Team:Sharon MA Aquila/Notebook

From 2012hs.igem.org


March

March 13: After looking through old iGEM projects for inspiration, we stumbled upon a project dealing with antifreeze protein (AFP) production. We began brainstorming possible applications of this protein, which prevents ice crystal formation in certain fish, plants, fungi, and bacteria. We discussed using AFP on roads, but after researching activation methods for road-thawing bacteria, we determined that we would have to introduce another material (arabinose, lactose, etc.) in order for our bacteria to begin producing the protein. Briefly, we researched cold activation methods, before determining that these methods were generally difficult to implement and only operational at temperatures above zero - not to mention that bacteria are usually nonfunctional below freezing.

March 20: After some discussion with Mr. Dixon and among the group members, we decided to take an idea from the 2008 MIT iGEM team, who created teeth-cleaning bacteria by transforming the bacteria used for yogurt cultures. We decided it might be a good idea to introduce the AFP gene into one of the yogurt bacteria, which would already be present in the food we were trying to affect. We additionally decided that the gene should be activated by lactose, because it was already present in the yogurt and because the bacteria already reacted to it to produce lactic acid.
After researching the two bacteria present in yogurt cultures, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, we determined that the former is more suitable for our experiment, because it generally remains in the yogurt following yogurt production - and because the MIT team wiki had detailed protocols regarding transformation of Lactobacillus bulgaricus bacteria.

March 27: Today, we started to plan out how we are going to execute our project. We plan on getting Lactobacillus buglarius bacteria from ATCC and our AFP gene from the Registry of Standard Biological Parts, hopefully from the source plate. The 2011 Yale team created a part called RiAFP (Rhagium inquistor antifreeze protein), which came from the Rhagium inquisitor beetle. More information about possible parts we can use can be found here.
Additionally, we also did some research on the procedures that we will need to complete in order to put our new DNA in the Lactobacillus delbrueckii subsp. bulgaricus. We will be using the 2008 MIT team's protocols for this task, including the Lactobacillus transformation and Lactobacillus miniprep. This is how we will culture our bacteria.

April

April 3: Today we wrote a parts list, which includes our bacteria in addition to the various DNA parts we need (the RiAFP part, a plasmid responsible for Erythromycin resistance, and the LacS promoter). We decided that we would introduce an antibiotic into our growth medium so that other bacteria would be unable to grow; therefore, we would need to make our altered bacteria resistant to the antibiotic. We also compiled a list of materials we will need for culturing and transforming the bacteria. Our lists and procedures can be located in the Project tab.

April 10: Because the registry is finally updated to accurately portray which genes are in which sample plates, we worked on locating the necessary parts on the plates. Unfortunately, we discovered that none of the BioBricks we need are in the source plates.
Additionally, we continued to work on the wiki, implementing dropdown menus.

May

May 1: Several people from iGEM came to our school to explain more about the organization, its purposes, and its growth over the years. They additionally introduced us to the procedure we will be performing next week, which will unite two segments of DNA in a single plasmid, thereby allowing the bacteria to express RFP. We also learned about the E, S, X, and P sites where the restriction enzymes would cut, allowing the DNA segments to attach to each other and the antibiotic immunity-providing backbone. Hopefully, this experiment will be good practice for the lab work we'll need to do for our own project.

May 8: We did the miniprep procedure today to practice extracting DNA from the bacteria it was being grown in. We ran into several problems with our centrifuges (we ended up having to use pliers to unstick the test tubes from their holders), but we were eventually able to proceed with the procedure. Unfortunately, in our last step we messed up the procedure and accidentally threw away the DNA while keeping the waste! The experience demonstrated quite vividly that we needed the practice before attempting the procedure on the bacteria for our project! We additionally tried to measure the concentration of DNA before determining that our spectrophotometers were insufficient for the task. We'll have to see if we can procure a new instrument or measure the concentration a different way.