BioWire Progress ReportWeek Six

“Halfway There”

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

Last Week

[Almost] Finished building Lux parts Conducted experiments with Lux sender test

construct Got cleanroom certification

Building the Circuits

Lux– All parts should be finished today (awaiting analytical

digest).– Received DH5alpha cells for transformation– Started moving parts onto Kan plasmids in order to

cotransform into cells. Las

– Still building parts.– Switched constitutive promoters, P(Bla)-->P(Cat) because

the old part just wasn’t working.– Moved within two cycles of finishing.

Building the Circuits

Cotransformation Plan– Putting CI repressor on kan plasmid enables us to control

the copy number, and thus repressor levels, through IPTG induction

KAN: Receiver Repressor Component (J06004)

AMP: Receiver Output+Propagation Component (J06007,8)

BioWire Cell

Experiments

Testing the Sender Reporter Construct

– Input: anhydrotetracycline (aTc) – Output: fluorescence (CFP)

Sender Reporter (eCFP), Ptet Test

J06003

Experiments

Experimental Design– Positive Control - constitutive GFP (gift from Yin)– Negative Control - no aTc added/DMF only– Negative Control - strain without CFP (LuxI

sender under Tet promoter)

Experiments

Experimental Design– Grow up overnight cultures of cells– Backdilute to 0.1 OD600– Add varying concentrations of aTc

25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.13 μg/ml, 1.56 μg/ml, 781 ng/ml, 391 ng/ml, 195 ng/ml

– Check for fluorescence after 3 hours using CFP filter on microscope

Experiments

Results– Fluorescence was observed in the Sender Reporter Test

Construct, visible with both CFP and GFP filters

J06003: 3.13 ug/ml aTc, 100X, GFP filter J06003: 3.13 ug/ml aTc, 100X, CFP filter

Experiments

Results– Cell density decreased with higher concentrations

of aTc (same volume of aTc solution was added to all samples)

– DMF only cells did not seem to be affected - is aTc toxic at higher concentrations?

J06003: no aTc (DMF only), 100X, GFP filter without UV J06003: 1.56 ug/ml aTc, 100X, GFP filter

J06003: 3.13 ug/ml aTc, 100X, GFP filter J06003: 6.25 ug/ml aTc, 100X, GFP filter

Experiments

Results– No aTc added negative control did not fluoresce.– No CFP negative control fluoresced at levels

comparable to experimental samples

J06001: 1.56 ug/ml aTc, 100X, GFP filter J06003: 1.56 ug/ml aTc, 100X, GFP filter

Experiments

Possible explanations for negative control fluorescence

– Faulty part - we will be rebuilding and retesting; indication that we should be sequencing our finished parts

– Autofluorescence

Sender Construct (LVA+): aTc -> AHL

J06001

Planned Experiments

Testing the AHL receiver Testing the Weiss pulse generator

contransformed with the receiver/receiver repressor construct

Testing the BioWire pulse propagator cotransformed with the receiver/receiver repressor construct

Photolithography

Making the Master– Completed general cleanroom training.– Will get tool-specific training as soon as Kit’s lab

gets their SU-8 2.– SU-8 2075 is being ordered this morning.

(Balance of low viscosity and good thickness.)– Talked with Microchem researcher about SU-8

types and protocols.

Photolithography

Stamps– Poured PDMS negative molds out of 33 rpm vinyl

records in Kit’s lab.– Continued to experiment with pouring stamps

from 96- and 384-well plates, using 3% and 5% high gel strength agarose.

– Stamped cells using last week’s stamps.

Stamp Picture

This Week

Building parts– Test constructs for Lux, finish Las parts.– Cotransform finished Lux parts.– Part validation/sequencing.

Experiments– Test receiver constructs– Reconstruct and test LuxI sender (unexpected

fluorescence) Photolithography

– Go into cleanroom to finish training and make a first master.

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; complete a cycle of

making a master mold, PDMS mold, and stamp

Week 8 (7/25): Experiments Week 9 (8/1): “ Week 10 (8/8): “ Week 11 (8/15): “ Week 12 (8/22): “