Team:Penn/Notebook

June 11 2013 - June 18 2013

Goals:

This week we wanted to transform some parts for our methylation idea that we ordered from addgene. We also wanted to attempt a bisulfite conversion to test methylation. We had some problems transforming some biobrick parts the previous week, so we wanted to troubleshoot our transformations as well. Another goal of ours was to make more competent DH5-alpha and Dam- cells as well as figure out methylation assays for our promoters.

Achievements:

We worked out the details of one of our potential projects: creating DNA binding domain-methyltransferase fusions. Like last week, we transformed and miniprepped parts that we would need for the quorum sensing project, like the LuxI system. We also transformed and miniprepped the standard biobrick backbones psb1A3, psb1C3, and psb1K3, which would be useful in almost any project we decide to pursue. We pinned down and ordered exactly the DNA binding domains that we thought we would be using and ordered the gene blocks from addgene. We methylated a biobrick part, c0051, with an M.sssI, and used our bisulfite conversion kit for the first time.

June 18 2013 - June 25 2013

Goals:

It was increasingly important that we decide on our project. At this point we were considering a quorum sensing/cell signaling project that would build upon previously created quorum sensing parts submitted in the registry. We were also considering a methyltransferase-DNA binding domain fusion project with the goal of creating a new methyltransferase. We wanted to begin cloning t9002, a GFP producer controlled by an AHL signaling molecule receiver into psb1C3 from psb1A3 in order to use it in a quorum sensing device we were imagining.

Achievements:

This week we got all of our minipreps submitted for sequencing, and we made headway on our first cloning reaction. We figured out how to add restriction sites for cloning reactions using PCR and completed our first cloning reaction according to our successful colony PCR. We experimented with different thermocycler conditions and polymerases for our PCRs that had previously failed, and began to perform PCR with more success.

June 25 2013 - July 2 2013

Goals:

This week we wanted to think about applications for selective methylation in E. coli and figure out how our project could be useful for future research. We brainstormed how we wanted to create our constructs and began thinking about the cloning reactions that would be required to create them. We also wanted to begin creating our wiki and start generating useful content.

We confirmed that we completed our first successful cloning reaction. We transformed and miniprepped the biobrick parts for the quorum sensing system that we envisioned creating, with one cell having a light-induced AHL producer, and another cell having a AHL producer downstream of an AHL activated promoter. We thought about creating a device that would spatially separate the cells, as opposed to having them in the same media. We used a simple restriction digest assay to test how BstUI, a methylation sensitive promoter, would behave.

July 2 2013 - July 9 2013

Goals:

We wanted to experiment with USER cloning for assembling our gene blocks and minigenes. We also wanted to characterize pdawn and mcherry, parts we got from a lab at the University that we could use in our quorum sensing project. We wanted to attempt to PCR assemble our MsssI and begin fabricating a device for our quorum sensing project.

Achievements:

We practiced measuring fluorescence using our pDawn and mcherry constructs. We successfully completed PCR assembly of our M.sssI. We performed our first cotransformation with I751250, a AHL sender that was downstream of pDawn and T9002. We redesigned primers for PCR reactions that had been giving us trouble, paying more attention to the annealing temperatures of the primers we were designing.

July 10 2013 - July 17 2013

Goals:

As the summer was nearing its halfway mark, we wanted to decide on the relative merits of the two projects and reach a decision. We also wanted to continue our cloning for the two projects and troubleshoot cloning-related problems. We now had all the parts we needed for Cas9, TALE, and zinc finger constructs and wanted to begin assembling them into constructs that we envisioned. These constructs would have a two-plasmid system. One plasmid would have the DNA binding domain downstream of a T7 promoter activated by tetracycline, and the other would be a reporter plasmid tot detect methylation.

Achievements:

We decided on the constructs that we would create and began the restriction digest and gel extract steps of our miniprepped components. We planned an experiment we could do with tetracycline induction to test whether our hypothetical methyltransferase-DNA binding domain fusions were actually being expressed.

July 18 2013 - July 24 2013

Goals:

This week our main goals were to attempt our tetracycline induction experiment, order primers for performing a COBRA assay, and refine our LIMS and retroactively manage our samples. We also wanted to finish the cloning experiments we began the previous week.

Achievements:

We completed our construct for our zinc finger fusion plasmid after a successful colony PCR and sequencing result. We performed our first induction experiment by inoculating cultures with a tetracycline-activated promoter. We redid a PCR assembly of our TALE and Cas9 systems that had been failed before.

July 25 2013 - July 31 2013

Goals:

We wanted to finish our remaining fusion protein constructs and begin thinking about an SDS PAGE to measure protein expression. We also wanted to dedicate more time to pursuing drylab—making advances on our human practices and continue reading the literature on selective methylation. We also wanted to look into trying out different biobrick parts for our quorum sensing system.

Achievements:

We made advances on our cloning of the constructs for both projects. We completed a LIMS system on a Google spreadsheet that would be used to manage our increasingly overwhelming primers box as well as fixing our minipreps and glycerol stock LIMS and retroactively adding samples. We performed a new induction experiment, inducing our cultures with aTc.

Aug 1 2013 - Aug 7 2013

Goals:

We wanted to make advances toward generating our device for the quorum sensing project. We wanted to better budget our remaining resources and stock up on supplies that we would need throughout the rest of the summer. We wanted to use our zinc finger clone in an induction experiment, similar to the ones we had performed earlier for the quorum sensing project. We wanted to generate a growth curve and see how the zinc finger cells would respond to induction.

Achievements:

We met with various professors at the University and made contacts who had knowledge about areas in both projects. We ordered more reagents and created the abstract for our project. We designed primers to attempt to Gibson assemble the Cas construct that had been giving us trouble during USER cloning. We hoped that the new cloning strategy would yield better results. We aliquot our antibiotics, making boxes full of Kan, Chlor, and Amp to last us the entire summer and well into the year.

Aug 8 2013 - Aug 14 2013

Goals:

We wanted to use our restriction digest-based test to analyze how our constructs were working with regards to methylation. We needed to decide on which project we were going to pursue as soon as possible, and we decided this week would be the deadline. We wanted to perform our first SDS Page, which had been in the brainstorming stage for a while. Additionally, we wanted to consult experts about designing better primers and see how we could improve our protocols.

Achievements:

We finally decided to fully pursue the project of constructing targeted methyltransferase fusions. While it was a hard decision, it was necessary to commit our efforts to one project. We also finished our TALE clone and began the induction experiment for testing the methyltransferase fusion. We consulted with designers from the School of Design about making our website and better presentations. We began transforming promoters for an additional screen for methyltransferases that we had envisioned, with the belief that methyltransferases could silence promoters.

Aug 15 2013 - Aug 21 2013

Goals:

We wanted to get our Cas construct working after additional drylab work in designing better assembly reactions. We needed to make a detail plan of where our funds were going to go for the rest of the summer and begin planning our slides for our iGEM presentation. We wanted to decide where we wanted to be by the competition and what we needed to do to get there.

Achievements:

We began creating a plan for creating a video about methylation in general and our project specifically. After consulting with our mentor we decided to change the direction of our methyltransferase-DNA binding domain fusion from a two-plasmid to a one-plasmid system that would incorporate a digestion-based assay within the plasmid.

Aug 22 2013 - Aug 28 2013

Goals:

Our main goal for this week was to finalize our ideal cloning constructs and figure out how to port what we had already created into the one-plasmid system. We wanted to elaborate more on the assay, as our mentor had emphasized that as one of the more important contributions that our project could make to the synthetic biology community.

Achievements:

We focused on drylab this week, as we needed to get our plans for the rest of the summer set. We worked on a human practices essay about the ethics of methylation in medical therapies. We also began work designing new primers for our revised cloning strategy.

Aug 29 2013 - Sep 4 2013

Goals:

Our goal was to once again begin a new cloning strategy focused on assembling our one-plasmid systems.

Achievements:

We began setting up our travel plans to get to the Jamboree. We began the initial stages of cloning our new systems and made substantial progress on cloning our zinc finger and TALE.

Sep 5 2013 - Sep 11 2013

Goals:

Our goal for this week was to make progress on cloning our constructs. We also wanted to continue our work on the human practices essay, begin creating a poster presentation for presenting at University research conferences. We also wanted to begin thinking about designing an application for analyzing gels.

Achievements:

During a marathon weekend coding session, we created a program that we called MaGellin that was designed to analyze our gels and characterize the amount of methylation. We attempted to improve our cloning efficiency by utilizing Antarctic Phosphatase within our reactions. We made substantial progress on finishing our TALE and zinc finger constructs, and selected and grew colonies that would be used to test our assay the following week.

Sep 12 2013 - Sep 18 2013

Goals:

We wanted to make substantial progress on the website this week. We also wanted to run our assay and test how well our methyltransferase-DNA binding domain fusions had worked. We wanted to work on animation for our video as well as make progress on our human practices essay.

Achievements:

We ran the assay on the TALE and zinc finger clones, and it yielded promising results. The expression of our fusion protein was a definite achievement. We made substantial progress on the website as well.

Sep 19 2013 - Sep 25 2013

Goals:

We needed to finish our website and various forms this week, as well as run our assay in triplicate on the constructs we had created thus far. We needed to plan our remaining time up to the Jamboree, and practice our presentation skills at various outreach events.

Achievements:

We attended several events aimed at recruiting future team members that showed substantial interest in our research and iGEM in general. Our assay returned promising results about the state of our methyltransferase-DNA binding domain fusions. Additionally, we finished the website and filled out all the forms we needed. We have a few experiments still in progress, but our time now is spent preparing for the Jamboree and being ready to present our results.