Difference between revisions of "Team:UMaryland/notebook"

Week 1

Our first step this summer was to finalize the design of our three plasmids (sMMO, Fructose, and Formate). Many of the genes needed to create our plasmids are offered in the iGEM registry, but we realized that we would need to order some genes as GBlocks from IDT. We began to design GBlocks for the genes not offered in the registry (MMOY & MMOZ subunits, MDH2, HPS, PHI), which was a new experience for most of our lab members, who had little experience creating parts for Gibson Assembly.

This summer, we moved into a new lab space, so we had to set up the lab with all our equipment. Once that was completed, we were able to train our very young team in the basic skills of genetic engineering, like using micropipettes and performing transformations.

week 1, wet lab, set up, lb, idt, gblocks, mmoy, mmoz, mdh2, hps, phi, smmo, fructose, formate

Week 2

We received the iGEM kit this week! After opening the kit, we all cloned the biobricks we had requested (MMO subunits B,D,C,X; FDH; FALDH) to create stocks of them. After cloning those genes, we grew overnight cultures that we miniprepped to isolate the plasmids with the biobricks.

During dry lab, we revised the design for our GBlocks (MMOY & MMOZ subunits, MDH2, HPS, PHI), to include restriction sites, and promoters and terminators where needed. We also scavenged the building for old lab equipment that other scientists wanted to dispose. In addition to scavenging for lab equipment, we scavenged for DNA and asked team Braunshweig for a copy of the sMMO plasmid they constructed in 2015.

week 2, wet lab, dry lab, gblocks, mmoy, mmoz, mdh2, hps, phi, restriction sites, re sites, promoters, terminators, braunshweig, smmo, plasmid, clone, overight, miniprep, fdh, faldh

Week 3

We started assembling parts together to reach our final goals of creating plasmids with their respective promoters, ribosome binding sites, enzymes, and terminators. We used the technique of 3A assembly - the digestion and ligation of an antibiotic resistant backbone with two inserts - to attach ribosome binding sites to each of the biobricks we requested.

We then transformed our competent cells with the assembled plasmids (after which they grew successfully on our antibiotic plates) and grew them overnight to then extract the plasmid DNA by doing a miniprep. We also contacted a professor on campus who works with methane to ask her about her safety protocol when handling the gas.

week 3, promoter, promoters, rbs, ribosome binding sites, terminator, terminators, 3A, 3A assembly, ligation, digestion, biobricks, transformation, transform, methane, professor, miniprep

Week 4

After digesting our minipreps, we screened our resulting plasmids using a gel. Our results from the gel matched up with our expectations, so we continued our process of assembling the plasmids using 3A. This time, we attempted to combine the RBS and FDH plasmid with the RBS and FALDH, RBS and MMOB with RBS and MMOD, and RBS and MMOC with RBS and MMOX. Additionally, we finalized our g blocks and met with several university employees to discuss funding.

week 4, minipre, gel, 3A, rbs, ribosome binding sites, FALDH, MMOB, MMOD, MMOC, MMOX, g blocks, GBlocks, funding