Team:BostonU/Collaborations

WPI Worcester Polytechnic Institute

This year, the BU iGEM team collaborated with the WPI-Worcester iGEM team.
The collaboration became possible at the NEGEM meet up in June, where six New England iGEM teams presented their project ideas to the rest. During one of our small group discussions, a team member from WPI talked about how lucky we, the BU team, were to get access to a flow cytometer for single cell fluorescence measurement. Right then, the two teams decided to collaborate: we would test some of their constructs on our flow cytometer and they would view cells expressing our devices with their confocal microscope.

They gave us two copies of the same construct, one in a chloramphenicol resistant backbone and the other in an ampicillin resistant backbone. The device has BclA-YFP , which is a cell surface targeted protein to express YFP on the cell surface. We also ran one of our own single color YFP controls, which is listed at "Internal YFP" as this is not targeted to the cell surface. It should be noted that this control is in a kanamycin backbone.

The results our shown below. For details on how we converted the arbitrary YFP units to MEFLs, please see the TASBE Tools page for information about controls required to do that conversion.

Fluorescent measurements of three devices expressing YFP. The BclA-YFP parts were provided by WPI while the Internal YFP is one of the control parts provided by Boston University.

The WPI team took micrographs of cells containing tandem promoter- RFP testing constructs. We gave them media with two concentrations of arabinose (1 mM and 100 mM) and aTp (100 ng/mL and 1000 ng/mL). The used the following methodology for microscopy:

The pTet-pBad-RFP or pBad-pTet-RFP expressing E. coli strains were cultured in LB with kanamycin plus the additives indicated overnight at 37°C overnight with shaking. 20µL samples of each culture were dropped onto microscope slides and allowed to air dry. Slides were then fixed in methanol for 10 minutes, dried, and wet mounted with a coverslip in a 50% glycerol solution. Slides were imaged on a Zeiss AxioVert microscope with an 100x oil immersion lens, and imaged with a Zeiss AxioCam MRm camera. Images were acquired using Zen software, and processed with Adobe Photoshop. All images were exposed for 2 seconds. Brightness and contrast adjustments made during processing were applied equally to all panels within the figure.

Micrographs showing the expression of RFP and GFP at different small molecule concentrations for pTet+pBAD+RFP Micrographs showing the expression of RFP and GFP at different small molecule concentrations for pBAD+pTet+RFP

Arabinose is clearly inducing RFP induction but aTc induction is weak at best. This may have been due to a mix up in the media we gave them. Likewise, the presence of GFP in some of the cells suggests we may have gave them a mixed culture since we didn't see GFP for the same constructs when we ran flow cytometry analysis. The change in RFP under arabinose suggests that there are cells with the correct construct growing in the culture. For a more detailed result analysis, click here .

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