iGEM 2018 — Team Tufts

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# Team:Tufts/Notebook

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# Notebook

**Project Timeline**

5/27-6/2: Ordered all necessary DNA materials

6/3-6/9: Worked on acquiring lab access for all members that would be researching in the summer

6/10-6/16: Developed protocols for how to execute the design of the project

6/17-6/23: Moved into new facilities, Tufts Science & Engineering Center

6/24-6/30: Prepped DNA and lab materials, including diluting the DNA

7/1-7/7: Attempted to assembly PCR the two overlapping gene blocks of Cas13a unsuccessfully

7/8-7/14: Collaborated with Josef Bober from the Nair lab in order to establish a better PCR protocol

7/15-7/21: After several failed assembly attempts, new primers were ordered in case the ones previously used had degraded

7/22-7/28: Pause in work before the new primers arrived. Ordered geneblocks and emailed iGEM organization about necessary missing DNA kit and current transformed bacteria. Extension for interlab project given.

7/29-8/4: Interlab procedure preparation was completed.

8/5-8/11: Interlab days 1-3 were completed.

8/12-8/18: Interlab cell counting was completed. Equipment was inventoried with refrigerator space shared with graduate student lab in facilities.

8/19-8/25: The interlab data was submitted. Assembly PCR with new primers was performed. This were somewhat effective, but produced a smear, leading us to conclude that the DNA was degraded. At this point, the primary project was abandoned, as there was not sufficient time to order new DNA and PCR the toehold.

8/26-9/1: Break prior to the start of the semester

9/2-9/8: Break during start of the semester - Tufts University classes begin

9/9-9/15: iGEM website

9/16-9/22: iGEM outreach with Student Campus Activity Fair. General interest meeting and presentations given on iGEM summer research and competition.


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# Team:Tufts/Experiments

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# Experiments

This page contains only the experiments that were carried out somewhat successfully, not those the theoretically would have followed those performed had they been fully successful.

### Assembly PCR of Cas13a

Oligonucleotides of the two parts of Cas13a, overlapping by 25 nucleotides, were diluted according to IDT manufacturer instructions. Several assembly PCRs were performed, the first several of which were unsuccessful due to primer degradation. In the PCR producing a somewhat smudged band of successfully assembled Cas13a DnA, the following protocol was used for an extension PCR:

11 micrometers of sigma water, 4 microliters 5x green buffer, .5 microliters dNTP's, 2 microliters Cas13a primer forward, 2 microliters Cas13a primer reverse, and .5 microliters of Tax polymerase were mixed thoroughly by vortexing and then by centrifugation. This was run in a standard PCR program with an annealing temperature of 65 degrees Celsius.

This was followed by an assembly PCR, which utilized a similar procedure, except was done in a larger total volume of 100 microliters.

### Gel electrophoresis

A 1.5% agarose gel was prepared, using SYBR safe instead of ethidium bromide for safety. The gel was run at 110 V for 45 minutes, at which point it was imaged, revealing the [following.](https://2018.igem.org/Team%3ATufts/Results)

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# Team:Tufts/Results

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[Human Practices](https://2018.igem.org/Team%3ATufts/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ATufts/Public_Engagement)

[AWARDS](https://2018.igem.org/Team%3ATufts/Awards)
[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=Tufts)

# Results

We discovered our DNA oligonucleotides had degraded before assembly could be completed.

### What happened?

We spent about one month over the Summer trying to assemble our gBlocks for PCR. During this time we modified the PCR heat cycle many times, believing optimization of the heat cycles to be the issue holding us back. We even reached out to a grad student, Jessica Stieglitz, for tips on PCR optimization.
Only after weeks of failed attempts (X PCR’s over X Gels), we approached Jessica Stieglitz again for help, explaining our problem and protocol more specifically, and realized that assembly PCR required one run of the thermocycle without polymerase. By the time we ran the corrected assembly PCR protocol, our DNA blocks were degraded already from so many freeze-thaw cycles and periods outside of refrigeration. Regardless, we did see some bands of properly assembled product, although it was too impure for any use.
After that month was over, Aiden Lewis, the treasurer head had to return home, since his stipend for summer housing only lasted one month. While he was gone, an order was placed to order more gBlocks for another trial of the project. However, earlier in the year, the wrong mailing address had been placed and this had not been corrected. Since Aiden was not around to pick up the shipment from the wrong address, and he was on vacation out of the country, the rest of the team failed to receive replacement gBlocks, as they were lost in the mail.

### As time was running out, we shifted our focus to completion of the interlab study to fulfill the bronze medal requirement

The interlab study was done successfully and data was submitted in time through the online form. Some data was not consistent with expected results, likely due to confusion over handwritten labels on some cultures.

### What went wrong?

* Lack of experience: most team members were Freshmen, or had only participated in one year previously. Tufts iGEM was unable to compete last year due to lab renovations.
* Reluctance to reach out to grad students: we should have asked grad students for a second opinion before assuming we knew what the issue in our protocol was.
* Lack of funding: we should be more proactive next year to try to secure more funding so we can order backup-reagents more quickly, and have longer lasting summer stipends.
* Not enough team coherence: we should have been more communicative with each other to prevent the issue with missed gBlock shipments.

This shows that the Gene Blocks have been degraded, evidenced by smudges:

![]( https://static.igem.org/mediawiki/2018/4/44/T--Tufts--GelResult.png)![]()