Team:US AFRL CarrollHS/Notebook

Week 1

Monday (06/04/18)

Lab1

The students brainstormed ideas on project goals and ideas. After briefing many ideas, the students and Mike ultimately decided to bring a “sense and attack” approach to the project. In theory, the module would be able to move naturally towards jet fuel biofilms via chemotaxis, and a higher concentration of biofilms would allow the module to swim towards biofilms before attacking the biofilm and nullifying it before it destroys the jet fuel. Researchers and students alike were excited at the idea and eager to begin the research.

Lab 2

The RX students eagerly began their first day of work by acquiring their base passes early in the morning. They then left to UES where they had a meeting with UES employees and they signed papers and contracts to solidify their positions. They then arrived on base where they were introduced to the colleagues they would be working with inside of the lab and were shown around the lab. With the assistance of previous year team member, Andrea Poole, they made 2 liters of LB Broth. They also made 2 liters of LB Agar of which they added Ampisilin and used the solution to pour plates.

Tuesday (06/05/18)

Lab1

In an effort to reintroduce the students the lab after the break, they began the day by making LB Media. This was also another way to ingratiate the new members of the team to become familiar with real world lab experience and excite them in pursuing the project. Later, the students spent time researching possible project ideas and ways of utilizing quorum sensing and chemotaxis. They spent the day researching in literature and beginning preliminary wiki work.

Lab 2

Wednesday (06/06/18)

Lab1

The students spent most of the day brainstorming ideas in order to host a meet-up for nearby iGEM teams. At the meeting with their mentor, the students discussed the viability of different “search and attack” mechanisms and current research being done to possibly implement into the project. The team brainstormed and looked into different ways of being able to sense fungi.

Lab 2

Thursday (06/07/18)

Lab1

The LabPats began the day by making plates with chloramphenicol resistance. The students plated their old E. coli cells from last year and letting it settle in incubation. Later in the afternoon, the students and mentors discussed the viability of their project idea and creating a basic plasmid outline to begin sequencing through IDT.

Lab 2

Friday (06/08/18)

Lab1

Early in the morning the students listened to a presentation given by Vanessa about fungal contamination on aircraft. The students were very excited as they themselves were working with biofilm contamination in aircraft fuel tanks and it was very eye-opening to listen to their own mentors talk about her own research in the area. The students were able to learn a lot and absorbed new information on how to approach their project and design their own plasmid module. Next, the plates with the old colonies from last year were put in the fridge to be grown as a cell culture on Monday. Afterwards the students began introductory MatLab courses on modelling and how to model synthetic biology in order to enhance their project this year.

Lab 2

Week 2

Monday (06/11/18)

Lab1

The day began with an early lab meeting. The goal of the meeting was to ingratiate the new members with basic lab rules and procedures. Afterwards, the students prepared a presentation about a current project proposal to present to the other half of the team as well as practicing to a few mentors who had been on vacation. Later in the afternoon, the students grew up the Prhl_GFP cells from last year and also streaked plates of a “RhlR” plasmid from Dr. Goodson.

Lab 2

Tuesday (06/12/18)

Lab1

Early the next morning, the students began by performing a mini-prep on the grown up Phrl_GFP cells in order to retrieve the plasmids. After viewing fluorescence under a UV light, they were able to determine that last year’s glycerol stock solution was still usable. Next, they picked colonies from Dr. Goodson’s “RhlR” in order to begin growing up in the incubator. After a quick lunch, the students headed to the meeting with their mentors and USAFA team in order to give a short briefing about their current project proposal and method of approach.

Lab 2

Wednesday (06/13/18)

Lab1

The LabPats began the day by performing a mini-prep on the RhlR cultures in order to extract the plasmid for later transformations and digestions. Before they could continue with their project, the students needed to find primers and utilize the Gibson Assembly in order to continue on with their plasmid design. After a long day in the lab, the students began research on the specifics of the Gibson Assembly and how to design their own primers. Annie also began preliminary Wiki work, Yazmin emailed a professor to acquire a certain E. coli strain with the CheZ gene knocked out. Jason continued learning about mathematical modelling.

Lab 2

Thursday (06/14/18)

Lab1

Dr. Breedon assisted the LabPats in designing primers and sequencing their plasmid for IDT to make. They discussed plans on trying to make their own plasmid from scratch via Gibson Assembly, as well as double checking the final product with the IDT construct. They ordered many primers and parts, like different ribosome binding sites attached to the CheZ gene.

Lab 2

Friday (06/15/18)

Lab1

Lab 2

Week 3

Monday(06/18/18)

Lab1

The students began bright and early with their first presentation to the lab. The lab meeting included many prominent scientists from around the department excited to hear about the summer intern’s development with a new iGEM project. The team heard a stellar presentation by USAFA iGEM before giving their own and receiving praise as well. The LabPats are still currently waiting for their sequenced parts and primers to arrive before proceding with further lab work. Thus, they took this opportunity to learn more about wiki development and modelling, with Annie deciding to implement bootstrap into their wiki design.

Lab 2

Tuesday (06/19/18)

Lab1

Early on the workday, the students took the overnight cultures of the “Rhl” plasmid and tested the fluorescence level to ensure the GFP still worked and that the plasmid wasn’t corrupted. It worked exactly as intended, and the students were able to obtain great fluorescence and pictures. They wanted to have a backup for later just in case, and created a glycerol stock of their “Rhl” plasmid. They then performed a genomic sequence protocol to extract the DNA from the Rhl plasmid so that they can amplify the CheZ gene later when designing the various plasmids.

Lab 2

Wednesday (06/20/18)

Lab1

Lab 2

Thursday (06/21/18)

Lab1

With DNA sequences still coming in, the students were out of the lab for today. Today they learned a lot about DNA sequencing programs and further modelling methods.

Lab 2

Friday (06/22/18)

Lab1

The students are still currently waiting for DNA sequences to come in. Mentor Dr. Varaljay thought it would be a perfect opportunity to use NCBI Blast as a method of analyzing DNA sequences with proteins and nucleotides. She gave a quick lesson on how to use it and how to discover more about unknown surface proteins pertaining to the project.

Lab 2

Week 4

Monday (06/25/18)

Lab1

The primers finally arrived! The students were ecstatic to be able to begin lab work again. They began by re-suspending the new primers before utilizing them to perform a PCR on Dr. Goodson’s “Rhl” plasmid and CheZ gene. After a gel confirmed the presence of expected band lengths, the team was prepared to tackle the next task of purifying the gel and utilize the PCR product for preliminary construction of the final plasmid. They also hoped to utilize the CheZ gene in many ways by attaching different ribosomal binding sites and testing them all within the iGem-Rhl plasmid.

Lab 2

Tuesday (06/26/18)

Lab1

After a very successful PCR yesterday and picture perfect yesterday gel, the LabPats were ready to begin the gel purification step. After the protocol was completed, the team was devastated to discover that the nano drop machine was out of commission and that it would be difficult to measure the concentration of the purified “Rhl”and “CheZ” bands. After jumping through some hoops, the labpats were able to utilize a neighboring lab’s machine and measure the concentration of both accurately. The measured results were a lot less than expected, and Dr. Breedon expressed her worries about a Gibson assembly protocol being successful. They decided to proceed anyways. In addition, the Dr. Breedon also talked to the students about attaching overlaps on the CheZ gene, and performing a PCR on the three ribosomal binding sites.

Lab 2

Wednesday (06/27/18)

Lab1

The students began the morning by redoing a PCR on the “Rhl” plasmid and “CheZ” gene to gather more concentration the second round. They also began their first Gibson Assembly, hoping to ligate the “Rhl” plasmid to the iGem backbone. Afterwards, the LabPats ran the “Rhl” and “CheZ” on a gel in order to perform a gel extraction. However, they were disappointed to find out that their “Rhl” plasmid did not produce a band on the gel in order to perform a gel extraction. Disappointed but not defeated, they knew that their plasmid worked, though they could’ve messed up on the PCR.

Lab 2

Thursday (06/28/18)

Lab1

After yesterday’s failed attempt at PCRing the Rhl and CheZ bands correctly, the students arrived in the lab with a new desire to accomplish what they didn’t yesterday. They meticulously followed the protocol, taking extra care this time to ensure everything was right. After gel electrophoresis, they were ecstatic to find out all of their PCR’s had gone according to plan and they were ready to perform a Gibson Assembly tomorrow. Even though the Gibson Assembly from Monday (with Rhl and iGem backbone) produced a shockingly low concentration, Amy decided to have the students attempt to grow up the cells in DH5a on Chloramphenicol plates anyways.

Lab 2

Friday (06/29/18)

Lab1

After a week of solid work, the LabPats were a little caught off guard when they realized there wouldn’t be much lab work today. It being the end of the work week and the lab closing on weekends, the students were not able to do overnights or anything requiring an extra day. Thus, the students started and finished the day checking to see if their “iGem-Rhl” plasmid with the iGem backbone grew up. There’s growth!” the students exclaimed as they opened their incubators and examined their plates. As it turns out, there was growth on the “iGem-Rhl” plates with chloramphenicol. The students knew for sure that their plasmid had been transformed correctly into the DH5a cells. Afterwards, the student had a quick meeting and final tune-up presentation before the Michigan State Meet-up.

Lab 2

Week 5

Monday (07/02/18)

Lab1

It was another short day in the lab as the LabPat’s main focus today was just to grow up cultures of the “iGem-Rhl” plasmid in DH5a cells. Their goal was to grow it up overnight, then perform a mini-prep to extract the plasmid in order to send it off for sequencing. After the labwork, the students contacted teams at University of Texas for collaboration, Wilbert from National University of Singapore for modelling, and the London School of Boys for Wiki Tips and Tricks.

Lab 2

Tuesday (07/03/18)

Lab1

After performing the mini-prep, the nano-drop machine displayed a large concentration of DNA. They sent the “iGem-Rhl” plasmid off for sequencing, with fingers crossed on achieving the desired sequence. Afterwards, a weekly meeting with the entire team was productive and informative, as everyone reset goals and reassigned tasks to finish the rest of the year out strong.

Lab 2

Wednesday (07/04/18)

Lab1

Lab 2

Thursday (07/05/18)

Lab1

The LabPats worked on logo design and the abstract as they waited for sequencing results to arrive. Today was the last day for the USAFA team, and the LabPats decided to go out for lunch to treat them before they left. After a tearful goodbye, the students were hopeful to see them later at the Jamboree. They reached out to MIT labs where they had been experimenting with biofilms within the human body. The sequencing results arrived later and Dr. Breedon explained that the backbone had most likely ligated to itself, allowing the cells to grow but not producing the desired results. The LabPats learn the hard way that not everything in SynBio always goes exactly as planned.

Lab 2

Friday (07/06/18)

Lab1

Early the next day, the LabPats were eager to re-do everything. They first performed the same Gibson Assmbly, ligating the iGem backbone with the Rhl plasmid before transforming the product within DH5a. Afterwards, they replated the cells and hoped that favorable results would come Monday.

Lab 2

Week 6

Monday (07/09/18)

Lab1

IT WORKED!!! Annie exclaimed as she retrieved the plates from the incubator. The plasmid had grown up exactly as planned. They then proceeded and extracted the plasmid from the host DH5a in order to send it in for sequencing.

Lab 2

Tuesday (07/10/18)

Lab1

The sequencing results arrived early in the day, and the students were both excited and anxious to have a peek at the results. The last time, the results were unfavorable, but this time, with everything going right, the LabPats opened the sequencing document and examined the results. Almost every base pair matched up! It was a huge win and morale booster for the team, as now the students could begin putting the “CheZ” insert into the plasmid finally.

Lab 2

Wednesday (07/11/18)

Lab1

Another big component of the project would be to test the motility of the cell. By testing cells with the CheZ knockout strain and ones without, the students can observe the motility of CheZ. The cells with the CheZ knocked out doesn’t move and grows a single colony, while the ones with the CheZ should be able to expand and grow many colonies all around the epicenter.

Lab 2

Thursday (07/12/18)

Lab1

At another early morning start, the Labpats began by digesting yesterday’s PCR. Next, they looked at the plates from the motility test, and were pleasantly surprised to see that they looked as expected and grew nicely. They were also able to conclude that the toothpick method worked a lot better than the drop method, as the motility was clearly shown by the expanding non-motile colonies. Aftwards, they made a gel for the PCR digest, ran the gel, and cut the bands. Finally, the students grew up the backbone-RhlR from yesterday’s plates.

Lab 2

Friday (07/13/18)

Lab1

Starting off, the LabPats completed a gel purification from the bands that were cut yesterday. The C4 test did not work as expected but the students deduced that it may have been too low of a concentration. Thus, the LabPats chose to re-do it, however; this time with the goal od getting a high concentration. To end off the week, they set up a Gibson Assembly with backbone-RhlR (everything in the plasmid except GFP) with CheZ to create our CheZ plasmid. The students were ecastatic, as the completion of this plasmid would mean they would be almost completely done with their project.

Lab 2

Week 7

Monday (07/16/18)

Lab1

Another day closer to finishing their project! The LabPats arrived at the lab with the goal of being able to send their finished Rhl-iGem plasmid with the CheZ in for sequencing. However, after checking the plates in morning, the LabPats were disappointed to find that the plates did not grow at all. Nevertheless, they decided to re-do the PCR, Gel Electrophoresis, Gel Extraction, and transformation again. After another long day, the students were able to complete the PCR, Gel Electrophoresis, and stored the gel in the fridge.

Lab 2

Tuesday (07/17/18)

Lab1

The day was hectic yet again. They checked their overnight cultures and discovered that no gFP had been expressed. For some reason, the bands on the PCR displayed a continuous long band down the middle instead of a clearly defined, horizontal band. The students were dismayed and disappointed, realizing all their work from yesterday was for nothing. However, they retraced their steps back to square one. At the beginning of their project, they had tested Dr. Goodson’s “Rhl” plasmid that clearly worked and decided to go back to the glycerol stocks they had come from and be able to regrow out the cells to work with them. They were able to prep overnight cultures and streaked them out on the plates. Meanwhile, the LabPats also wanted to re-do the PCR once again of the overnight cultures just to make sure that they hadn’t just been things wrong the days prior. They discovered, yet again, that the singular band had not shown up but instead a long band ran down the length of the gel. There was also a team meeting later in the day that the entire team congregated at to discuss project progress as well as possible human outreach “field trips” to the Cleveland Becket Oil and an oil subsidiary of Marathon in Cincinnati.

Lab 2

Wednesday (07/18/18)

Lab1

Today’s plan included a digestion protocol, purification, and ligation on the IDT order with the with experience after doing so many for so long. They were able to complete everything and then head off to the team photo shoot at the United States Air Force Museum, where they took team pictures in front of many iconic planes.

Lab 2

Thursday (07/19/18)

Lab1

The students were ready for another busy day in the lab! They discussed with mentors about the status of their project, and decided to proceed with using IDT parts in order to ligate their Rhl Insert, iGem backbone together in order to perform a transformation and grow them up in DH5a cells. In addition, they were tasked with performing another Gibson assembly, this time joining the CheZ inserts with various ribosomal binding sites to the insert and backbone. Both transformations went into the incubator overnight, with the LabPats hoping for its success. Late in the day, Annie performed the Flow Cytometry portion of the Interlab.

Lab 2

Friday (07/20/18)

Lab1

The next day, the LabPats looked at the transformations from the previous day. They were disappointed to see that none of the transformations worked. Saddened, they continued to persist and discuss new ways to approach it. First, they settled on re-doing the PCR, as there may have been an error early on in the process before extracting the “Rhl” plasmid. Thus, they started again, completing a PCR and running it on a gel. After looking at the bands, however; the students were still disappointed to discover that the bands were popping up in the wrong place and the backbone, which is supposed to be at 3,000 base pairs, did not appear as so. Looks like it’s back to the drawing board!

Lab 2

Week 8

Monday (07/23/18)

Lab1

After discussing all of their options, they discussed ways their PCR might have messed up. Thus, they began by double checking their primers, before agreeing that it was indeed correct. Next, they concluded that there may have been a problem with the digest that was performed, as there were multiple cut sites for the DpN1 digest that may have messed up the bands from cutting in the right places. Armed with this knowledge, Mike suggested them re-doing the PCR, this time leaving the digest out and see if there are the right bands.

Lab 2

Tuesday (07/24/18)

Lab1

The LabPats began another PCR this time with the exact same backbone and primers as yesterday, except skipping the Digestion step with DpN1 directly after PCR completion. Unfortunately, after a full day of diligently performing the PCR and running the gel, the correct band was still missing from the final gel. The students were visibly upset, but very ready to continue to persist until the desired result was achieved.

Lab 2

Wednesday (07/25/18)

Lab1

Today the LabPats tried a new PCR method, using gradient temperatures to test and see if the PCR would work and the primers would be able to bind using different temperatures. Unfortunately, it did not work again, and the LabPats decided that there may have been a problem with the primers. They reviewed their suspicions with their mentors and Mike confirmed that they may need new primers.

Lab 2

Thursday (07/26/18)

Lab1

The LabPats re-completed the Interlab after realizing that they were missing a set of wells from the last time. They spent the day completing the CFU protocol, working with the plate reader, and calibrating the flow cytometer for use. Later in the day, Mike helped the students locate primers and sequences in order to order more primers in time. After a long day with the Interlab, the students were happy to proceed forward with their project.