Joshua Browning and Dr. Allen Buskirk, Department of Chemistry and Biochemistry
The TAT two hybrid system for discovering novel stalling peptides met with some immediate challenges that ultimately resulted in the abandonment of this project. The feasibility for using this method to discover new peptide sequences that would cause the stalling of translation within an elongating ribosome became unlikely.
The initial idea for this project was to use the typical two hybrid system to have two protein subunits fuse together when a peptide stalled. The bacteria cells would be in an environment where they would only survive if there was stalling along with this fusion. The protein that fused would carry out of the cell a protein that would protect the cell against the antibiotic in the media we were growing the cells.
This was a clever new way to delve deeper into the mystery of how certain peptide sequences cause ribosomes to stall, and how recovery comes via the curious nucleic acid polymer called tmRNA. The initial, and ultimately defining, problem with this method was the manner in which we set up the experiment. Unfortunately, the selection process for insuring that our plasmids (long DNA polymers that can be artificially made with desired protein coding regions, promoters, etc.) were taken by the cell would not allow for the experiment to work. We used an ampicillin based antibiotic in the media to insure that only cells who uptook the plasmid would survive. However, the problem with this is that the selection process we had developed for determining if the cell’s ribosome had stalled at a particular sequence used an antibiotic that is considered almost identical to ampicillin, only considered much more highly pungent. Had we continued with the project, we would have seen too many false positives to determine what were real cells with stalling sequences and what were just figments.
Dr. Buskirk determined that the likelihood of altering our selection processes to make them fit was slim. Choosing another antibiotic for our plasmid selection process might work, but unfortunately we were using a variety of other antibiotics as selectable markers to insure that other plasmids were uptaken by the cell. The conclusion was that this very clever means of identifying new stalling peptide sequences would have to be abandoned.
The result was that I was moved to another project. I was to test, optimize, and use a protocol developed by Dr. Griffits to knock-out bacterial genes. The method is via recombination, and I was assigned to use this method to knock out the trmD gene in a certain e. coli strain.
Working in tandem with another lab at John Hopkin’s we began working with the trmD and trm5 genes to determine their exact function. The lab at John Hopkin’s wanted a trmD knock-out strain of e. coli for their experimentation, and so I was assigned to use the method developed by Dr. Griffits.
My experience with these projects, and specifically the TAT two hybrid system taught me a great deal. It showed me that in preparation for a project it is best to check, and recheck, every method you are going to employ so as to not allow for your methods to contradict or inhibit one another. Preparation would have been key in determining if this would have been successful and we did not prepare effectively. We found after several trials that the system would not work because of the overabundance of false positives, and so we determined to move on to a different project.