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

2. Last Week [Almost] Finished building Lux parts Did experiments with Lux senders Got cleanroom certification

3. Building the Circuits Lux – All parts finished (except receiver test construct). – Started moving plasmids in order to cotransform. 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.

4. Experiments

5. Lux System Constructs Sender Construct (LVA+): aTc -> AHL J06001 Sender Construct (LVA-): aTc -> AHL J06002 Sender Reporter (eCFP), P tet Test J06003 Receiver Test Construct: AHL -> YFP J06000 Receiver Repressor Component: AHL -> cI J06004 Receiver Construction Intermediate: J06006 Receiver Output Component (Weiss): AHL -> YFP, cI repressible J06005 Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+ J06007 Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA- J06008

6. Lux System Constructs: Status Receiver Test Construct: AHL -> YFP Sender Construct (LVA+): aTc -> AHL Sender Construct (LVA-): aTc -> AHL Sender Reporter (eCFP), P tet Test Receiver Repressor Component: AHL -> cI Construct Status Completed Done Completed J06001: P tet +luxI(LVA+) J06002.x: P tet +luxI(LVA-) J06003: P tet + ECFP J06000: LuxPL + luxR + luxPR + YFP J06004.x (vary RBS) Notes (R0040+B003x) + (C0161+B0015) Building I13272 missing. (R0063+I0462) + (R0062+E0432) Done Lig./Trans. 7/15, miniprep today F2621 initially missing. ([(R0063+I0462) + R0062] + [B003x + (C0051+B0015)])

7. Lux System Constructs: Status Receiver Output Component (Weiss): AHL -> YFP, cI repressible Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+ Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA- Receiver Output Construction Intermediate: Construct Status NO! Done Notes Done J06006 (R0065+B0034) + E0032 (R0065+B0034) + (E0032 + B0015) J06005 J06007.x J06008.x (((R0065+B0034) + (E0032 + B003x)) + (C0161 +B0015)) x=1,2,4 (((R0065+B0034) + (E0032 + B003x)) + (C0061 +B0015)) x=1,2,4

8. Las System Constructs Receiver Test Construct: AHL -> YFP Sender Construct (LVA+): aTc -> AHL Sender Construct (LVA-): aTc -> AHL Receiver Repressor Component: AHL -> cI Receiver Construction Intermediate: Receiver Output Component (Weiss): AHL -> YFP, cI repressible Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+ Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA- Sender Reporter (eCFP), P tet Test

9. Las System Constructs: Status Receiver Test Construct: AHL -> YFP Sender Construct (LVA+): aTc -> AHL Sender Construct (LVA-): aTc -> AHL Sender Reporter (eCFP), P tet Test Receiver Repressor Component: AHL -> cI Construct Status Building Completed J06101: P tet +lasI(LVA+) J06102.x: P tet +lasI(LVA-) J06103=J06003: P tet + ECFP J06100: PBla + lasR + PLasR + YFP J06104.x (vary RBS) Notes (R0040+B0034) + (C0178+B0015) Building (I14033 + (B0034+C0079+B0015 + R0079)) = J06400 (I14033 + (B0034+C0079+B0015 + R0079)) + E0432 = J06100 Building Done Lig./Trans. 7/15, miniprep today J06400 + ( B003x+C0051+ B0015) x=4,1,2 (R0040+B0034) + (C0078+B0015)

10. Las System Constructs: Status Receiver Output Component (Weiss): AHL -> YFP, cI repressible Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+ Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA- Receiver Output Construction Intermediate: Construct Status NO! Building Notes Building J06106 ((I14016 + B0034) + E0032) J06105 J06107.x J06108.x x=1,2,4 (I14016+B0034+E0032+B003x) + (C0178 + B0015) x=1,2,4 (I14016+B0034+E0032+B003x) + (C0078 + B0015) (I14016 + B0034) + (E0032 + B0015)

11. 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-8 2075. (Balance of low viscosity and good thickness.) – Talked with Microchem researcher about SU-8 types and protocols.

12. 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.

13. Stamp Picture

14. This Week Building parts – Test constructs for Lux, finish Las parts. Experiments Photolithography – Go into cleanroom to make a master.

15. 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): “