Sharyu C Barapatrey 01:16, 10 February 2014 (EST)
Created lab notebook.
Over the past few weeks, completed the Golden Gate experiment, which was successful: multiple colonies (both red and green) were grown on the KanR plate. Red colonies meant that at least the kanamycin oligo transformed into the competent cells, while green colonies meant that both the kanamycin and the GPF oligo transformed into the competent cells.
For the main project of the semester, the current objective is to come up with an organism that is a bacteria (prokaryotic thermophile) and whose genomic DNA is easily available for the experiments to be performed over the course of the rest of the semester. This is to be done by Thursday.
Over the semester, each person will make at most three parts and the gene that we will be cloning out is a tRNA synthetase.
Sharyu C Barapatrey 13:29, 18 February 2014 (EST)
So over the last few days we have been going over the exact procedures that we will be following in order to conduct the experiments, focusing on the oligos and the plasmid (pBAD) that we will be using. Currently we have been told to start working on figuring out the oligos that we need in order to clone out the synthetases.
This is to be done by first finding the protein/nucleotide sequence of each tRNA synthetase for each organism chosen (Thermus thermophilus and Thermotoga mantima) on the NCBI website and then by figuring out what restriction enzymes recognition sites are present in each sequence. From here we have to figure out how to get the NcoI/BsaI/BsmBI site into each oligo and fix up any other restriction sites. Also, we have to fix some of the sequences so that they start with a methionine after the NcoI/BsaI/BsmBI site, and thus may have to have amino acid substitutions or insertions.
Sharyu C Barapatrey 13:34, 27 February 2014 (EST)
This week on Tuesday, we finished creating the oligos for tRNA synthetases for alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, lysine, and leucine in Thermus thermophilus. These were submitted to the professor and we will hopefully be able to start experiments soon.
Today, we are learning how to pour a gel and then run a miniprep (we are using a Qiagen kit for this).
Sharyu C Barapatrey 20:38, 7 May 2014 (EDT)
I feel like I learned a lot this semester, namely with basic synthetic biology techniques, since I had never done any of them before in a wet lab setting. Unfortunately, my group was not successful in transforming E. coli with the tRNA synthetases for our assigned amino acids from Thermus thermophilus correctly, which was a disappointment.
One of the biggest issues we had was that the digested vector and the digest PCR product didn't ligate together properly. This was seen through a gel where all of the lanes had the same bands show up, and indicated strong vector bleed-through. Though the rest of the experiment went off with minor difficulties, once we saw this gel, my group realized that something was very wrong.
So we went back to designing and ordering new oligos for alanine and isoleucine, and tried religating all the PCR products with a new vector digest (we were able to get some from Karim). Still because we were not able to fix the alanine and isoleucine PCR products, we had to go on without them in order to attempt the transformation again. This was also not a success and when I went back to culture the colonies only E, F, G, and H had colonies. This before the last class, and so we did not continue.
Also, this is a Google Doc that my team used in order to keep track of some notes (although why we used a Doc instead of this is something I don't remember). It has notes and some pictures of gels, so that it is easier to understand what we did over the semester.
While these notes are not complete, they do give insight into the issues my group and I ran into all semester.