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

Last Week 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.

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

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

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 Building 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 F1611 missing. (R0040+B003x) + (C0161+B0015) Building I13272 missing. (R0063+I0462) + (R0062+E0432) Building Done Lig./Trans. 7/9, miniprep today F2621 initially missing. ([(R0063+I0462) + R0062] + [B003x + (C0051+B0015)])

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! Building Notes Building J06006 (R0065+B0034) + E0032 (R0065+B0034) + (E B0015) J06005 J06007.x J06008.x (((R0065+B0034) + (E B003x)) + (C0161 +B0015)) x=1,2,4 (((R0065+B0034) + (E B003x)) + (C0061 +B0015)) x=1,2,4

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

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 (I (B0034+C0079+B R0079)) = J06400 (I (B0034+C0079+B R0079)) + E0432 = J06100 Building Done Lig./Trans. 7/9, miniprep today J ( B003x+C0051+ B0015) x=4,1,2 (R0040+B0034) + (C0078+B0015)

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 ((I B0034) + E0032) J06105 J06107.x J06108.x x=1,2,4 (I B0034) + (E B003x) + (C B0015) x=1,2,4 (I B0034) + (E B003x) + (C B0015) (I B0034) + (E B0015)

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.

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.

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?).

Photolithography

This Week Parts scheduled to be finished today (still need analytical digestion for confirmation) – J Receiver with Repressor Component – J LVA+ Receiver Construct with propagation – J 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.

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): “