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 • Circuit Construction • Sequencing • Sequencing results back for some test constructs • Experiments • Senders and receivers in liquid and solid media • Photolithography

3. + Building the Circuits • Currently building • Lux receiver with the hybrid lux box/CI promoter for use as a positive control on experiments with J06007 and J06008 parts (2 cycles) • Lux->Las and Las->Lux crosslinkers with CFP (3 cycles) • J06007 and J06008 constructs with R63/I0462 ligated, for comparison with our constructs that have R63/I0462 cotransformed (1 cycle) • J06004ilator construction is continuing with some delays due to ligation failure (2 cycles)

4. Sequencing • Sent in parts for sequencing • Constitutive sender (2 tubes) • 1 correct sequence, 1 incorrect; sequencing results contradicted gel results – tube mixup? • In any case, we know which sender tube is working from experiments • 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 6301.4A (lux->las crosslinker) • 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: 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. Experiments: Cotransformants • Conclusions • Similar results to last week - either constitutive on or off • Results may be invalidated by confusion revealed through sequencing • Need to look at positive control with the hybrid lux box CI promoter (being built) • Will look at receiver constructs with repressor component since repressor will affect expression levels

11. 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

12. Experiments: S/R Liquid Media • Experimental Design • Overnight cultures were backdiluted to 0.1 OD600 • Senders grow to .5 OD600. • 10ml sender spun down and pellet resuspended in 1ml of receiver • Cultures were incubated for 2 hr before imaging • Controls • (-) Receiver alone, sender alone, receiver with random cells • (+) Receiver with AHL

13. Experiments: S/R Liquid Media • Results • Positive control worked as expected • Negative control worked as expected • Receivers fluoresced with addition of sender cells!

14. Sender / Receiver GFP Sender / Receiver Phase Receiver Phase Receiver GFP

15. Experiments: S/R Liquid Media • Conclusions • Sender receiver induction works. • An additional experiment confirmed that we need to have a high cell density of sender cells in order to set off any induction.

16. 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!

17. 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

18. 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

19. 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

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

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

22. 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

23. 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

24. 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

25. Photolithography • Made a final cleanroom cycle • Features over 1mm • 240 micron range (960 – 1,200) • 2 x 200 micron range (1,100 – 1,300; 1,040 – 1,240) • 105 micron range (1,140 – 1,245) • Two were distorted by feature detachment • Worked on stamping technique • Time course of stamping density

26. 1 hour 2 hours 3 hours 4 hours

27. 5 hours 6 hours 7 hours

28. 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

29. 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): “