BioWire Progress Report Week Seven

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

2. Last Week • Finished off most major Lux and Las system constructs • Repeated experiments with Lux sender test construct • Conducted preliminary experiments with Lux final components • Continued certification, visited SU-8 factory, and made first master

3. Building the Circuits • Lux • Constructed and gel verified versions of each major component. • Successfully co-transformed nine Lux components into DH5alpha cells. • Sent all gel verified Lux parts for sequencing. • Las • Constructed and gel verified versions of almost all major component. • Planning for Las co-transformations. • Sequencing of Las parts pending

4. AMP: Receiver Output+ Propagation Component (J06008) Building the Circuits • Successful Cotransformations KAN: Constitutive luxR producer DH5alpha Cell

5. Experiments • Retesting the Sender Reporter Construct with more negative controls • Testing Receiver Constructs (cotransformations)

6. Sender Reporter (eCFP), Ptet Test J06003 Experiments: Sender Reporter • Retesting the Sender Reporter Construct with more negative controls • Last week, negative controls for this experiment fluoresced • Input: anhydrotetracycline (aTc) • Output: fluorescence (CFP)

7. Experiments: Sender Reporter • Experimental Design • Positive Control - constitutive GFP (gift from Yin) • Negative Control - no aTc added • Negative Control - J06001 (has tet promoter, no CFP), J06004 (no tet promoter, no CFP) • Negative Control - untransformed top10 cells • Procedure same as before

8. Results: Sender Reporter • All -aTc negative controls did not fluoresce, as expected • All +aTc negative controls still fluoresce, including the untransformed top10 cells • Adding aTc causes cells to fluoresce • TetR[atc–Mg]2 complex is fluorescent (Schubert et al 2004) and shares similar absorption and emission wavelengths to ECFP

9. top10 -ctrl, no aTc 100x GFP filter top10 -ctrl, no aTc 100x, no UV top10 -ctrl, 6.76 uM aTc 100x, no UV top10 -ctrl, 6.76 uM aTc 100x GFP filter

10. Experiments: Sender Reporter • Building a different sender reporter using YFP rather than CFP (tetR-atc fluorescence is significantly less in YFP than CFP/GFP) • Putting sender, sender reporter cells on DH5alpha cells

11. R0063 I0462 Experiments: Cotransformation • Can R0063/I0462 produce LuxR? • R0063 - Lux PL, weak constitutive promoter • I0462 - produces LuxR • Can Lux PR promoter respond to LuxR-AHL? • Input: acyl-homoserine lactone (AHL) • Output: YFP fluorescence J06008 R0063/I0462

12. R0063 I0462 Experiments: Cotransformation • Experimental Design • R0063/I0462 on Kan plasmid was cotransformed with J06008 on Amp plasmid in DH5alpha cells • R0063/I0462, which produces LuxR, was ligated to J06008, which has YFP regulated by Lux PR promoter in Top10 cells. • Positive Control: Glowing Green Yin in Top10 • Negative Control: R0063/I0462 on Kan plasmid in Top10 • Negative Control: J06008 on Amp plasmid in Top10 • Negative Control: No AHL added to cells J06008 R0063/I0462

13. Experiments: Cotransformation • Experimental Design • Overnight cultures were backdiluted to 0.1 OD600 • Various concentrations of AHL were added • 0 nM, 14.8 nM, 49.2 nM, 148 nM, 492 nM • After 40 minute incubation, cells were imaged with YFP filter • Note: Cells with Kan plasmid were IPTG induced for 3 hours prior to backdilution and addition of AHL.

14. Experiments: Cotransformations • Results • R0063/I0462/J06008 ligation failed to fluoresce • R0063/I0462 cotransformed with J06008 on DH5alpha cells died (no fluorescence) • Positive control, Glowing Green Yin, fluoresced • Negative controls did not fluoresce • IPTG induced R0063/I0462 on Kan in Top10 had normal growth

15. R0063/I0462 ligated to J06008 on top10 492 nM AHL, 100X no UV R0063/I0462 ligated to J06008 on top10 492 nM AHL, 100X GFP filter IPTG induced R0063/I0462 on Kan in top10 492 nM AHL, 100X no UV R0063/I0462 cotransformed with J06008 on DH5alpha cells 492 nM AHL, 100X no UV

16. Experiments: Cotransformations • Conclusions • Unsure if R0063/I0462/J06008 ligation succeeded. • Will be retransforming into DH5alpha cells to test • IPTG induction may have killed DH5alpha cotransformants • Should backdilute cells in fresh media prior to IPTG induction • Will test cells with and without IPTG induction • We will be transforming all test constructs onto DH5alpha

17. Planned Experiments • Repeating cotransformant tests (today) • Test receiver constructs for Lux and Las, Weiss circuit as they are completed • Begin tests on solid media • Quantify fluorescence to create time curves

18. 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.

19. 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): “ • Week 10 (8/8): “ • Week 11 (8/15): “ • Week 12 (8/22): “ • Week 13 (8/29): “