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 • Determined that our YFP reporter (used in the receiver) is defective • Re-ordered a working YFP and began reconstruction using the new YFP • Received the receiver test construct from BioBricks, conducted experiments • AHL to receiver • aTc to sender+receiver • Sequencing results • Photolithography

3. Oh YFP… • YFP Biobrick Parts: • E0030 (YFP, no degradation tags) • E0032 (YFP, LVA+) • E0034 (YFP, AAV+) • We had been using E0032, since we want degradation of YFP for temporal analysis • Turns out that E0032 does not glow when added to a constitutive promoter (degradation tag too strong?) • Reporter control experiments

4. 100x Phase 100x YFP Filter I14033 + E0430 [Const. P(cat) + YFP] I14033 + E0434 Const. P(cat) + YFP AAV I14033 + E0432 [Const. P(cat) + YFP LVA]

5. REPORTER (Re)Building the Circuits • Now rebuilding circuits with E0030 (YFP), E0034 (YFP AAV), and mCherry (LVA+ and LVA-) • Thanks Biosketch • Lux and Las components with YFP, YFP AAV, and mCherry LVA- will be ligated together today • Also building mCherry sender reporter • Retransforming into MC4100 (LacI-) cells, since they seem hardier than DH5alpha cells Receiver Output+Propagation Component (J06008) = +

6. Sequencing • Sent in completed Lux parts for sequencing • Total of 9 parts sent in (29 sequences) • 4 readable sequences • 3 okay, 1 missing an RBS • Parts are being rebuilt with new reporters anyways • All others had issues with “multiple priming”

7. Experiments • AHL to receiver • Is AHL working? • Is the AHL receiver promoter working? • aTc to sender+receiver • Is the sender (AHL producer) working? • Is aTc induction working (the tetR promoter)? • Is luxI producing AHL? • Solid media

8. Experiments: AHL to receiver • Does the Receiver Test Construct fluoresce with addition of AHL? • Input: acyl-homoserine lactone (AHL) • Output: EYFP fluorescence • I13272 (receiver) • Produces luxR constitutively • Has EYFP after promoter lux pR, which is upregulated by the luxR-AHL complex

9. Experiments: AHL to receiver • Experimental Design • Grow up overnight cultures • Backdilute to 0.1 OD600 • Add appropriate concentration of AHL (0, 15, 50, 150, 500 nM) • Incubate 1 hr • Place on M9 agar slides and image • Controls • Positive: Constitutive YFP producer (I14033+E0430) • Negative: Untransformed MC4100 cells

10. Experiments: AHL to receiver • Results • All controls worked as expected • Fluorescence was observed at all levels of AHL (except 0 nM) under the GFP and YFP filters • Fluorescence levels at different concentrations of AHL were qualitatively comparable

11. PHASE GFP FILTER 0 nM AHL 15 nM AHL 500 nM AHL

12. Experiments: Receiver Construct • Conclusions • All parts in the receiver construct are functional • Fluorescence can be observed at even low levels of AHL

13. Experiments: aTc to sender/receiver • Does the AHL sender, when induced by aTc, cause the receiver to fluoresce? • Input: aTc to sender cells • Output: EYFP fluorescence of receiver cells • J06001 (AHL sender part) • Produces luxI, which in turn produces AHL, in response to aTC

14. Experiments: aTc to sender/receiver • Experimental design • Grow up both types of cells overnight; backdilute both to OD600 = 0.01 • Add 1.56 ug/mL aTc to sender cells and incubate all cells in 37C shaker for 3 hours • Add different concentrations (same volume) of sender cells to tubes of 2mL of receiver cells • 1,5,10,20x sender:receiver ratios • Incubate mixtures in 37C shaker for 40min • Controls • +: 500nM AHL+receiver; 20x mixture+500nM AHL+ receiver • -: LB+receiver, sender+receiver w/o aTc, receiver+aTc

15. Experiments: aTc to sender/receiver • Results • Controls behaved as expected • Fluorescence of sender/receiver + aTc mixtures was not high relative to negative controls (aTc/tetR fluorescence) • Potential explanations • Sender is not producing enough AHL (need to have higher sender:receiver ratios) • Not adding enough aTc/tetR promoter for inducible aTc expression • Sender part defective (promoter bad or luxI coding part bad)

16. 100X Phase 100X CFP 100X GFP 100X YFP 20x Sender to Receiver ratio Positive Control (receiver + AHL) Positive Control (receiver + sender + AHL + aTc)

17. Experiments: aTc to sender/receiver • Potential explanations • Sender is not producing enough AHL (need to have higher sender:receiver ratios) • Not adding enough aTc/tetR promoter for inducible aTc expression • Sender part defective (promoter bad or luxI coding part bad) • Promoter appears to be working (aTc inducible)

18. 100X Phase 100X YFP R0040/E0434 -aTc R0040/E0434 +aTc 1.56 ug/mL

19. Planned Experiments • Replicating Sender/Receiver experiment • Higher sender cell concentration • Creating a time course for AHL induction with receiver construct • Testing Propagation Constructs with new YFP (to be cotransformed with LuxR producers)

20. 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.

21. 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

22. 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

23. 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?

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

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

26. This Week • Building parts • Test constructs for Lux, finish Las parts. • Move finished Lux parts onto DH5alpha cells. • Part validation/sequencing. • Experiments • Test receiver constructs • Reconstruct and test LuxI sender (unexpected fluorescence) • Photolithography • Go into cleanroom to make 150m and 1000m master.

27. 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): “ • Week 11 (8/15): “ • Week 12 (8/22): “ • Week 13 (8/29): “