1. BioWire Progress Report Week 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. 1:1 ratio, 100X, GFP1:1 ratio, 100X, phase - control, 100X, GFP- control, 100X, phase

11. 2:1 ratio, 100X, GFP2:1 ratio, 100X, phase - control, 100X, GFP- control, 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 plasmidOn 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 AHLNo AHL

18. J6007.4A: LuxI (LVA+), YFP (AAV-) 500 nM AHLNo AHL

19. J6007.4B: LuxI (LVA+), YFP (AAV+) 500 nM AHLNo AHL

20. J6008.4A: LuxI (LVA-), YFP (AAV-) 500 nM AHLNo AHL

21. J6008.4B: LuxI (LVA-), YFP (AAV+) 500 nM AHLNo AHL

22. Experiments: Cotransformants Conclusions – AAV tag on YFP is too strong to visualize fluorescence – LVA tag on LuxI (produces more AHL to propagate signal) significantly reduces efficacy of AHL propagation; LVA+ strain had significantly weaker fluorescence – Weak constitutive activity of LuxPR is problematic Will test constructs with weaker RBS to reduce amount of noise Also test constructs with repressor

23. 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

24. 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.

25. 150 micron master 8/2 – “150 micron”, second round 85-110 micron range 100 um 85 um 90 um 85 um 90 um 85 um 90 um 110 um 90 um 85 um 90 um 85 um 90 um 85 um

26. 1mm master 8/5 – 1 ”millimeter”, second round, 90 sec. exposure 715-975 micron range 910 um 870 um 875 um945 um 905 um 955 um 970 um 715 um 725 um 715 um 780 um 790 um 775 um 795 um 890 um

27. 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?

28. Stamps 1mm wide perimeter 1mm wide lines500 micron lines

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

30. 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

31. 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 Week 8 (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): “