BioWire Progress ReportWeek Nine

Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush

Last Week

Rebuilt parts with new YFP reporters Experiments

– Constitutive senders + AHL receivers– AHL + cotransformed receivers

Sent parts in for sequencing Photolithography

Building the Circuits

Rebuilt all major Lux parts with new YFP reporters

Cotransformed AHL receiver with propagation constructs

Nearly complete with major Las constructs

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

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

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?

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

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

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

1:1 ratio, 100X, GFP 1:1 ratio, 100X, phase

- control, 100X, GFP - control, 100X, phase

2:1 ratio, 100X, GFP 2:1 ratio, 100X, phase

- control, 100X, GFP - control, 100X, phase

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

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 variedCotransformed in

MC4100 cells (LacI-)

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+)

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

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

Positive Control: Receiver Test Construct

500 nM AHL No AHL

J6007.4A: LuxI (LVA+), YFP (AAV-)

500 nM AHL No AHL

J6007.4B: LuxI (LVA+), YFP (AAV+)

500 nM AHL No AHL

J6008.4A: LuxI (LVA-), YFP (AAV-)

500 nM AHL No AHL

J6008.4B: LuxI (LVA-), YFP (AAV+)

500 nM AHL No AHL

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

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

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

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

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.

150 micron master8/2 – “150 micron”, second round

85-110 micron range100 um 85 um

90 um90 um

90 um

90 um

85 um90 um

90 um

85 um90 um

110 um

90 um85 um

90 um85 um

90 um

90 um

90 um

85 um

1mm master8/5 – 1 ”millimeter”, second round, 90 sec. exposure

715-975 micron range910 um 870 um

875 um945 um

905 um

955 um

970 um

715 um

725 um725 um

715 um

715 um780 um

790 um

775 um

795 um

890 um

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?

Stamps

1mm wide perimeter

1mm wide lines 500 micron lines

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

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

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): “