BioWire Progress Report Week Six “Halfway There” Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush

Last Week [Almost] Finished building Lux parts Conducted experiments with Lux sender test construct Got cleanroom certification

Building the Circuits Lux – All parts should be finished today (awaiting analytical digest). – Received DH5alpha cells for transformation – Started moving parts onto Kan plasmids in order to cotransform into cells. Las – Still building parts. – Switched constitutive promoters, P(Bla)-->P(Cat) because the old part just wasn’t working. – Moved within two cycles of finishing.

Building the Circuits Cotransformation Plan – Putting CI repressor on kan plasmid enables us to control the copy number, and thus repressor levels, through IPTG induction KAN: Receiver Repressor Component (J06004) AMP: Receiver Output+ Propagation Component (J06007,8) BioWire Cell

Experiments Testing the Sender Reporter Construct – Input: anhydrotetracycline (aTc) – Output: fluorescence (CFP) Sender Reporter (eCFP), P tet Test J06003

Experiments Experimental Design – Positive Control - constitutive GFP (gift from Yin) – Negative Control - no aTc added/DMF only – Negative Control - strain without CFP (LuxI sender under Tet promoter)

Experiments Experimental Design – Grow up overnight cultures of cells – Backdilute to 0.1 OD600 – Add varying concentrations of aTc 25 ug/ml, 12.5 ug/ml, 6.25 ug/ml, 3.13 ug/ml, 1.56 ug/ml, 781 ng/ml, 391 ng/ml, 195 ng/ml – Check for fluorescence after 3 hours using CFP filter on microscope

Experiments Results – Fluorescence was observed in the Sender Reporter Test Construct, visible with both CFP and GFP filters J06003: 3.13 ug/ml aTc, 100X, GFP filterJ06003: 3.13 ug/ml aTc, 100X, CFP filter

Experiments Results – Cell density decreased with higher concentrations of aTc (same volume of aTc solution was added to all samples) – DMF only cells did not seem to be affected - is aTc toxic at higher concentrations?

J06003: no aTc (DMF only), 100X, GFP filter without UVJ06003: 195 ng/ml aTc, 100X, GFP filter J06003: 3.13 ug/ml aTc, 100X, GFP filterJ06003: 6.25 ug/ml aTc, 100X, GFP filter

Experiments Results – No aTc added negative control did not fluoresce. – No CFP negative control fluoresced at levels comparable to experimental samples

J06001: 1.56 ug/ml aTc, 100X, GFP filterJ06003: 1.56 ug/ml aTc, 100X, GFP filter

Experiments Possible explanations for negative control fluorescence – Faulty part - we will be rebuilding and retesting; indication that we should be sequencing our finished parts – Autofluorescence Sender Construct (LVA+): aTc -> AHL J06001

Planned Experiments Testing the AHL receiver Testing the Weiss pulse generator contransformed with the receiver/receiver repressor construct Testing the BioWire pulse propagator cotransformed with the receiver/receiver repressor construct

Photolithography Master – Completed general cleanroom training. – Will get tool-specific training as soon as Kit’s lab gets their SU-8 2. – Ordered (will order?) SU (Balance of low viscosity and good thickness.) – Talked with Microchem researcher about SU-8 types and protocols.

Photolithography Stamps – Poured PDMS negative molds out of 33 rpm vinyl records in Kit’s lab. – Continued to experiment with pouring stamps from 96- and 384-well plates, using 3% and 5% high gel strength agarose. – Stamped cells using last week’s stamps.

Stamp Picture

This Week Building parts – Test constructs for Lux, finish Las parts. – Cotransform finished Lux parts. – Part validation/sequencing. Experiments – Test receiver constructs – Reconstruct and test LuxI sender (unexpected fluorescence) Photolithography – Go into cleanroom to make a 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 Week 8 (7/25): Experiments Week 9 (8/1): “ Week 10 (8/8): “ Week 11 (8/15): “ Week 12 (8/22): “