Last Week Determined that our YFP reporter (used in the receiver) is a defective part Re-ordered a working YFP and began reconstruction using the new YFP Received the receiver test construct from BioBricks, conducted AHL->receiver experiments and aTc- >sender+receiver experiments Sequencing results Photolithography stuff

BioWire Progress Report Week Eight Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush

Oh YFP… YFP Biobrick Parts: – E0430 (YFP, no degradation tags) – E0432 (YFP, LVA+) – E0434 (YFP, AAV+) We had been using E0432, since we want degradation of YFP for temporal analysis Turns out that E0432 simply does not glow (degradation tag too strong?)

I E0432 [Const. P(cat) + YFP LVA] I E0430 [Const. P(cat) + YFP] 100x Phase 100x YFP Filter

Building the Circuits Lux – Rebuilding major parts with new reporters – Will be transforming into MC4100 (LacI-) cells, since they seem hardier than DH5alpha cells – Sequencing data is indecipherable – issues with “multiple priming” Las – Rebuilding major parts with new reporters

Experiments SOMETHING ABOUT SENDER HERE

Experiments: Receiver Construct Does the Receiver Test Construct fluoresce with addition of AHL? – Input: acyl-homoserine lactone (AHL) – Output: EYFP fluorescence LuxPL: weak constitutive promoter LuxPR: promoter activated by LuxR-AHL complex YFP: E0434 (eYFP)

Experiments: Receiver Construct Experimental Design – Grow up overnight cultures – Backdilute to 0.1 OD600 – Add appropriate concentration of AHL (0, 15, 50, 150, 500 nM) – Incubate 1 hr – Place on M9 agar slides and image Controls – Positive: Constitutive YFP producer (I14033+E0430) – Negative: Untransformed MC4100 cells

Experiments: Receiver Construct Results – All controls worked as expected – Fluorescence was observed at all levels of AHL (except 0 nM) under the GFP and YFP filters – Fluorescence levels at different concentrations of AHL were qualitatively comparable

PHASE GFP FILTER 0 nM AHL15 nM AHL500 nM AHL

Experiments: Receiver Construct Conclusions – All parts in the receiver construct are functional – Fluorescence can be observed at even low levels of AHL

Planned Experiments Creating a time course for AHL induction with receiver construct Testing Propagation Constructs with new YFP (to be cotransformed with LuxR producers)

Photolithography Made a 5 μm master and two μm masters (more on that later). Made PDMS negatives. Learned how to plasma oxiginate PDMS in order to reduce air bubble distortion. Made agarose stamps from the PDMS.

Photolithography Issues in the cleanroom: – Non-uniformity in photoresist… – caused wafer to bake onto the mask… – changed our soft-bake and development times… – and caused variations in feature height between 150 μm and 350 μm. (We were aiming for 150 μm features.) Also, the agarose stamps we made had some minor issues (levelling, user error).

Photolithography With current PDMS negatives, will remake agarose stamps to start stamping cells. Will remake 150 μm master to increase uniformity. Will begin process of making 1mm master.

This Week Building parts – Finish Lux constructs with new YFP reporter – Cotransform constructs into MC4100 cells – Send new parts in for sequencing Experiments – Create and test propagation constructs Photolithography – Go into cleanroom to make 150  m and 1000  m master.

Updated Schedule Week 1 (6/6): Project Choice and Design Week 2 (6/13): Got parts and set up tests Week 3 (6/20): Began building test constructs, finished sender Week 4 (6/27): Finish receiver, receiver w/repressor; CAD a mask Week 5 (7/4): Continued building parts, received mask Week 6 (7/11): Finished Lux, Tested senders, made PDMS molds Week 7 (7/18): More experiments, finish Las, make first master/PDMS/stamp, eating pizza courtesy of Alain Week 8 (7/25): More experiments, Meeting Their Master Week 9 (8/1): Continue experimentation, Danny and Orr-ish things Week 10 (8/8): “ Week 11 (8/15): “ Week 12 (8/22): “ Week 13 (8/29): “