"
Team:Northwestern/Notebook
From 2014.igem.org
| Line 111: | Line 111: | ||
<li><a href="https://2014.igem.org/Team:Northwestern/Team">Team</a></li> | <li><a href="https://2014.igem.org/Team:Northwestern/Team">Team</a></li> | ||
<li><a href="https://2014.igem.org/Team:Northwestern/Safety">Safety</a></li> | <li><a href="https://2014.igem.org/Team:Northwestern/Safety">Safety</a></li> | ||
| - | <li class=" | + | |
| - | + | <li class="active"> | |
| - | + | <li class="dropdown"> | |
| + | <a href="https://2014.igem.org/Team:Northwestern/Notebook">Notebook<span class="caret"></span></a> | ||
| + | <ul class="dropdown-menu" role="menu"> | ||
<li><a href="#">June</a></li> | <li><a href="#">June</a></li> | ||
<li><a href="#">July</a></li> | <li><a href="#">July</a></li> | ||
<li><a href="#">August</a></li> | <li><a href="#">August</a></li> | ||
<li><a href="#">September</a></li> | <li><a href="#">September</a></li> | ||
| + | </ul> | ||
</li> | </li> | ||
<li class="dropdown"> | <li class="dropdown"> | ||
Revision as of 19:39, 2 September 2014
Caption 1
Caption 2
Caption 3
Week 1
6/17 We started our iGEM bootcamp by shadowing Will Bothfeld (our primary graduate student TA) during ligating, transforming and working with DNA software. In addition, we learned the fundamentals of primer design, colony PCR and gel electrophoresis. We ended by continuing research and discussing all of our projects of interest.
6/18 To start our day, we narrowed down on project ideas to CRISPR-Cas system, quick quench GFP, and non-model organisms; we then discussed their individual pros and cons. During our meeting with Professors and graduate students, we had an in depth discussion on this topic as well as laboratory logistics such as lab equipment and our summer budget. With this information and guidance, we were able add to our initial thoughts on each of our potential projects by starting corresponding flowcharts of events.
6/19 All team members finished necessary safety training modules online to partake in wet lab work. We then cleaned the lab and organized materials/equipment. Will proposed a problem to us regarding primer design that we worked on first individually and then collaboratively to compiled an answer to for later review.
6/20 After learning the basics of and observing plate reading, we compiled a series of protocols that we felt would be important to have before getting starting on DNA cloning first-hand. We also updated our lab shelves and freezer inventory thoroughly to get a sense of what we should be ordering and asking in our sponsorship emails.
Week 2
6/23 In the morning, we had a meeting with a TA, Karthik Sekar, to discuss the iGEM registry and parts along with how to use them. Following that, we had a meeting our professors and grad students in which we learned more about the project of non-model organisms. After our meeting, we made LB Mix and discussed which project to pick- non-model organisms or quenching GFP. In the end, we came to agreement to do our project on non-model organisms by having E Coli and yeast as our controls and other strains of bacteria and fungi as our non-model organisms. We would test out how promoters would differ in their activity across all these strains by the use of GFP.
6/24 Throughout the day, we sent out emails to sponsors for lab supplies. We also worked on a problem that Karthik gave us to do in which we had to construct a plasmid with an origin of replication and a PhoP promoter controlling PFP expression as the insert using iGEM registry parts. We also started cloning with Will.
6/25 We took an inventory of the chemicals we had from before in our lab and of the contents in our freezers. We met with Karthik again to discuss the solution we came up to the problem he gave us to do. In addition, we brainstormed project titles and created a tentative timeline for the summer. Lastly, we read a paper by Paul Freemont of the Imperial College of London that talked in detail about cell free systems being accurate representations of E Coli cells.
6/26 This day had relatively more wet lab work. We started by running PCR, and then running a gel for 1000 base pairs to confirm if the PCR worked. After analyzing our results, we researched promoters that we could potentially use to test for our project. We also searched for professors and labs around campus from which we could get strains of non model organisms. The final task we did was shadow Dr. Jian Li, a member of Jewett lab who showed us how to collect cells for lysate preparation and observation.
6/27 We started our day at the Jewett lab where a grad student, Rey Martin, showed us how to lyse E Coli cells and perform extract. We made another gel and tested for ladders. Meanwhile, some members of the team made M9 media and then autoclaved it. Two of our members met with a representative of IDT to talk about buying their products. Lastly, we continued researching promoters and reaching out to labs asking for non-model organisms.
place images here
Week 3
6/30 - We met with the professors to further discuss our project and our overall timeline. We gained information about cell-free systems from the meeting. We also decided not to use fungus as a non-model organism as eukaryotes would make the project overly complex. After, we had a meeting with Joe to talk about mathematical modeling. In our discussion, we also tried to refine and narrow our project idea.
7/1 - We started the morning in the Jewett Lab shadowing a grad student who was carrying out a cell-free reaction. We decided to split the team in two. One team focused on harvesting three strains of E. coli cells and the other dealt with the initial steps of cloning. All of us continued researching promoters since the cell-free system uses T7 bacteriophage and the promoters we had initially selected from the Chris Anderson list were incompatible. For cloning we used the 5 promoters which we originally planned to use. The T7 compatible promoters from the iGEM registry were not present in the distribution kit, so we need to order from IDT or some other similar company. We also obtained a sample of Pseudomonas cells (one of the non-model strains) from the Wang lab which was incubated overnight.
7/2 - Mitch and Sharon teamed up to harvest cells. We calibrated the spec by making serial dilutions and making a linear curve. We finished growing the starter culture and inoculated appropriate concentrations in 2x YTPG media. We grew these for a few hours and collected the cells by cycles of centrifugation. We used S30 buffer for extraction. We also had a meeting with Anthony, a grad student, who agreed to help us out by providing the non-model organism, streptomyces.
7/3 - We transformed plasmids into competent cells and then plated them. We designed primers for the inserts that we obtained the day before. We put the Pseudomonas cells in a frozen stock, while Karthik gave us an article about spinach aptamer to read.
Week 4
7/7 - We continued the lysate making process by resuspending and sonicating cell pellets. We made frozen stock of successful colonies from previous plating, but started another round of cloning using the same 5 plasmids and DH5alpha cells as before in the hopes to get better cell colonies. We also made a RAM chart to keep tasks and priorities organized.
7/8 - Tiffany and Adam had a meeting with Joe to talk more about mathematical modelling. All of us also had a meeting with the professors in which we talked in depth about outreach, T7 promoters, and judging criteria for the various medals. We continued to do cloning and cell free protein synthesis, and we had a review of primers with Will.
7/10 - We started the day by autoclaving beakers and flasks. David, Adam and Tiffany had a meeting with Joe to further discuss modelling. We grew two cultures of Pseudomonas cells in parallel from a frozen stock to establish a growth curve by checking OD every half an hour. We also had a discussion about how we will measure our results and we didn’t come to a clear conclusion, but some ideas that were thrown out were to measure strength by how much protein or mRNA is produced and how quickly protein is produced. We plan to test different RBS’s as well.
7/11 - Our team worked together to create a powerpoint to present to our advisors and update them on our progress. We then re-stocked our shelves with autoclaved materials and more media. We made our final edits to our g-block design and added it to our powerpoint. Afterwards, we headed to Chicago to meet with the UChicago iGEM team to discuss our respective projects and potential collaboration. The meeting proved very helpful- once we heard about their project, we found that their promoter could be tested in our lysates. We hope to get this promoter in plasmid form the next time we meet.
Week 5
7/14 - Abdullah and Kristi started working on a presentation on synthetic biology for the Center for Talent Development (CTD) high school program. We met with our advisors and found points of our initial testing plan that needed revision. We also got Streptomyces fluorescens from the Kelleher lab and prepared a survey for outreach.
7/15 - In the morning, we made LB and then autoclaved it. We also procured some glucose from Will and autoclaved that too. From Professor Yun Wang, we got a BSL1 strain of Pseudomonas in the afternoon. Abdullah, Kristi, and Adam gave a presentation of synthetic biology to a biomedicine class for highschoolers. In addition, we continued to grow strep as it takes two days while searching for different RBS sequences for our non model organisms.
7/16 - Abdullah and David went to Roycemore School to meet a CTD teacher to talk about having a synthetic biology presentation for his honors biology class. Adam, Sharon and Kristi went to a BioExcel event where they advised students on their application based projects, for example, how to prevent HIV. We also got N. Europea from Freddy from the Wells lab. After getting ISP ingredients from Jian, we made ISP and plates. We also analyzed the surveys that we handed out to CTD students the day before.
7/17 - We reached out to UChicago informing them that we can provide them with some E Coli strains. We did extensive research on RBS of the non model organisms we are experimenting out. We also ordered a gBlock but it had some issues so we decided to fix it the following day.
7/18 - We started to make a growth curve of strep, which was proving to be complicated as the OD kept on oscillating. We also revised our gBlock and finally ordered it to IDT. For the meeting with our professors on Monday, we prepared a powerpoint of what we’ve done this past week and questions we have. Lastly, we continued researching on RBS’s.
