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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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