BioWire Progress ReportWeek Five

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

Last Week

Continued building Lux and Las test constructs.

Reorganized BioBrick information database. Got our mask back from Dr. Lu. Started pouring agarose stamps. Ordered CFP filter.

Building the Circuits

Sasha created a REALLY HOT DATABASE OF LIGATIONS OMFG HOT!!

Higher success rate with transformations - now within 2 cycles of finishing all of our parts

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

Building

Completed

J06001: Ptet+luxI(LVA+)

J06002.x: Ptet+luxI(LVA-)

J06003: Ptet+ ECFP

J06000: LuxPL + luxR + luxPR + YFP

J06004.x (vary RBS)

Notes

F1611 missing.(R0040+B003x) + (C0161+B0015)

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

Building

Done Lig./Trans. 7/9

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!

Building

Notes

Building

Building

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

(I14018 + (B0034+C0079+B0015 + R0079) ) = J06400

( I14018 + (B0034+C0079+B0015 + R0079) + E0432) = J06100

Building

Done Lig./Trans. 7/9

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

Mask– We have our mask. Cost: Free.– Next step: Ed (from Kit’s lab) will make us two

masters from the mask. Time frame: end of the week.

– Step after that: Go to Kit’s lab and make PDMS negative molds from the two masters.

Photolithography

Master– Because we will be stamping by hand, we really

want our features to have tangible depth. But SU8, the photoresist that we will use, is normally used up to 250 microns.

– We have a protocol from the Whitesides group for making features 1 mm deep, and we’re also in contact with cleanroom personnel about different types of SU8 and protocols.

Photolithography

Stamps– To make negative molds, we cut up a 96 well plate using a

heated spatula. This was done under a hood.– We placed the negatives into small beakers and poured 5%

agarose in PBS to make stamps. We separated the stamps from the molds.

– It turns out that the 96 well plates have tiny writing between wells to indicate the row and column. We found this accidentally by looking at the stamps, meaning that the agarose has clear definition at ~200 microns (or less?).

Photolithography

This Week

Parts scheduled to be finished today (still need analytical digestion for confirmation)

– J06004 - Receiver with Repressor Component– J06007 - LVA+ Receiver Construct with propagation– J06008 - LVA- Receiver Construct with propagation

Conduct experiments on Sender Reporter Test Construct with CFP filter

Plan and conduct experiments on Receiver Constructs Finish building lux, las constructs Practice inking with our stamps.

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): Test constructs, finish building parts, get master mold,

make PDMS molds Week 7 (7/18): Construct IFFL Week 8 (7/25): Test it Week 9 (8/1): “ Week 10 (8/8): “ Week 11 (8/15): “ Week 12 (8/22): “