BioWire Progress Report Week Five

1. BioWire Progress ReportWeek Five Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush

2. Continued building Lux and Las test constructs. Reorganized BioBrick information database. Got our mask back from Dr. Lu. Started pouring agarose stamps. Ordered CFP filter. Last Week

3. Building the Circuits • Sasha created a REALLY HOT DATABASE OF LIGATIONS OMFG HOT!! • Higher success rate with transformations - now within 2 cycles of finishing all of our parts

4. 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

5. Lux System Constructs: Status Lig./Trans. 7/9 Done Construct Status Notes Sender Construct (LVA+): aTc -> AHL Completed J06001: Ptet+luxI(LVA+) Building F1611 missing. (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 Building F2621 initially missing. [(R0063+I0462) + R0062]+[B003x + (C0051+B0015)] J06004.x (vary RBS)

6. Lux System Constructs: Status Construct Status Notes NO! Receiver Output Construction Intermediate: (R0065+B0034) + E0032 J06006 Building (R0065+B0034)+ (E0032 + B0015) Receiver Output Component (Weiss): AHL -> YFP, cI repressible J06005 Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+ Building ( (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- Building J06008.x

7. 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

8. Las System Constructs: Status Lig./Trans. 7/9 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 (I14018+ (B0034+C0079+B0015 + R0079) ) = J06400 ( I14018+ (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)

9. 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

10. Photolithography • Mask • We have our mask. Cost: Free. • Next step: Ed (from Kit’s lab) will make us two masters from the mask. Time frame: end of the week. • Step after that: Go to Kit’s lab and make PDMS negative molds from the two masters.

11. Photolithography • Master • Because we will be stamping by hand, we really want our features to have tangible depth. But SU8, the photoresist that we will use, is normally used up to 250 microns. • We have a protocol from the Whitesides group for making features 1 mm deep, and we’re also in contact with cleanroom personnel about different types of SU8 and protocols.

12. Photolithography • Stamps • To make negative molds, we cut up a 96 well plate using a heated spatula. This was done under a hood. • We placed the negatives into small beakers and poured 5% agarose in PBS to make stamps. We separated the stamps from the molds. • It turns out that the 96 well plates have tiny writing between wells to indicate the row and column. We found this accidentally by looking at the stamps, meaning that the agarose has clear definition at ~200 microns (or less?).

13. Photolithography

14. This Week • Parts scheduled to be finished today (still need analytical digestion for confirmation) • J06004 - Receiver with Repressor Component • J06007 - LVA+ Receiver Construct with propagation • J06008 - LVA- Receiver Construct with propagation • Conduct experiments on Sender Reporter Test Construct with CFP filter • Plan and conduct experiments on Receiver Constructs • Finish building lux, las constructs • Practice inking with our stamps.

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): Test constructs, finish building parts, get master mold, make PDMS molds • Week 7 (7/18): Construct IFFL • Week 8 (7/25): Test it • Week 9 (8/1): “ • Week 10 (8/8): “ • Week 11 (8/15): “ • Week 12 (8/22): “