BioWire Progress Report Week Six “Halfway There”

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 Did experiments with Lux senders Got cleanroom certification

Building the Circuits

Lux– All parts finished (except receiver test construct).– Started moving plasmids in order to cotransform.

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.

Experiments

Lux System ConstructsSender Construct (LVA+): aTc -> AHL

J06001

Sender Construct (LVA-): aTc -> AHL

J06002

Sender Reporter (eCFP), Ptet Test

J06003

Receiver Test Construct: AHL -> YFP

J06000

Receiver Repressor Component: AHL -> cI

J06004

Receiver Construction Intermediate:

J06006

Receiver Output Component (Weiss):AHL -> YFP, cI repressible

J06005

Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+

J06007

Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA-

J06008

Lux System Constructs: Status

Receiver Test Construct: AHL -> YFP

Sender Construct (LVA+): aTc -> AHL

Sender Construct (LVA-): aTc -> AHL

Sender Reporter (eCFP), Ptet Test

Receiver Repressor Component: AHL -> cI

Construct Status

Completed

Done

Completed

J06001: Ptet+luxI(LVA+)

J06002.x: Ptet+luxI(LVA-)

J06003: Ptet+ ECFP

J06000: LuxPL + luxR + luxPR + YFP

J06004.x (vary RBS)

Notes

(R0040+B003x) + (C0161+B0015)

Building I13272 missing.(R0063+I0462) + (R0062+E0432)

Done

Done Lig./Trans. 7/15, miniprep today

F2621 initially missing.([(R0063+I0462) + R0062] + [B003x + (C0051+B0015)])

Lux System Constructs: Status

Receiver Output Component (Weiss):AHL -> YFP, cI repressible

Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+

Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA-

Receiver Output Construction Intermediate:

Construct Status

NO!

Done

Notes

Done

Done

J06006

(R0065+B0034) + E0032

(R0065+B0034) + (E0032 + B0015)

J06005

J06007.x

J06008.x

(((R0065+B0034) + (E0032 + B003x)) + (C0161 +B0015))x=1,2,4

(((R0065+B0034) + (E0032 + B003x)) + (C0061 +B0015))x=1,2,4

Las System Constructs

Receiver Test Construct: AHL -> YFP

Sender Construct (LVA+): aTc -> AHL

Sender Construct (LVA-): aTc -> AHL

Receiver Repressor Component: AHL -> cI

Receiver Construction Intermediate:

Receiver Output Component (Weiss):AHL -> YFP, cI repressible

Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+

Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA-

Sender Reporter (eCFP), Ptet Test

Las System Constructs: Status

Receiver Test Construct: AHL -> YFP

Sender Construct (LVA+): aTc -> AHL

Sender Construct (LVA-): aTc -> AHL

Sender Reporter (eCFP), Ptet Test

Receiver Repressor Component: AHL -> cI

Construct Status

Building

Building

Completed

J06101: Ptet+lasI(LVA+)

J06102.x: Ptet+lasI(LVA-)

J06103=J06003: Ptet+ ECFP

J06100: PBla + lasR + PLasR + YFP

J06104.x (vary RBS)

Notes

(R0040+B0034) + (C0178+B0015)

Building

(I14033 + (B0034+C0079+B0015 + R0079)) = J06400(I14033 + (B0034+C0079+B0015 + R0079)) + E0432 = J06100

Building

Done Lig./Trans. 7/15, miniprep today

J06400 + ( B003x+C0051+ B0015)x=4,1,2

(R0040+B0034) + (C0078+B0015)

Las System Constructs: Status

Receiver Output Component (Weiss):AHL -> YFP, cI repressible

Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+

Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA-

Receiver Output Construction Intermediate:

Construct Status

NO!

Building

Notes

Building

Building

J06106

((I14016 + B0034) + E0032)

J06105

J06107.x

J06108.x

x=1,2,4(I14016+B0034+E0032+B003x) + (C0178 + B0015)

x=1,2,4(I14016+B0034+E0032+B003x) + (C0078 + B0015)

(I14016 + B0034) + (E0032+ B0015)

Photolithography

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

gets their SU-8 2.– Ordered (will order?) SU-8 2075. (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.

Experiments

Photolithography– Go into cleanroom to make a 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, make first

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