Aug
22

Good to be back!

I arrived back on campus a week ago, and I jumped straight into my research again! I have a lot of projects going on, so bear with me.

  1. HopZ adhesin expression and regulation: As a recap, I am looking at mutations in the poly-adenine (poly-A) tract and poly-CT tract and how they affect hopZ expression. Mutating the poly-A tract (+/- 5 adenines) hypothetically increases RNA polymerase binding affinity and mutating the poly-CT (+1 CT) puts the gene in frame for complete transcription and translation. J99WT H. pylori has a hopZ poly-A tract length of 15 A’s and poly-CT tract length of 6 CT’s. Our mutants are A9, A19, and CT7. Previously, preliminary results showed that these mutations increase hopZ expression. However, at the end of last summer, Dr. Forsyth and I discovered that all along, out CT7 mutant had a duplication of the poly-CT tract! As this is a confound, we have to generate a new CT7 mutant. In addition, we are going to try and examine the additive effects of the two mutations (poly-A +/- 5 adenines and poly-CT +1 CT) on hopZ expression. We hypothesize that the two mutations put together will further increase hopZ expression. Now to my work for the past week- I have been working on creating a new CT7 mutant as well as creating double mutants A9CT7 and A19CT7. To do this, a previous research ligated in the hopZ gene into a PCR4 TOPO cloning vector and designed mutagenic primers to mutate the poly-A tract to A9 and A19. I have taken these A9, A19, and WT A15 plasmids and used a poly-CT7 mutagenic primer to introduce a second mutation. Using an antibiotic resistance screen, I selected for E. coli colonies that hopefully contain these new mutant plasmids. I prepped these plasmids and ran a sequencing reaction. Hopefully I will get good news on this project next week!
  2. SabA poly-T tract adhesion studies: As another recap, in a previous study, I showed that increasing or decreasing the sabA promoter poly-T tract by 5 thymines leads to increased expression of sabA. I now want to look at whether this increased sabA expression leads to increased adherence of H. pylori to human gastric adenocarcinoma (AGS) cells. At the end of the summer, I got to work with this tissue culture (with the help of Dr. Hinton) and infect them with my poly-T mutant and a control. J99WT has a poly-T tract length of T18 and my mutant (with increased sabA expression) has a poly-T tract length of T23. Digging through some sequencing files from our own, as well as external, databases, Dr. Forsyth and I found out that the strain we’ve been working with, J99, actually has sabA phase-off with a poly-CT tract of CT8… Because we are now doing studies that require functional SabA protein, this poses a problem because a phase-off gene will not be transcribed and translated completely. Although the strains I used to infect the AGS cells had sabA phase-off, this experiment acted as a good control. Preliminary results of my adhesion assay show that the T23 mutant adhered slightly more than the T18 control. A small percentage of the H. pylori population may have sabA phase-on due to slip-strand mispairing (an error that occurs during replication), which would explain these results. However, during our assay, we noticed H. pylori colonies only growing at the less dilute concentrations (10^-1 through 10^-3), which is unusual for this assay. I hypothesize that this could be due to the fact that I grew up my H. pylori strains in a cholesterol supplemented broth for 24 hours before infecting the AGS cells. 1X PBS with 0.1% saponin was used to lyse the AGS cells. Saponin complexes with cholesterol within the AGS cell membranes, creating pores and thus lysing the AGS cells. H. pylori does not normally have cholesterol integrated into its membrane. However, the incubation of H. pylori in a cholesterol-supplemented broth may have led to the incorporation of cholesterol into the H. pylori membrane. Therefore, the addition of saponin may have lysed not only the AGS cells, but some of the H. pylori as well. I am changing my protocol to grow the H. pylori on blood agar plates, rather than broth, and then infect the AGS cells. This past week, I have been taking care of my AGS cells and growing my T18 and T23 strains from freezer stock to prep for another adhesion assay. I have also received my primer for mutating the sabA poly-CT tract to put it in frame. I will be working on a mutagenesis reaction to put the poly-CT tract in frame this coming week. *I will need to run my adhesion studies before I can move on to examining CagA translocation and IL-8 production*
  3. SabB expression regulation and function studies: sabB is sabA‘s paralogous gene. Little is known about SabB’s function. I will be mutating sabB‘s poly-thymine tract similarly to how I mutated that of sabA. I will be also putting the sabB poly-CT tract in frame. I will then insert this mutant plasmid into a J99 sabA knock out strain and run adhesion studies to see if increasing sabB expression rescues sabA‘s function as an adhesin. Right now, I am working on constructing my sabB plasmid. I think I have successfully ligated in the sabB gene into the plasmid (from gel electrophoresis results), but I ran a sequencing reaction this past week to confirm (again, hopefully I’ll hear good news next week!)

TO DO:

  1. Create A9CT7, A19CT7, and CT7 hopZ plasmids, transform J99WT, run qRT-PCR
  2. Create T18CT8 and T23CT8 sabA plasmids, transform J99WT, run qRT-PCR
  3. Run adhesion studies on T18, T23, T18CT8, and T23CT8
  4. Run CagA and IL-8 studies on T18, T23, T18CT8, and T23CT8
  5. Create sabB plasmid as well as T5, T15, T5CT10, and T15CT10 mutants, transform J99WT, run qRT-PCR
  6. Run adhesion studies on T5, T15, T5CT10, and T15CT10

It looks like I have a hard road ahead of me for this upcoming school year, but I am always ready for a good challenge!

Until next time,

Catherine