Team:Cadets2Vets/Collaborations

Cadets2Vets Adviser Judy Nguyen and Cadets2Vets Collaborator from Center for Urban Waters Joel Baker had a conference call with two team members from iTesla-SoundBio: Anna Vasyura and Advisor Zach Mueller. They shared information about each others’ projects and the status of their experiments.

Anna and Zach are looking for help regarding the analysis of the degradation of polychlorinated biphenyls (PCBs). Joel had previously collaborated with a lab that performed this type of research and had a lot of valuable information to share. One of the significant bits of advice was to pivot the analysis from PCBs to PCEs (tetrachloroethene).

PCBs take a really long time to degrade and the analysis is not fast or easy to do. PCEs degrade a little quicker and make more sense to measure for the iGEM timeline. Joel will meet face to face with iTesla-SoundBio to work out more details of what the analytical experiments would involve. Judy has also joined the team’s Slack workspace and is available to help with any other technical questions or assistance with laboratory space.

The Cadets2Vets team had a Skype call with Dallas McDonald from the Lab Pats (AFRL) team. Our teams had Skype’d previously during the summer and we wanted to talk a little more about the Interlab Measurement Study.

We are sharing the results from our Interlab Study and wanted to see if Lab Pats had measured data that was similar to ours. Cadets2Vets had graphed the fluorescence values from the 6 Devices and positive and negative controls, and found that the Devices all had variable performance, although there were a few Devices that had very low GFP fluorescence. We also found that some Devices performed at or below the LB+Chloramphenicol blank, which means that the previously known autofluorescence of LB was being picked up by the plate reader. The Devices that performed worse than LB+Chloramphenicol are promoter/RBS sites that we would want to avoid in the future when trying to express GFP, because the amount of GFP expressed was unable to rise higher than the background autofluorescence under the given experimental conditions.

Cadets2Vets will work with Dallas to establish a collaboration where Cadets2Vets will send Dallas the plasmids we have created so that he can help us characterize the GFP fluorescence. We will base the assay off the InterLab study and we will consider these questions:

Do our plasmids produce GFP as they are expected to?
How does our plasmid compare to the Interlab positive and negative controls?
What is the appropriate density of cells and duration of growth to get significant GFP fluorescence?
Can other labs reproduce our data?

Members of the Cadets2Vets team had a teleconference with Yoshi Goto from the UW-Seattle iGEM team. The Washington team has a really interesting and complex project that involves the creation of a bioreactor and manipulation and development of a violacein reporter system to monitor metabolite production from chemical reactions made by bacteria or yeast.

The violacein pigment is produced through a series of 5 different genes and altering the synthetic pathway can produce different colors. This project is also developing an analytical system that monitors the turbidity and color of the culture in the bioreactor. There are many possible applications of this technology for monitoring metabolic reactions while maintaining affordability.

We discussed the possibility of sharing some reagents to help each other characterize some Parts. Cadets2Vets is looking for collaborators to measure the GFP fluorescence from the plasmids we made and Washington is looking for ways to characterize some of its plasmids in a similar way.

Cadets2Vets had a Skype call with Dallas McDonald from the Lab Pats team.

The results obtained from the Lab Pats Interlab study were different from Cadets2Vets. At the 6 hour time point, the plasmids with highest to lowest GFP fluorescence were:

LB + Chloramphenicol
Negative Control
Device 6
Positive Control
Device 1
Device 4
Device 5
Device 3
Device 2

After sharing these results, Cadets2Vets were curious why the results were so different. The Lab Pats team speculated that the difference may have been due to pipetting errors or differences in the instruments. Te Lab Pats used a SpectraMax M5, which uses monochromators to excite the GFP instead of filters, which is what Cadets2Vets had in their FilterMax F5 instrument. Cadets2Vets went back to their transformation plates and tried to assess whether the bacterial colonies sill expressed GFP and if the intensity was similar to the pattern gain from the plate reader.

Cadets2Vets’ one-month old transformation plates were still fluorescent, and we used a hand-held blacklight to qualitatively observe the colonies. The order of highest to lowest fluorescence using this method was:

Positive Control
Device 2
Device 4 = Device 1
Device 6 = Device 3
Negative Control.

Device 5 was omitted due to the few colonies present were on the edge of the plate and difficult to see clearly.

The sensitivity of using a blacklight was much lower than using a plate reader, and so it was hard to see the differences between some of the plates. However, it appears that the pattern of fluorescence we observed in the plate reader is consistent with the bacteria in the plates. Washington also shared the results of their Interlab study. Summarizing their data in the same way we looked at ours and Lab Pats, the ranking of high to low GFP for Washington was:

Device 2
Positive control = Device 4
Device 5 = Device 1
Negative control = Device 3 = Device 6 = LB + Chloramphenicol

Team:Cadets2Vets/Collaborations

WELCOME TO OUR COLLABORATION PAGE

CONTRIBUTORS TO THE CAUSE

First Inter-Team Meeting (07/14/2017)

iTesla-soundbio team meeting (09/28/2017)

Lab Pats First Interlab Meeting (10/05/2017)

FOURTH INTER-TEAM MEETING (10/05/2017)

Interlab collaboration with lab pats air force research lab

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