Difference between revisions of "Team:RHIT/DesignNotebook"

Click here to edit this page!

Notebook

What should this page have?

• Chronological notes of what your team is doing.
• Brief descriptions of daily important events.
• Pictures of your progress.
• Mention who participated in what task.

6/8/15-6/12/15

6/8/15

Our team spent time discussing project ideas, establishing the overall goal to create an on/off “switch” for the mitochondria in S. cerevisiae. We also spent time clarifying each of the medal requirements, inquiring from iGEM headquarters on questions that arose. We began discussing possibilities for parts submissions, including the ORF for mRPS12, a promoter, and a reporter. Since yeast are able to survive without mitochondria on fermentable media, we will test the efficacy of our construct using a growth test on fermentable and nonfermentable carbon sources. The strains with their mitochondria turned off are expected to only grow on fermentable media. We also discussed Kozak sequences and determined that the Kozak sequence listed on the registry on the yeast page is actually the mammalian sequence. Our team began researching the consensus Kozak sequence in yeast.

6/9/15

Today the group discussed two constructs that will be developed for our project. The first will be composed of the wild type mRPS12 sequence, preceded by the native Kozak sequence. We discussed the use of an ADH1 promoter. Our team designed and ordered two parts from IDT. The first sequence (construct 1 optimized) was generated by optimizing the wild type sequence of mRPS12 from S. cerevisae for expression in yeast. Following optimization, two serine codons were modified from TCT to TCA in order to eliminate prohibited Xba1 restrictions sites. The mRPS12 ORF was then flanked by 30 nucleotide overhangs homologous to the prefix and suffix listed in the registry to allow for insertion into pSB1C3 via homologous recombination. This part will be used as our new BioBrick part for the second silver medal requirement.

In addition to the mRPS12 ORF construct, our team also designed and ordered a transcriptional unit to submit to the registry as a new part (construct 2 optimized). This part consists of the optimized mRPS12 ORF following the native Kozak sequence. This composite part was also flanked by 30 nucleotide overhangs homologous to the registry’s prefix and suffix.

Lastly, our team discussed possibilities for promoters that could be used for our translational unit and concluded that a tetracycline operator system (TetO) could be useful for regulation of our construct. With this system, we would also need to utilize a construct consisting of a constitutive promoter (perhaps ADH1) and the TetR-VP64A coding region in order to produce the regulatory element (TetR-VP64A) required for the operator.

6/10/15

Today our team began discussing methods for visualizing yeast mitochondria as well as methods for comparing the mitochondria between the on and off states. Several methods were proposed including staining with dyes, tagging with fluorescence, and viewing under a light microscope.

It was also realized that the MRPS12 ORF part order previously (construct 1) was designed with the prefix for non-coding regions rather than the prefix for coding regions, and must be reordered with the correct prefix sequence.

6/11/15

6/12/15

6/15/15-6/19/15

6/15/15

6/16/15

6/17/15

6/18/15

6/19/15

6/22/15-6/26/15

6/22/15

6/23/15

6/24/15

6/25/15

6/26/15

6/29/15-7/2/15

6/29/15

6/30/15

7/1/15

7/2/15

7/6/15-7/10/15

This week was taken off by the team

7/13/15-7/17/15

7/13/15

7/14/15

7/15/15

7/16/15

7/17/15

7/20/15-7/24/15

7/20/15

7/21/15

7/22/15

7/23/15

7/24/15

6/27/15-7/31/15

7/27/15

7/28/15

7/29/15

7/30/15

7/31/15

8/3/15-8/7/15

8/3/15

8/4/15

8/5/15

8/6/15

8/7/15