

MCDB 310 Lecture Notes - Lecture 2: Endonuclease, Restriction Enzyme, Sticky And Blunt Ends

Molecular, Cellular and Developmental Biology
Course Code
MCDB 310
Kenneth Balazovich

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Assignment 2: Plasmid Not Working?
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. (1 point) We want to ensure the correct gene was actually cloned and sent to us. What
method could you use to show that the specific and complete DNA sequence we require is
present in the product we were sent? This method also has to verify that the gene is free of
Automated DNA Sequencing
2. (1 point) The result from you 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 method do you suggest to use to accomplish this? Use a different method than the one
used for Q1.
Northern Blotting
3. (1 point) 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.
Major Groove
4. (2 points) Using only methods described in MCDB 310, 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 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.
We can first use separation by charge via cation exchange which will bind protein to the anionic resin.
We can further purify the protein using separation by affinity, using DNA column which will bind the
5. (3 points) Now you want to find the gene for this lactase regulator. You have purified the
protein and you have access to a database containing the complete genomic DNA sequence of
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