Notebook

Week 1: February 11-February 17

This week Dylan and Muriel started their 3A Assembly. They copied the protocol and materials into lab notebooks and comprised an Excel spreadsheet of the four plates in the Spring iGEM 2012 kit. They also made a stock solution of ampicillin. Muriel and Dylan streaked plates and grew media cultures. The plasmid miniprep was slow because they misread the directions, but it turned out well! Using the NanoDrop, they quantified the DNA. Muriel also installed the Lecia LAS EZ microscope photographer.

Week 2: February 18-February 24

Muriel and Dylan completed the 3A Assembly. Tuesday, Muriel ligated and combined the parts onto the linearized plasmid backbone. Wednesday was transformation day. They also grew some basic cells to make them competent. The transformations went well! Dylan and Muriel had plates with 1, 6, 22, and 46 colonies. The colonies were light pink because the plasmid backbone makes a red florescent protein. Also, they made the RFP control for 3A Assembly.

Week 3: February 25-March 3

After letting the plates grow more over the weekend, they extracted the plasmid DNA and then transformed it using fresh competent cells that Muriel made. The plates had large pink colonies as expected. Muriel learned about wiki programming software for our iGEM page. Later, with an open flame flickering, Dylan and Muriel streaked the 3A plates already spotted with large pink bacteria.

Week 4: March 4-March 10

Since the project thesis for iGEM is due next week, Muriel organized a brainstorming meeting. Our project will be more competitive if we build on another team’s project and have at least some experimental data instead of choosing a more ambitious project. However, the project should be scalable so the next year’s team can continue. So far, making microbes survive in saline and extreme temperature environments is our best option. Because of its application with biofuels, we can have an elaborate community outreach program with energy conservation and environmental issues.

Week 5: March 11-March 17

With the deadline for iGEM coming close, it is time to focus time and efforts on the development of iGEM ideas. At first, our iGEM project was to use properties of extremophiles, specifically halobacteria, to make the microbes more suitable to biofuel reactors. After seeking local professionals in the field, we finally met with Dr. Fenton from KU Med to talk about our iGEM project. He guided us away from biofuels because there are so many variables that need to be considered and the best conversions do not match the efficiency of normal gasoline.

Week 6: March 18-March 24

For iGEM, we located the plasmid shuffling vector from the 2008 Hawaii iGEM team and we have it with the 2012 distribution kit. Muriel sent an email to the team describing our project and asked about the part, but we have not heard back.

Week 7: March 25-March 31

For iGEM, we have been preparing to transform the cells. This week Muriel and Dylan made the components of STET solution that will extract the plasmid from the cell. The most challenging component of this solution is making EDTA which requires several days to go in solution with the help of sodium hydroxide, a strong base. Discussing about this year’s iGEM project, Muriel and Dylan brought up the pros and cons of the idea. Although it is not the most challenging project, it can be expanded on with by adding other environmental controls. One of the problems with this, and other projects, is the time limitations. We need to have our data by the end of May so that we can analyze, update the wiki, and make our poster.

Week 8: April 1-April 7

For iGEM, Muriel have been helping Dylan with the poster. We grew up a nice batch of E. coli with the broad host range plasmid inside, so they are ready for the next step, plasmid extraction. Since normal, kit-based extractions do not work for this large plasmid, we have to follow a longer protocol from the designers of the plasmid. This protocol requires a particularly dangerous chemical, phenol. We have reached out to Dr. Fenton through KU Medical Center because we do not have phenol. Another challenge that arose with the gene itself is that the coding sequence contains a standard restriction site.

Week 9: April 8-April 14

Because Dr. Fenton explained that biofuels have so many variables, he directed us to pursue a project that dealt with the overexpression of a protein. After doing some preliminary research on the production of phenylalanine, we realized that that metabolic pathway is more complex than we thought. We chose to up-regulate the pyruvate kinase gene because of its production of fatty acids and their application in biofuels.

Week 10: April 15-April 21

Meanwhile, Muriel have been in contact with a researcher in Hawaii that helped develop the broad host range plasmid and other parts for cyanobacteria that may be of interest to our project. He warned that this plasmid was difficult to work with and required non-standard protocol. Nevertheless, it’s all we have so far that is cheap and easily accessible.

Week 11: April 22-April 28

Arrived in the mailbox is the 3A Assembly package from iGEM! At CAPS today Alec, Amy, and Mason opened the box and started reading the directions of the procedure! We grew up cell cultures today by using an inoculating loop to pick up a colony from our agar plate and into the LB broth. Continuing the long and complicated procedure, the next few days Alec, Amy, and Mason started our first 3A Assembly plasmid mini prep! During the process we used our new and expensive micropipettes to re-suspend our cell pellets! Enjoy doing wet labs in class!

Week 12: April 29-May 5

Today Alec, Amy, and Mason checked our work. We used this special tool called the nanodrop machine which calculates the purity of a substance. Thank you Mr. Whalen for introducing this to us! Mason and Alec put a tiny drop of our plasmid onto this machine and found out that it is ultra-pure! What a great feeling! Later in the week, we learned that patience is key. We are moving on to the next step of this procedure. Alec, Amy, and Mason are making seed stocks right now! Afterwards, was our first iGEM team meeting! Even though we were missing a Corinne and Dylan, everyone else including Austin and Muriel discussed about the 3A Assembly and its purpose. One step closer!

Week 13: May 6-May 12

Instead of working on iGEM or the 3A Assembly, Muriel, Alec, Amy, and Mason are privileged to listen to Dr. Barney Graham's work about a vaccine for respiratory syncytial virus fusion. We are fortunate to have these amazing connections. Also, Mr. Kessler mailed off our news team members and mentors consent forms. It feels like iGEM is becoming the real deal. We can't wait for what iGEM has in stores for us! During our second meeting today, Corinne, Dylan, Muriel, Alec, Austin, Amy and Dylan discussed what happens when the gene of interest has a standard cut site contained within the coding sequence. Muriel is explaining this concept to the group, this time we have a full team!

Week 14: May 13-May 19

Still in the process of the 3A Assembly, but we are learning many new lab techniques and machines. We used the incubator and the nanodrop machine again. The transformation process takes a long time, literally a full day just ask Alec. While waiting we talked about how complex plasmids are. Also we took our first group picture with all of us, Corinne, Dylan, Muriel, Alec, Austin, Amy and Dylan! For the wiki a few of us wrote each other’s biography. Later, Alec plated the 3A Assembly transformation and controls. A fun fact we learned was synechocystis cyanobacteria was the first photosynthetic autotroph to have its entire genome sequenced.

Week 15: May 20-May 26
Who knew that making an abstract can be challenging? With the help of Mr. Kessler, Dylan, Muriel, Mason, Corinne, and Amy are trying to edit and correct our information without any distractions. It is a struggle. It is also a tedious task for wording things. Thanks to the iGEM Headquarters, they tweeted us an important requirement for the abstract. Good thing Amy created a twitter account! We found out that the abstract has a limit of 150 words… oops! Onto more editing for the deadline…

Week 16: May 27-June 2

Our last few days of school! Instead of taking an hour long exam, Mason, Corinne, Alec, Muriel, and Amy learned about metabolic pathways. We also began our discussion of primer design to make a biobrick. It is hard to create a primer under certain circumstances, but Corinne has an idea.

Week 17: June 3-June 9

Even though it is summer time now, we still go to CAPS to talk about iGEM material. We are dedicated students! Today Austin, Corinne, Amy, Mason, and Alec debated between the two different routes for producing a part. Which option should we take? With the assistant of Mr. Kessler, Mr. Whalen, and Mrs. Tuel, we can decide which option to take! Using the tinker cell software for creating the metabolic pathways from CO2 to alkanes, Mason is almost finished! Mason, Corinne, Amy and Austin all chipped in to help with the wiki too, and we are making additions to meet the wiki requirements. It is turning out well, thanks to Austin!

Week 18: June 10-June 16

Mr. Kessler, Corrine, and Amy all traveled down to Dr. Fenton’s lab to talk about our iGEM project. We learned more about plasmids, restriction sites and more. We are working on creating specific primers for our DNA site directed mutagenesis. It is tedious work and it is the longest primer Qingling has created. We looked over the sequence for pyruvate kinase many times to be exact. We may be getting closer to having a part for the registry.

Week 19: June 17-June 23

This week we will be losing Mr. Whalen because he is going to Paris. But we asked him about the protocols for our next steps in PCR. He showed us where all the materials are located. Everyone has been working hard on completing our iGEM wiki. There are so many parts to be done that Mr. Kessler divided the parts amongst us. Corinne is editing the method and safety, Austin is formatting the wiki, Amy is editing the notebook, Muriel is working on problem and solutions, and Alec is deciphering many articles from Dr. Fenton with the help from Mrs. Tuel.