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BioWire Progress Report Week Six “Halfway There”

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 Did experiments with Lux senders Got cleanroom certification. Building the Circuits. Lux

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BioWire Progress Report Week Six “Halfway There”

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

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

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

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

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

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

  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): “

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