Monroe Update 2

It’s been awhile since my last post! I ended my last post describing the cloning reaction that allowed me to make a large number of copies of the plasmid that contained the region of interest. I had to then extract the plasmid from the bacteria grown up in the broth cultures using a procedure called a Mini Prep to continue working with the region of interest. This is done by lysing the bacteria to release the genomic material and is followed by purification steps to separate the desired DNA from proteins and other impurities. After performing the Mini Prep, I now had plenty of samples of the plasmid with the region of interest to work with.

To clarify the overall goal of my project, I am working towards modifying this region in such a way that I can remove sections of the sequence leading up to the promoter region of the napA gene. By removing sections of this sequence, I can determine the site that crdRS binds to in order to regulate napA. When I delete the site that crdRS binds to in order to regulate napA, I would expect to see a change in napA activity since it would not be regulated. There are several successive steps that must be performed to make this possible.

I began by creating a mutation in my region of interest for a BglII restriction site. BglII is restriction enzyme that cuts the sequence at a specific palindromic site (shown below). However, the BglII restriction site doesn’t actually exist within my region of interest, so I have to first induce a mutation to create the site. To create this mutation, I designed large primers (nearly fifty nucleotides long) that were complementary to the 26695 sequence with the exception of three nucleotides that were changed to encode the BglII site. This high degree of similarity allowed the primer to hybridize with the template sequence during PCR, causing the resulting copies to contain the mutation for the BglII site. These PCR products are then run through an agarose gel to make sure the entire plasmid was copied before being transformed into E. coli. Following standard procedures, individual colonies from the transformation are selected and then grown in broth culture to rapidly make a large number of copies of the plasmids containing the BglII mutation. Again, after these broth cultures are grown, a Mini Prep is performed to extract the plasmids.

BglII restriction site     BglII

There are two ways to check that a restriction site is introduced into a sequence, and I performed both on my mutant plasmids. One method is to sequence the region of the plasmid to confirm that the mutation is present in the new plasmids. Another method is to perform a simple digestion reaction. When the BglII restriction enzyme cuts the sequence at its restriction site, the circular plasmid is linearized. This digested plasmid is then run on a gel to compare its size to the uncut plasmid. If the linearized, digested plasmid is shown to be larger than the supercoiled, uncut plasmid on the gel, then we know that the mutation for the restriction site was successful because the enzyme was able to function. Since my mutants passed both tests, I know that the mutation was successfully added to my samples.

After confirming that the BglII site was added to my region of interest, I was able to begin attempting to insert the a gene for chloramphenicol acetyltransferase (cat) into my plasmid. The cat gene allows bacteria to be resistant to the antibiotic chloramphenicol. I need to insert the cat gene into my region of interest because H. pylori will not replicate the entire plasmid like E. coli. The gene for ampicillin resistance that I mentioned in my first blog post is included within a different region of the plasmid, and consequently won’t be transferred or functional in H. pylori. However, the region of interest that I’ve been working with will be capable of recombining with the H. pylori genome because of its high similarity, since it was originally taken out of strain 26695. Thus, when my modified region is recombined back into H. pylori, the cat gene will also be transferred, as it is within the region.

Inserting the cat gene into my region of interest is a fairly involved process. I first have to open the plasmid by digesting with BglII. BglII makes a sticky-end cut; there are overhangs from the strands because the cuts do not line up with each other. To ligate the cat gene in, which has blunt-ends, I have to add blunt-ends to my digested plasmid. This protocol simply fills in the overhangs, which allows the cat gene to be directly ligated in. After allowing a molar excess of the cat gene to incubate with the blunt-ended digested plasmid, this ligation reaction is added to E. coli for another transformation reaction.

Unlike my previous transformation reactions, this one yielded only one colony. Although this was discouraging, it was still possible that the colony had a successful ligation of the cat gene, so I grew it in broth culture, performed a Mini Prep to extract the plasmid, and had the region of interest sequenced to see if the cat gene was inserted properly. However, sequencing did not provide any clean proof of the sequence, indicating that the ligation and transformation reactions were unsuccessful. I retried the blunting, ligation, and transformation with higher efficiency E. coli bacteria, but this time I got no successful colonies. I retried the whole process again with a different sample of the cat gene, but this also yielded no colonies. Several other people in my lab have had a lot of trouble working with the cat gene, so these problems are somewhat expected.

Since all of my attempts to insert the cat gene have failed so far, I will try generating my own new sample of the cat gene in the hopes of producing a more pure sample to work with.