BioWire Progress Report Week Ten

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

2. Last Week • Uhhhh… what did we build? • Sequencing results back for some test constructs • Experiments • Senders and receivers in liquid and solid media • Photolithography

3. Building the Circuits • Stuff about circuit building here • J06004ilator construction is continuing with some delays due to ligation failure

4. Sequencing • Sent in parts for sequencing • Constitutive sender – one was assembled correctly, although sequencing results contradicted gel results – tube mixup? • Propagation constructs (J06007.4 A/B, J06008.4A/B, J06108.4 A/B) • Results showed that our freezer tubes were mixed up • Have assembled J06008.4 A/B and what other ones? • Will be sending other major parts in this week

5. Experiments • Cotransformants • Can LuxR receivers cotransformed with receiver constructs respond to addition of AHL? • Senders and Receivers • Liquid media • Can the constitutive senders AHL induce the receiver test construct? • What sender/receiver ratio is necessary for this to occur? • Solid media • Can AHL induction occur on solid media?

6. Experiments: Cotransformants • Can LuxR producers cotransformed with receiver constructs respond to addition of AHL? • Input: AHL • Output: YFP fluorescence On KAN plasmid On AMP plasmid Degradation tags on YFP and LuxI varied Cotransformed in MC4100 cells (LacI-)

7. Experiments: Cotransformants • Experimental Design • Positive Control: Receiver Construct + AHL • Negative Control: Cells without YFP + AHL • Negative Control: No AHL added to cotransformants • Experimental Strains: • J06007.1A/B: LuxI (LVA+), medium RBS, YFP (AAV-/+) • J06008.1A/B: LuxI (LVA-), medium RBS, YFP (AAV-/+) • J06007.2A/B: LuxI (LVA+), weak RBS, YFP (AAV-/+) • J06008.2A/B: LuxI (LVA-), weak RBS, YFP (AAV-/+)

8. Experiments: Cotransformants • Experimental Design • Overnight cultures were backdiluted to 0.1 OD600 • 500 nM AHL was added to each culture • Cells were imaged after 40 minute incubation

9. Experiments: Cotransformants • Results – very similar to last week • Positive Control worked as expected • Negative Control: No YFP cells did not fluoresce • Cells with YFP (AAV-) fluoresced even without AHL addition • Cells with YFP (AAV+) did not fluoresce even with AHL addition • Exception: J06008.1B: LuxI (LVA-), medium RBS, YFP (AAV+) constitutively flouresces

10. PICTURES

11. Experiments: Cotransformants • Conclusions • FAILURE!!!

12. Experiments: S/R Liquid Media • Can the constitutive senders AHL induce the receiver test construct? • Input: Combining LuxI senders with receiver cells in varying ratios • LuxI produces AHL, which binds with LuxR to activate LuxPR promoter • Output: Fluorescence

13. Experiments: S/R Liquid Media • Experimental Design • SASHA FILL THIS IN

14. Experiments: S/R Liquid Media • Results • Positive control worked as expected • Negative control worked as expected • It worked!!!! More here from Sasha

15. PICTURES

16. More pictures

17. Experiments: S/R Liquid Media • Conclusions • YAAAAAAAAAAAY IT WORKS

18. Experiments: S/R Solid Media • Can AHL induction occur on solid media? • Input: Constitutive LuxI senders • Output: Fluorescence • Using Danny and Orr’s stamp to lay down cells!

19. Receiver cells Senders or AHL Experiments: S/R Solid Media • Experimental Design • Positive Control: Receiver Construct + AHL • Negative Control: Sender Cells (no fluorophore) • Experimental Strain: Receivers stamped with Senders STAMP SCHEMATIC 1 mm ?? 1 mm 1 mm

20. Experiments: S/R Solid Media • Experimental Design • Overnight cultures were backdiluted to 0.25 OD600 • Receivers were concentrated 10 fold, senders were concentrated 100 fold. • 5 ul of Receivers were inked onto the “line,” 1 ul of Senders (or 5000 nM AHL for control) were inked onto the “post” • Cells were stamped onto M9 agarose slides • Slides were incubated for 1 hr before imaging

21. Experiments: Solid Media • Results • Positive Control worked as expected – Receivers glowed brightly with AHL • Negative Control: Sender cells did not fluoresce • Receivers stamped with senders fluoresced, with fluorescence decreasing as distance from senders increased

22. Receivers, 100X phase Receivers, 100X GFP Senders, 100X phase Senders, 100X GFP

23. Receivers far from senders, 100X phase Receivers near senders, 100X phase Receivers far from senders, 100X GFP Receivers near senders, 100X GFP

24. Experiments: S/R Solid Media • Conclusions • AHL induction with senders works! • Stamping works! • Signal travels quickly • Had to go almost to other end of wire to find nonfluorescent receivers • Should quantify this – gridded slides? • Time course of fluorescence

25. Experiments: S/R Solid Media • Conclusions • Problem with stamping: stamp depresses agarose, forming an air bubble when coverslip is placed • Reduces visual uniformity of cells when visualized • Should try stamping with 500 micron lines • 1 mm a bit large for field of view under 100X

26. Planned Experiments • Cotransforming propagation constructs with receiver/repressor component • Addition of repressor will affect noise levels • Should be performed on solid and liquid media • The microscope lamp is broken! • We need it working for solid media • Can use FACS for cotransformant experiments if necessary

27. Photolithography • Made 4 rounds of masters • 90 micron; really good uniformity (+/- 10 um) • Unknown, practice at 1mm protocol • 4 wafers, 600 – 900 microns • 1 mm • Really good uniformity • All features stayed on! • PDMS and agarose • Stamped from 100 micron and most recent 1mm.

28. 85 um 85 um 100 um 90 um 85 um 90 um 90 um 90 um 90 um 85 um 90 um 90 um 85 um 90 um 90 um 85 um 90 um 90 um 110 um 90 um 8/2 – “150 micron”, second round 85-110 micron range 150 micron master

29. 8/5 – 1 ”millimeter”, second round, 90 sec. exposure 715-975 micron range 1mm master 870 um 910 um 905 um 890 um 970 um 945 um 955 um 875 um 725 um 790 um 725 um 795 um 715 um 780 um 715 um 775 um 715 um

30. Photolithography • Issues in the cleanroom: • Still not getting perfectly level surfaces. • Wafer still sticks to mask. • Haven’t been able to spin a final coat for uniformity as the spinners have been down. • Only other step requiring work is actual stamping • Still not very precise; can we blot?

31. Stamps 1mm wide lines 500 micron lines 1mm wide perimeter

32. Photolithography • Practice stamping for precise cell growth • A few more cleanroom cycles to increase stamp depth, fix final uniformity issues

33. This Week • Building parts • Uh what were we doing again? • Send parts in for sequencing • Experiments • Test cotransformants with repressor component • FACS • Solid media experiments • Photolithography • STAMP STAMP STAMP STAMP STAMP

34. 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, eating pizza courtesy of Alain • Week8 (7/25): More experiments, Meeting Their Master • Week 9 (8/1): More experiments, construction with new reporters • Week 10 (8/8): More experiments, STAMP STAMP STAMP • Week 11 (8/15): Ahhhh we’re on week 11!! Panic!! • Week 12 (8/22): “ • Week 13 (8/29): “