Team:UCSC/Notebook

Overview - Day 1 was mutiny. Five students had met with a sponsor in the morning and when the two captains returned to the room, the students were milling around, whispering under their breath, and fidgeting. The problem was with motivation towards the project, as some students felt that it wasn’t novel enough. We had found a 1998 paper by Duport et al. that explained how their team developed yeast to produce pregnenolone and progesterone - the things we wanted to make. It turns out though that the paper didn’t lead to anything, the strain has been lost, and it was a good proof of concept for our idea. From the uprising we learned to communicate thoroughly.

Resolved issues - The overview for this week sums up our resolved issues relatively well; people expressed their concerns about the novelty of the project and directed their energy to come up with a solution.

Experiment Progress:

Week of July 2nd

Overview - Our yeast strain is going to have all the genes to make progesterone, as well as the lactase enzyme so that we can feed it lactose and it break it down to glucose and galactose. We figured out after much deliberation how we could have our friends in developing nations get the accurate dose of progesterone. We learned from our mentor that we could use a technique to measure the weight of an object using a coat hanger, and depending on weight, calculate the amount of lactose inside that container. This would be the dairy that the people have on hand. Depending on weight, we will have a chart that says how much lactose is in that container. The people will be guided to portion a certain amount of the lactose into an ice-cube-like container and grow the yeast in there. Because there will be a specific amount of lactose in each section, and our promoters are sensitive to galactose, our yeast will stop producing progesterone after it consumes all of the lactose.
We have found multiple ways to insert our genes into Y.lipolytica and we made pros and cons lists for each method to decide which one would be the most effective and time-efficient. The methods are regular homologous recombination using homologous arms on our plasmid, EasyCloneYALI: CRISPR/Cas9‐based technique for engineering Y.lipolytica specifically, and Drag and Drop cloning.

Resolved issues - We determined that we could create an easy-to-use method to provide the correct amount of food to yeast in developing nations.

Experiment Progress:

Week of July 9th

Overview - We confirmed that using a constitutive promoter would be better than an inducible one, at least initially, because it will use up energy to keep producing progesterone after the growth media has been consumed. Our website has gone through several prototype designs, but it seems like we may be getting close to a final design we would like to use to represent our work. One fun feature that is a work in progress is an interactive map that will, when moused over, provide information about different countries and our outreach there. We have also been working on finalizing our logo, which will include the name of our project: PoPPY. PoPPY stands for Portable Progesterone Production in Yeast, an accurate description of the goal of our project. A low this week for the team was realizing that most of the survey responses received from one of the groups we reached out to about contraceptive accessibility were unreliable, since many answers between supposedly different women were the exact same, word for word. However, we hope that this was a problem specific to the group, and not the norm. We have some more legitimate groups who are willing to send us photos of the women filling out the surveys, and we hope to see good results. A high for the team this week was finally understanding how Cre-Lox recombination, a method that we will use in one of our experiments to integrate our gene cassette into the yeast genome, works. We now expect to run three parallel experiments to get to our end goal: a control using Gibson and homologous arms to create our gene cassette and integrate it into our Y. lipolytica genome; assembly of the plasmid inside S. cerevisiae using yeast-mediated cloning, isolation and insertion of the plasmid into E. coli for amplification, and insertion of the gene cassette into Y. lipolytica using lox sites; and assembly of gene cassette/plasmid inside Y. lipolytica and integration into genome using lox sites. For each of the three experiments, we will begin by inserting lox sites into the Y. lipolytica genome using homologous recombination.

Resolved issues - This week, we resolved a few issues, including picking a promoter and terminator and understanding the Cre-Lox system.

Experiment Progress:

Week of July 16th

Overview - About half the team spent the week drafting our thesis. While some of us were writing, others were meeting with professors on campus to discuss our ideas and to pick up some S. cerevisiae strains to use as our experimental organism. We learned from Rohinton Kamakaka that we could insert Cre recombinase into our yeast using a plasmid, so now we are in pursuit of a method to both express and remove Cre recombinase once we no longer need it. Our yeast parents have been growing our Y. lipolytica and amplifying the plasmids that we have received. Our wiki boys have almost finished formatting the website to their (or our) liking and are now adding more content about our project itself. A few of our team members scheduled an interview with a representative from Family Planning 2020, but due to timing issues, had to reschedule.

Resolved issues - We figured out how we would both express and remove Cre recombinase when it was no longer needed!

Experiment Progress:

Week of July 23rd

Overview - This week, our team wrote a thesis! Or, more accurately, finished writing it. The majority of us put our brains together to create a draft and we chose one excellent editor to take control of the paper. She enlisted grammar police, style monitors, and LaTeX formatters to help her revise our drafted ideas into one cohesive overview of our work to-date. Each of us also wrote about our own contributions to the project and personal reflections describing our feelings towards the journey thus far. Also, our two captains and our PI met Dean Wolf, Dean of Baskin School of Engineering at UC Santa Cruz, Roger Trippel, Senior Director of Development and Individual Giving, and Abigail Kaun, Special Assistant to the Dean. We discussed the project to-date, our outreach efforts, and shared ideas for managing both our and future iGEM UCSC team’s funds.

Resolved issues - Writing a thesis with 17 people is tough! Somehow we managed to pull it off!

Experiment Progress:

Lab Work Begins

We received our Yarrowia lipolytica strain this week. We then took it to the lab and confirmed it was growing nicely.
We also received our S. cerevisiae and set it up to grow.
Inoculated E.coli cells and plasmids (pUC19 and pXR).

Designing (Test)

Week of July 30th

Overview - This week, the captains held check-ins with each individual on the team to hear concerns and alleviate worries, since we are now halfway through the summer. While the captains were busy with meetings, the rest of the team was working hard as always. Our modeling team started a growth curve for Yarrowia lipolytica so we can better understand the yeast’s growth rate, our riboswitch team began lab work, our plasmid team started linearizing plasmids, and our outreach team continued to update the wiki page and started creating a presentation for high schoolers. Four of our team members and our PI took a vacation to Minneapolis that wasn’t much of a vacation at all. The small group toured Medtronic's Operational Headquarters, Physiological Research Laboratories, and the University of Minnesota's Visible Heart Lab. Almost every other second of the trip was spent on the BMES Coulter College Conference, which required teams to come up with a medical solution to a need. Our team focused on hypertension in resource-constrained settings and developed a solution in the form of a blood-pressure-monitoring phone case and app. Even after the conference, the fivesome was hard at work; they met with the University of Minnesota iGEM team, toured their lab, and gave advice about human practices, outreach, and fundraising.

Resolved issues - Our team fixed the layout of our office and achieved maximum feng shui. We also corrected several small procedural mistakes by checking our protocols with our awesome TA.

Experiment Progress:

Week of August 6th

Overview - So many people are in the lab now! Because so many of our small lab teams have transitioned to lab work, the “Yeast Parents”, or team in charge of maintaining the yeast and the lab, gave everyone a tour of the space to keep everything organized (and keep us all in line).
Unfortunately, with great numbers of people in lab comes great margin for mistake. We burnt through a ridiculous amount of Q5 polymerase this week because we miscommunicated the concentrations needed to perform PCR. However, the team took this as a learning experience and practiced concentration math and master-mix-making. We are also going to begin a crowdfunding campaign to supplement the team funds when/if errors arise in the future.
Outreach is really cool! Three of us, plus our PI (of course), spoke to Martyn Smith, Managing Director of the Family Planning 2020 Secretariat, over the phone. He nearly gave us heart attacks when he offered to help us reach local agents familiar with the regulatory environments in the countries we wanted our project to extend to. Aside from the FP2020 call, two of our members have been diligently working on outreach at home. They’ve started to develop a game as an interactive way for high schoolers to join in when we present our project to them later this month.

Resolved issues - It turns out our original design to insert lox sites into the yeast genome would not work; luckily, we also realized we had never ordered the original design. Lose-lose-win? The new and better design has been ordered.
Many of our PCR attempts failed and we used almost our entire supply of Q5 in one week. We realized the issue was in miscommunication about master mix concentrations, so we stepped up our communication game, had a concentration practice session, and are fundraising to offset our costly mistake and any future mistakes.

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Week of August 13th

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Week of August 20th

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Week of August 27th

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Week of September 3rd

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Week of September 10th

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Week of September 17th

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Week of September 24th

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Week of October 1st

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Week of October 8th

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Week of October 15th

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