BioWire Progress ReportWeek Ten

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

Last Week

Circuit Construction Sequencing

– Sequencing results back for some test constructs

Experiments– Senders and receivers in liquid and solid media

Photolithography

Building the Circuits

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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)

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

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?

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:

J06007.1A/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-/+)

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 – 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

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

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

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

Experiments: S/R Liquid Media

Results– Positive control worked as expected– Negative control worked as expected– Receivers fluoresced with addition of sender cells!

Sender / Receiver Phase

Sender / Receiver GFP

Receiver Phase

Receiver GFP

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.

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!

Experiments: S/R Solid Media

Experimental Design– Positive Control: Receiver Construct + AHL– Negative Control: Sender Cells (no fluorophore)– Experimental Strain: Receivers stamped with

Senders

Senders or AHL Receiver cells

STAMP SCHEMATIC

1 mm

1 mm

1 mm

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

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

Senders, 100X phase

Senders, 100X GFP

Receivers, 100X phase

Receivers, 100X GFP

Receivers near senders, 100X phase

Receivers near senders, 100X GFP

Receivers far from senders, 100X phase

Receivers far from senders, 100X GFP

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

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

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

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

1 hour

2 hours

3 hours

4 hours

5 hours

6 hours

7 hours

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

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): Ahhhh we’re on week 11!! Panic!! Week 12 (8/22): “ Week 13 (8/29): “