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
+
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): “