Monday, June 29th
On Monday we continued PCRing the Gblocks and resuspended the new GBlock we received. We also got training to image the gels we ran on Friday. The kill switch team found that the second ligation transformation didn’t work and ran a gel of the digests to problem solve.
Tuesday, June 30th
On Tuesday we got gel results. Our PCR reactions did not have the expected results. Instead of having 1 band, the gels had smeared bands. Also, our digest gel only had one band per column, meaning the DNA was never cut. This would be why the ligation and transformation didn’t work.
Wednesday, July 1st
On Wednesday we learned that our PCR was not working because the thermo cycler was mislabeled. We were using the wrong sides of the machine. This was also the cause of the digest’s failure. We restarted PCR for all the Gblocks, doing it correctly this time. The kill switch team made more LB with Chloramphenicol to inoculate the promoter for miniprep. We received the terminator from the registry and transformed the lysing agent and terminator parts into E. Coli.
Thursday, June 2nd
On Thursday we continued doing correct PCR and running gels to confirm results. Additionally, we minipreped the promoter, RBS, and terminator parts for the kill switch. Also, we found that our terminator and lysing agent transformations had worked and inoculated some LB with Chloramphenicol so we would be set to miniprep and assemble on Monday.
Monday, June 22nd
On Monday, we spent the day researching alternative assays to perform, as well as investigating alternative killswitch pathways. We also inoculated the E. Coli test colonies in LB+Chloromphenocol broth to determine the success rate of our promoter plasmids. Before plating the liquid culture, we also wrote out and clarified many of the procedures we’ve performed and others yet to come.
Tuesday, June 23rd
On Tuesday (upon the arrival of more buffers), we miniprepped the killswitch’s RBS, Backbone, and Terminator DNA sequences, preparing them for assembly. Also, our practice yeast transformation was discovered to be a failure, as our yeast broth was contaminated through unknown means. Bottlenecked by the lack of IDT DNA, we decided to re-write the majority of our procedures in paragraph form, for easier addition to our wiki.
Wednesday, June 24th
On Wednesday, we discovered yesterday’s miniprepping to be a failure, prompting a second effort, as well as miniprepping the promoters for eventual transformation. Also completed today was the first preparation of our switchgrass sample via milling. We began the transformation of our RBS+Terminator genes into competent E. Coli cells. Outside of the lab, we began brainstorming fitting titles for our project. Considering the Pirate theme, “Dead Lignin Tell No Tales” seems to be the current leader.
Thursday, June 25th
On Thursday, our G-Blocks arrived from IDT! We spent the afternoon diluting and re-suspending the DNA samples, and began PCR in the late afternoon. We also started our second attempt at the RBS+Terminator assembly (as Wednesday’s failed… the use of competent cells places our DNA under suspicion.). We also began electrophoretic gel construction, in order to more fully check our results and discover the source of the error.
Friday, June 26th
On Friday, we underwent gel imaging training, in order to obtain clearer results from the gels (the ethidium bromide used in the gel fluoresces under UV light, allowing easy identification of DNA bands). Then, we ran gels for last night’s PCR products. We also poured more LB/LB+Chloromphenocol broth, for use when testing the transformations of E. Coli ligations (also performed today). We also discovered that the assembly of the RBS to the terminator was a failure, prompting the research and purchasing of new killswitch components. Also, due to our Graduate advisor running a workshop next week (rendering BIND134 unusable), we moved supplies downstairs for easy access.
Monday, June 15th
On Monday we started our day greeting our buddy high schoolers that came from the MASI program at Purdue. We showed them through basic lab sterile technique and got their aid on our second, revised mini prep, which gave a high yield of our E.coli. Backbones. In addition, we finally managed to tie up loose ends with our gene problems and ordered all the parts we needed from the registry and IDTDNA.
Tuesday, June 16th
On Tuesday we discussed our project progress with our PI, Jenna Rickus, and got her input on how to assay our enzymes that were transformed in our yeast. We then began running a growth curve test on our wild type yeast cells to set a standard to compare our modified yeast to. However, complications began to rise when the machine we used to count or cells experienced issues. Due to technical difficulties, we had to postpone this test for a later time. The high schoolers got the chance to learn more about lab technique when we decided to start our first E.coli transformations with our promoters, RBS, amp backbone, lysis agent, and terminator genes.
Wednesday, June 17th
On Wednesday we went through a second round of E.coli. Backbone mini prep in order to stockpile on DNA for future experiments. However, due to restraints on mini prep supplies at the end of our second round, we resorted to creating our own buffers in the lab. In addition, only one of the transformations that we did yesterday went through successfully. Because of this, we were really bottlenecked for tasks and experiments we could do. Instead, we turned to coming up with possible themes for our lignin breakdown project, writing up more of our wiki information, and started on our yeast death curve.
Thursday, June 18th
On Thursday, we decided to attempt another E.coli backbone mini prep with our new buffers, but faced some lackluster results as the buffer we made was not optimal enough to extract the DNA properly. Also, the re-cultured E.coli. Were submersed in broth that didn’t have antibiotic resistance, so selective pressures reduced the amount of DNA that we could collect from a sample. Since we had to wait for our transformed cells to grow in their plates, the only lab related activity we had left was to create more competent cells for future experiments. The MTT assay kit we had bought also had very vague and confusing instructions, so we needed to refer to our graduate assistants and advisors for help and alternatives for the assay.
Friday, June 19th
The majority of the work day involved running additional transformations with our yeast parts and mini prepping the promoter plasmids from E.coli liquid cultures. However, due to selective pressures and inefficient buffers, the mini prepping needed to be put on hold until more company buffers could be ordered. The rest of the time was spent working on the wiki, organizing our timeline, and researching future assays.
Monday, June 8th
On Monday we helped Soo pour plates to prepare for her projects and worked on protocols. Additionally, we began to organize our pages for our wiki and started protein profiles. We also discussed our killswitch ideas and worked to find the gene sequence for our second killswitch promoter.
Tuesday, June 9th
On Tuesday we worked on G-Blocks and promoters and met with Sam to figure out how we would make our primers. We worked on our G-Blocks, started on primers, and continued working on our materials list. We also continued work on our protein profiles.
Wednesday, June 10th
On Wednesday we met with Sam and Janie to discuss our “finalized” G-Blocks. We learned that our proposed method of gblock construction was not the best as having the prefix and suffix before each part would stop us from being able to submit our minal design as a full part., They both discussed our tagging methods for cellulase and xylanase and at the meeting we decided to use antibody tagging for these proteins. We also learned that we would need to redesign our gblocks as yeast is unable to produce multiple proteins with only one RBS. We redesigned our system to have a Kozak sequence before both the lignin breakdown enzyme and the helper enzyme. Finally, we decided to cut cellulase and xylanase from our design because of cost and just order the enzymes for testing.
Thursday, June 11th
On Thursday we helped Soo with her GERI students and epidemics presentation. It was a fun and rewarding experience and was good outreach for the iGEM team. Unfortunately we were forced to cancel our G-Block order as we had chosen a green florescent protein that was incomplete. We researched nduring the day to find a new GFP and possibly a new secretion tag in case we found a workable GFP designed for a different terminus. We ended up finding a possible replacement but the documentation was unclear and we decided to hold off on ordering until we talked to Dr. Rickus.
Friday, June 12th
Friday was factually the best day of the week. In the first hour we redesigned our primers and had them ready to go. Early in the day Dr. Rickus stopped by to talk to us about our progress and we were able to discuss our GFP progress. She was able to help us select our GFP and talked to us about linkers which we then researched and added to our parts list. We learned that our primers were no longer god as we needed to use a different iGEM prefix to meet reguloations and so we changed our G-Block design. Shortly thereafter we began to construct our final G-Blocks and then moved on to our primers. We started miniprep but some issues arose and we moved the miniprep to Monday. Before concluding for the day we ran our primers and Gblocks through a series of checks to ensure we would be able to complete PCR and that our sequences were correct. We ordered our DNA and ended the day on a good note.
Greetings fellow iGEM members and enthusiasts, hope you’re all having a great summer! This week has been a busy week for the summer intern-team back over here, but we’re getting close to finally conducting our tests!
Monday, June 1st
On Monday we all worked as a team to develop our Microbial Fuel Cell (MFC) project as much as we could before we presented our ideas to the iGEM members and faculty advisors still located on campus on Tuesday. We developed presentations for the MFC project and the Lignin Project and ensured that both were as equally developed as one another by the end of the day.
Tuesday, June 2nd
On Tuesday we presented our project ideas to our advisors and other members located on campus. After receiving feedback on both projects we came to the conclusion that we would be the most successful working on the lignin project, so long we ensured that our work was accurate and thorough. We put the MFC project on the shelf for next year’s jamboree because we would have more time to work and develop the project than if we worked on it this summer.
For the rest of the day we continued to work on the lignin project and split into three teams. An assay team, killswitch team, and enzyme team. The assay team researched assays we would use to collect data for our project. The killswitch team found promoters and killswitches we would use on our engineered yeast strains, and the enzyme team collected and organized the enzymes we would test. We decided to test four different enzymes, two from termites and two from fungi, and two different helper enzymes.
Wednesday, June 3rd
On Wednesday we have concluded that we’d test the enzymes/helper enzymes in combination and estimated about 120 tests to be conducted. We met with our graduate advisor, Sam Lee, and worked with her to develop our plasmids and G-Blocks. Originally, we assumed we could finish designing our plasmids and blocks by the end of Wednesday, but ran into some obstacles. We continued to work on developing our plasmids and had to continue working on them through Thursday and Friday.
Thursday, June 4th
On Thursday we continued working on our plasmids and blocks and found “errors” on NCBI, the website we used to collect the sequences for our enzymes, and found R’s and S’s in the DNA of our termite enzymes. After emailing Dr. Scharf, our Termite Guru, he cleared up the reasons for the R’s and K’s and gave us the correct nucleotide sequences.
Friday, June 5th
After three days of grueling work we finally finished designing our genetic constructs and believe that we can finally order our DNA on Monday the 8th, of next week.
Apologies for the late report, these should come out on Fridays in the future, but welcome to the first weekly report for iGEM Summer 2015! Our goal with these reports is to keep everyone in iGEM in the loop on the development of the project this summer.
So, starting things off with last week. On Tuesday, after getting our desk space and lab approval to Bindley, we dug into the biodigester project, starting with researching the enzymes we would need to do all of the breakdown in our preprocess. Things were looking good when on Wednesday, we met with Dr. Mosier (ABE professor who does research on biodigester pretreatment) to ask about how our solution would compare to current practices.
Dr. Mosier informed us that current industrial pretreatments essentially pressure cook the plant matter, exposing it to high temperature and high pressure. This melts the lignin, because lignin is hydrophobic, it wants to reduce its surface area as much as possible, so it clumps into small spheres. This allows the other enzymes (cellulases and hemicellulases) to go in and breakdown those polysaccharides (which happens in the biodigester). It turns out that lignin monomers actually inhibit one type of cellulose activity so our solution of just breaking apart the lignin may have a counter-productive effect on the efficiency of the biodigester. Dismayed with this news, we felt as though we had hit a dead end Wednesday afternoon. On Thursday, we resurrected the microbial desalination cell project as a back-up and began looking at details pertaining to that one. We found some great potential for a project.
On Friday, Arren discovered a paper in which a group of synthetic biologists had engineered a yeast cell to perform all of the biodigester processes except pretreatment. Suddenly, our lignin-breakdown idea was relevant again because we would breakdown the lignin but it would not inhibit the cellulase activity because the cellulases are only present inside the cell while the lignin is only outside of the cell. We also continued to research background on the fuel cell project and found a number of different areas we could focus on, including the production of electron conducting filaments called nanowires, focusing on increased electron shuttling between cells, and the application of this to a desalinating fuel cell (which would be new for iGEM).
A lot has happened over the past few months, and we’re closing in our final project idea for this year’s jamboree. The time between Spring Break and the end of Spring semester a lot will be happening, but starting now to the jamboree, we (me and the rest of the executive board) will be keeping you updated.
Thanks very much!!
-Sean (Director of PR)
After four weeks of work this summer, we have made some substantial progress on our project. Here’s what’s going on:
1. Project Name
After a brainstorming session, we have settled on “Minecrobe” as our project theme. Minecrobe combines the power of microbes in soil with a Minecraft twist, relating to the way in which mine carts shuttle ore from one location to another, as our bacteria will release phytosiderophores that will shuttle iron from the soil into plant roots. Expect all of our deliverables to have a Minecraft element to them.
This week we obtained the supplies necessary to begin our first round of plants. We are planting 102 plants, both corn and rice, in ideal growth conditions with the exception of iron availability. We are going to create a gradient of applied Sprint 330, which contains an iron-chelating compound called DTPA, which will enable us to assess numerical values for iron deficiency, sufficiency, and toxicity for both species of plant. We are scheduled to plant on Monday of next week.
While the first round of plants are growing, we will be shifting our focus to growing our bacteria. Our orders of three different strains of Bacillus subtilis have come in from the Bacillus stock center at Ohio State University and just today we have cultured them on LB plates. We are also in the process of determining the exact sequence of DNA base pairs we are going to send in for synthesis. Today, we ran our desired genes through an optimizing program to raise their level of success once the plasmids are transformed into the bacteria.
This year the iGEM Team is tackling the problem of global malnutrition! More than 870 million people are malnourished, according to the United Nations World Food Program. We plan to utilize the realm of synthetic biology to combat this problem by engineering a microbe, Bacillus Subtillus, to increase plants’ ability to uptake nutrients from the soil. Essentially we are optimizing bacteria that naturally live around plants to help plant take in more nutrients from the soil. If the plants take in more nutrients, their nutritional value increases, ultimately leading to more nourished plants into the global food chain. With diligent science and human practices committees, we plan to execute a project that is bigger and better than ever!
The time has well passed since our team competed in the iGEM jamborees. Since then, we have been busy eagerly preparing the team for the upcoming year, but don’t worry, we haven’t forgotten about the great successes that occurred during the jamborees and are excited to share those successes with you!
The regional jamboree, consisting of the North American iGEM teams, took place at the University of Toronto (which looked unbelievably like Hogwarts). At this competition we were named finalists, ranking third in the undergraduate division! This honor granted us the opportunity to present in front of all 64 teams that attended the event. Beyond this, we also received the award for Best New Natural BioBrick Part and advanced to the World Championship at MIT.
Although we didn’t win any additional awards at the international competition, we were more than satisfied with our performance and had an unforgettable experience in Boston, which included attending the Red Sox victory parade!
We are harnessing our enthusiasm from the previous competition to continue this success and reach for even greater heights!