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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

The team discussed the creation of a new part, a modified version of the p416 plasmid which would be biobrick compatible and would be used in our experiments with our created translational unit. We are still unsure of what assembly method to use to create this modified plasmid.

6/12/15

Our team started today by having a meeting to establish what needed to be finished for the week’s end, as well as goals for the following week. A checklist for achieving the Gold medal was also created. Enzymes and materials for Gibson Assembly were ordered, and began planning nucleotide sequences of parts. Our team also requested to time to work with middle school teachers in order to help them understand what they can do to teach kids about synthetic biology. Construct 1 arrived from IDT.

6/15/15-6/19/15

6/15/15

Our team performed minipreps to recover p416-GPD. Lab safety protocols were covered to Rose-Hulman’s standards. We discussed an outline for our parts documentation and submission status to the registry. The “About Our Lab” and “About Our Project” questionnaires had their first drafts completed. The TET system was investigated by our team as a potential candidate for an on/off switch. Construct 2 arrived from IDT.

6/16/15

Our team spent more time researching the tet-o system. We discussed regulating the tet-o system using the sugars galactose and glucose. This system would be regulated using varying sugars in the media.

6/17/15

Upon further looking into the tet-o system we found that there may have been potential copyright issues with using a tet-o system for our project. In order to avoid any potential conflicts, we started looking into new promoter systems that could potentially be used in our project.

6/18/15

Our team found three new potential promoter systems that may be used in the project: MET-25, FET-3, and FIG-1. Each promoter system was then discussed and thrown out due to issues with potential ligands needed to activate the systems and lack of information available that would prevent implementation.

6/19/15

Upon continuing review, the team found a new promising promoter system using copper. There are three possible promoters to use the two repressible CTR1 and CTR3 promoters and the inducible CUP1 promoter. Additionally, in the reviewed paper a copper chelator bathocuprione disulfonate (BCS) is added to remove copper which derepresses the system. We decided to continue research into this system as it is the most promising we have found.

6/22/15-6/26/15

6/22/15

We decided on continuing our examination of literature about the CTR1 and CTR3 promoter systems. The CUP1 promoter was ruled out since it is an inducible promoter and we prefer to use a repressible promoter. Upon further reading, it was found that many laboratory strains of yeast possess a transposon in their CTR3 promoters, preventing function. Thus, we chose to proceed with the CTR1 promoter. We read that these copper systems require Copper Recognizing Elements, CuREs, in their sequences in order to bind Mac1 protein. The activation of the system involves a binding event between the CuREs, Mac1 protein, and copper. Our sequence of CTR1 was then analyzed for these CuREs and three were found. Lastly, we found that a base concentration of 16nM of copper is necessary for the function of the promoter and the repression occurs around a copper concentration of 1µM. Additionally, we began consulting the literature to delineate the mechanism by which Mac1 protein interacts with the promoter.

6/24/15

The team planned for our human practices project with the middle schoolers. We plan on introducing them to synthetic biology and the idea of how genes are turned into proteins. A powerpoint presentation was made to accomplish this task and additionally a game or challenge was created to aide in this discussion. The challenge is for the middle schoolers to put together varying biological circuits in increasing order. The children will have access to various, promoters, genes, and terminators in order to accomplish these tasks. The students will begin with a generic promoter, gene, terminator system. This system will be used to explain the basic components in a general sense to the group before having them delve into the more advanced challenges.

6/25/15

Today the team had its synthetic biology education seminar with the middle schoolers. The students enjoyed the presentation and activity and were extremely interested in learning about synthetic biology and the field of biology in general. Each group was able to complete the challenges in a reasonable amount of time and were able to explain the purpose of each part in the biological circuit that they had constructed.

6/29/15-7/2/15

6/30/15

6/29/15

The team began a literature review regarding leader sequences and their function for mrps12. It was found that there are four common signal motifs that are found in leader sequences. The proteins involved in entry are mitochondrial peptidases for cleaving the signal peptidase after entry and various TIM and TOM proteins that mediate entry.

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