BioWire Progress Report Week Nine

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

2. Last Week • Rebuilt parts with new YFP reporters • Experiments • Constitutive senders + AHL receivers • AHL + cotransformed receivers • Sent parts in for sequencing • Photolithography

3. Building the Circuits • Rebuilt all major Lux parts with new YFP reporters • Cotransformed AHL receiver with propagation constructs • Nearly complete with major Las constructs

4. Building the Circuits • Started building new circuit (thanks to Ira) • Based off of “repressilator” – transcriptional cascade of repressors • Puts a time delay between AHL induction and CI repression, thus ensuring a pulse • Parts were ordered from MIT; within two cycles of completion

5. Building the Circuits • Current Design • CI, LuxI, and reporter induced at same time • New Design • Cascade causes CI to be induced later than LuxI and reporter, eliminating “competition” between propagation and repression

6. Experiments • Constitutive Senders to Receivers • Does the LuxI sender part work? • How much LuxI is needed to activate the receivers? • AHL to Cotransformed Receiver Constructs • Can the contransformed constructs be induced by AHL?

7. Experiments: Constitutive Senders • Does the Receiver Test Construct fluoresce when combined with constitutive LuxI senders? • Input: Combining LuxI senders with receiver cells in varying ratios • LuxI produces AHL, which binds with LuxR to activate LuxPR promoter • Output: Fluorescence Constitutive promoter is Plambda, thanks Biosketch

8. Experiments: Constitutive Senders • Experimental Design • Overnight cultures were backdiluted to 0.1 OD600 • Sender cells were backdiluted, IPTG induced for 2 hours, then backdiluted again • Sender and receiver cells were combined in varying ratios by volume • 1:1 (sender:receiver), 2:1, 4:1 • Controls • Positive: AHL + Receiver • Negative: Receivers alone • Cells were imaged after 40 minutes of incubation

9. Experiments: Constitutive Senders • Results • Positive control worked as expected • Negative control showed a significant amount of background fluorescence observed under YFP and GFP filters • Fluorescence was observed at all sender:receiver ratios under YFP and GFP filters

10. - control, 100X, GFP - control, 100X, phase 1:1 ratio, 100X, GFP 1:1 ratio, 100X, phase

11. - control, 100X, GFP - control, 100X, phase 2:1 ratio, 100X, GFP 2:1 ratio, 100X, phase

12. Experiments: Constitutive Senders • Conclusions • Fluorescence seems qualitatively stronger with addition of senders, but still unsure if it is caused by senders or background • LuxPR promoter (controls YFP) has weak constitutive expression – noise problems with construct • Complicated because senders and receivers are mixed – unsure what percent of receivers are actually fluorescing • Solid media may be better way of experimenting • Using stamp, can separate senders and receivers while allowing for induction

13. 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-)

14. Experiments: Cotransformants • Experimental Design • Positive Control: Receiver Construct + AHL • Negative Control: Cells without YFP + AHL • Negative Control: No AHL added to cotransformants • Experimental Strains: • J06007.4A: LuxI (LVA+), strong RBS, YFP (AAV-) • J06007.4B: LuxI (LVA+), strong RBS, YFP (AAV+) • J06008.4A: LuxI (LVA-), strong RBS, YFP (AAV-) • J06008.4B: LuxI (LVA-), strong RBS, YFP (AAV+)

15. 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

16. Experiments: Cotransformants • Results • 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

17. Positive Control: Receiver Test Construct 500 nM AHL No AHL

18. J6007.4A: LuxI (LVA+), YFP (AAV-) 500 nM AHL No AHL

19. J6007.4B: LuxI (LVA+), YFP (AAV+) 500 nM AHL No AHL

20. J6008.4A: LuxI (LVA-), YFP (AAV-) 500 nM AHL No AHL

21. J6008.4B: LuxI (LVA-), YFP (AAV+) 500 nM AHL No AHL

22. Experiments: Cotransformants • Is system being triggered? • Yes: LVA tag on LuxI (produces more AHL to propagate signal) significantly reduces efficacy of AHL propagation; LVA+ strain had significantly weaker fluorescence

23. Experiments: Cotransformants • AAV- YFP: Positive feedback loop is causing “auto-firing” even in absence of AHL • AAV+ YFP: tag on YFP is too strong to visualize fluorescence (YFP gets degraded too quickly)? • AAV+ YFP was visible in positive control (I13272), but that is under slightly different promoter (lux vs lux+CI) • Having propagation should only increase signal • Need to build positive control with lux+CI promoter

24. Experiments: Cotransformants • Weak constitutive activity of LuxBox is problematic • Will test constructs with weaker RBS to reduce amount of noise • Also test constructs with repressor

25. Planned Experiments • Testing cotransformants with varying RBS strengths • Testing receivers cotransformed with repressors (aka pulse generator) • Testing senders with receivers on solid media • Using the FACS for more accurate, quantitative measurements • Using the wicked cool stamps

26. 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.

27. 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

28. 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

29. 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?

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

31. Photolithography • Practice stamping for precise cell growth • Nutrient media stamps to cut down on remaking stamps, inking • A few more cleanroom cycles to increase stamp depth, fix final uniformity issues

32. This Week • Building parts • Continue cotransforming Lux test constructs • Build revised circuit design • Finish building Las parts with new reporters • Experiments • Test cotransformants with different RBS, repressor component • FACS • Solid media experiments • Photolithography • STAMP STAMP STAMP STAMP STAMP

33. 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): “ • Week 12 (8/22): “ • Week 13 (8/29): “