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Team:BU Wellesley Software/Notebook/EvelynNotebook
From 2011.igem.org
Contents |
Week 6/1/11-6/3/11
Participated in iGEM bootcamp. For the week we we learned bio-basics, CAD basics, Tuberculosis overview, digital logic design and clotho. For clotho we built an application that would show hello world and then we modified the app so that another user could input a part sequence that would be stored in the clotho database. Finally we had a group outing at Jillian's.
Week 6/6/11-6/10/11
On the last day of bootcamp we learned more information on synthetic biology and gave the Wellesley team a tour of the wetlab and our liquid handling robot. The next day I worked with Kyle Jones to look for constitutive promoters in the MIT Registry of Parts. We decided to use BBa_J23100, BBa_J23101, BBa_J23109, and BBa_I14053. The first three are part of the Anderson Collection and are Amp Resistant. These promoters had RBS inside their plasmids. The last constitutive promoter is Kan resistant. Vanessa Yanez and Alberto looked for induced promoters and Shannon and Margueax looked for RBSs. After locating our parts we transformed them and let them sit over night. Unfortunately the parts that we transformed did not work after the plasmid prep. We had low values for the concentration and realized that the cultures that did grow in the LB and amp solution was not E coli. In order to confirm our suspicions we looked at the culture under a light microscope. My cultures seemed to have string like cells which we concluded to be contamination. Only 1 culture worked out of the 18 that were performed that week. Finally we met together to figure out how we wanted to split up the work and what exactly we wanted to do as a team. Margeaux and Alberto did mini prepreps, restriction digest, gel extraction, ligations, and finally transformations for GFP and terminators. The rest of us redid all the old procedures in search for the reason for the contamination.
Week 6/12/11-6/17/11
In order to decrease contamination we autoclaved the pipet tips and used aseptic techniques where applicable. We redid the plasmid preps for the same parts. This time we grew the Ecoli in just LB solution and then in LB with Amp. The next morning we discovered that the cells in the LB and Amp solutions did not grow while the cells in the LB solution did. This proved our suspicion that the plates were our problem. On Monday we prepped the UV inducible promoter for the QiaCube. The UV promoter was the only transformation and plasmid prep that worked. We did the transformation on the existing Amp plates but this time we used a negative control. After incubating the transformed cells we discovered that the negative control did in fact grow colonies. This lead us to making new Amp plates and redoing transformations and plasmid preps for the chosen parts. For our transformations we had two of our Anderson Constitutive promoters grow red colonies. When we did plasmid preps however they did grow in the LB and AMP solution. These parts had concentrations values ranging from 9.6 to 56.6 ng/microliter. In the meanwhile, Alberto and Margeaux cut and ligated more GFP and terminator B0015.
Timeline
Week 6/19-6/24 Create the various constructs using the parts we have finally made plasmid preps of using GFP. Once we can prove that the construct in fact worked we will test them using the RFP, EYFP, and BFP. This way we will know how to order the various fluorescent proteins using various promoters and RBS.
Week 6/27-7/1 Figure out how to use invertases and figure out how to incorporate them into our various constructs. Trouble shoot any problems we run in to and fix them. Then we can try to flip the gene with the invertase and see if it actually works.
Week 7/4-7/15 Start to organize all the parts in the order in which the comp team has chosen to build them. It will be our task to figure out how the part will work together and what we will need in order to allow the process we want it to do to happen.
Week 7/15-8/1 Finish up the desired construct and possibly explore other forms using the TB gene.
Week 6/20/11-6/24/11
A restriction digest was performed on EYFP, BFP, ECFP, RBS: BBa_J61124, RBS: BBa_J61100, ligated GFP and terminator B0015,
