From 2014.igem.org

Collaborations

Team WPI met the members of the Boston University and MIT teams at NEGEM this year, and established several collaborations.

We gave the BU team two versions of our BclA-YFP (BBa_K1423005) cell surface expression device: one in the Cam backbone and one in the Amp backbone. Team BU performed flow cytometry on the samples as well as a control line expressing YFP internally from a Kan backbone. The results indicate good, though reduced, expression of BclA-YFP compared to the internal YFP.

In return, we performed microscopy on two devices built by the BU team: pTet-pBad-RFP (BBa_K1401009) and pBad-pTet-RFP (BBa_K1401010). The pTet-pBad-RFP or pBad-pTet-RFP expressing E. coli strains were cultured in LB with chloramphenicol 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 miscoscope 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.

The results indicate good induction of the pBad-pTet device. The concentrations of aTc used for induction of the pTet promoter may have been incorrect, leading to uninterpretable results.

Team MIT used their confocal microscope to take an image of our BclA-YFP device. The image below is a single Z plane, in which the membrane localization of BclA-YFP is not obvious. Our microscope is a simple fluorescence scope, and localization looked significantly membrane-associated in our hands, an experiment we repeated and confirmed on several occasions.