BioSketch The bacterial sketch pad.

BioSketchBioSketchThe bacterial sketch pad.The bacterial sketch pad.

Chris Doucette, Thomas Noriega, Hing EngYves Wang, Jennifer Gao, Yin Li

Group Meeting2005-08-15

BioBricks TeamBioBricks Team

BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005

33

LacI SwitchLacI SwitchConstructed J06961 – LacI (mut 241) switch with EYFP

Failed to make J06962 – LacI (mut 265) switch with EYFP


44

LacI (mut241) SwitchLacI (mut241) Switch

After overnight Growth:


55

LacI (mut241) SwitchLacI (mut241) Switch

After 4 h in 40°C:


66

LacI (mut241) SwitchLacI (mut241) SwitchSuspect that the ligation actually failed

=

No cI production, no LacI geneSupported by an analytical digest

Redid digestion and ligationAnalytical digest shows appropriate bandsMicroscope lamp brokenSending out for sequencing


77

A Cautionary TaleA Cautionary TaleWhat I was trying to do: Use miniprep isolated from a ligation mixture to transform JM2.300 cells for experiments.

Mini Prep:Decided to useJ06961 mini 4J06962 mini 2


88

When I transformed the JM2.300 cells with the minis they did not act as expected under the microscope. Confused, hurt, and betrayed I picked ten colonies from the transformation and looked at their plasmids:

Minis 1, 2, 3, 5, 8, and 9were CONTAMINATEDMinis 4, 6, 7, and 10Were GOOD


99

Sadly, the other plasmid transformation did not yield any good results…


1010

MORALMORALBeware of contaminated minis, especially if you plan on using them to transform bacteria that you are going to experiment on directly.

CollinsMod TeamCollinsMod Team


1212

What has been done: What has been done: CollinsModCollinsMod

Experimenting with UV irradiation of cells on plastic substrate

Replicating last week’s results with Collins Switch

Finding temperature at which ThermoSwitches are bistable

Examining apparent mutations in CI-repressed P(L*)


1313

Irradiating Cells on Plastic Irradiating Cells on Plastic SubstrateSubstrateLogic:

Eliminate the agar bits that might be mistaken for cellsSubstrate:

3-cm polysterene petri platesCells:

JM 2.300 cells with amp resistanceExperiment (UV killing curve):1. Dilute OD600 = 0.5 cells 1:107.2. Pipet 50 or 100ul cells into 3-cm dish; spread evenly.3. Irradiate.4. Pipet another 100ul LB into dish.5. Resuspend cells and pipet onto standard selective LB

plates; spread.6. Grow overnight.


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UV (Killing) CurveUV (Killing) Curve

0

20

40

60

80

100

120

0 20 40 60 80 100 120UV (J/m2)

# co

lon

ies

0

5

10

15

20

25

30

35

100ul

50ul

Agar


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UV Induces Signal in Collins UV Induces Signal in Collins SwitchSwitch

Day 1: 37C Day 1: 37C Day 2: 2mM IPTG Day 2: 2mM IPTG Day 3: UV irradiation, o.n. @ 37C Day 3: UV irradiation, o.n. @ 37C Day 4: Assay Day 4: Assay

JM 2.300JM 2.300 GFP reporterGFP reporter

Collins – 0 J/m2Collins – 0 J/m2Collins – 6 J/m2Collins – 6 J/m2

Collins – 12 J/m2Collins – 12 J/m2




1616

Finding Thermo-BistabilityFinding Thermo-BistabilityExperimental design:

Expected results:If cells are incubated (Day 3) at a temperature that permits bistability (Tb), then

Cells previously incubated @ 30C would remain ON.Cells previously incubated @ 40C would remain OFF.In other words: the state of the cell at Tb should be history-dependent.

30C30C

40C (OFF)40C (OFF)30C (ON)30C (ON)

30, 32, 35, 37, 40C (stay ON?)30, 32, 35, 37, 40C (stay ON?) 30, 32, 35, 37, 40C (stay OFF?)30, 32, 35, 37, 40C (stay OFF?)

Day Day 11

Day 2Day 2

Day 3Day 3


1717

ThermoSwitches Exhibit ThermoSwitches Exhibit Bistability at 30°CBistability at 30°C

JM 2.300JM 2.30030C 30C T Tbb

GFP reporterGFP reporter30C 30C T Tbb 40C 40C T Tbb

40C switch40C switch30C 30C T Tbb 40C 40C T Tbb

37C switch37C switch30C 30C 30C 30C 40C 40C T Tbb

30C30C

32C32C

35-38C35-38C

37C37C

40C40C


1818

A Closer Look at Behavior at A Closer Look at Behavior at 30°C30°C

M1

M1

M1

M1

40C Switch40C Switch 30C Switch30C Switch

30C 30C 30C 30C

40C 40C 30C 30C

A Closer Look at Behavior at A Closer Look at Behavior at 30°C30°C

76.3%76.3%23.7%23.7% 77.0%77.0%23.0%23.0%

62.5%62.5%37.5%37.5% 57.9%57.9%42.1%42.1%


1919

Mutations in CI-repressible Mutations in CI-repressible PPL*L* Promoter PromoterpWG (GFP reporter)

"Correct" sequence w.r.t. file sent from Collins labCanonical polymerase-binding regions (-10, -35)Noncanonical CI-binding site OL1

pTS (Collins switch)Mutations are in -10 TATA box and OL1 binding site.

Should make LacI expression weaker and CI repression stronger.

P(L*) from pTS/241/265P(L*) from pTS/241/265

5’-GCGTCCTGCTGATGTGCTC5’-GCGTCCTGCTGATGTGCTCAAGGTATTATCCACCGCCAGTGGTAACCGCCAGTGGTATTTATTTATGTCAATGTCAACACCGCCAGAGATAATTCACCGCCAGAGATAATTTATCACCGCAGATGGTTTATCACCGCAGATGGTTATCTGT-3’ATCTGT-3’3’-CGCAGGACGACTACACGAG3’-CGCAGGACGACTACACGAGTTCCATAATAGGTGGCGGTCACCATAAATTGGCGGTCACCATAAATACAACAGTTGTTGTGGCGGTCTCTATGTGGCGGTCTCTATTAAATAGTGGCGTCTACCAATAGACA-5’TAAATAGTGGCGTCTACCAATAGACA-5’

P(L*) from pWGP(L*) from pWG

5’-GCGTCCTGCTGATGTGCTC5’-GCGTCCTGCTGATGTGCTCAATTTATTATAAACCGCCAGTGGTAACCGCCAGTGGTATTTATTTATGTCAATGTCAACACCGCCAGAGATAATTCACCGCCAGAGATAATTTATCACCGCAGATGGTTTATCACCGCAGATGGTTATCTGT-3’ATCTGT-3’3’-CGCAGGACGACTACACGAG3’-CGCAGGACGACTACACGAGTTAAATAATATTTGGCGGTCACCATAAATTGGCGGTCACCATAAATACAACAGTTGTTGTGGCGGTCTCTATGTGGCGGTCTCTATTAAATAGTGGCGTCTACCAATAGACA-5’TAAATAGTGGCGTCTACCAATAGACA-5’

TATTATCCACCGCCAGTGGTAACCGCCAGTGGTA :O:OLL11

GTTGTGGCGGTCTCTATGTTGTGGCGGTCTCTAT :O :OLL22

OOLL3: 3: TATCACCGCAGATGGTTTATCACCGCAGATGGTT

-35: -35: ACAGTTACAGTT-10: -10: TTAAATAATATTlacIlacI


2020

ThermoSwitches: Results ThermoSwitches: Results and Implicationsand ImplicationsTurning ON: Weak

Basal level of reporter expression at 30C is weakly elevated (compared to OFF state).Would a lower temperature further increase GFP inductin?Does UV irradiation increase GFP induction above baseline?

Turning OFF: StrongReporter expression is well-suppressed by temp > 37C.

Maintaining state: WeakReporter expression is moderately history-dependent at 30C, with tendency to be ON.Reporter expression is history-independent at 32C, with weak suppression of signal.Are the promoters leaky?Is CI activity reduced at 30C?

In a previous experiment, IPTG-based repression at 30C did not work well. Was this due to decreased LacIts sensitivity to IPTG or decreased CI activity at 30C?


2121

Achieving Better ON StateAchieving Better ON State

o.n. @ 40C (OFF)o.n. @ 40C (OFF)

0, 12, 24, 48 J/m20, 12, 24, 48 J/m2

4h @ 25C (ON?)4h @ 25C (ON?) 4h @ 30C (ON?)4h @ 30C (ON?)

Day 1:Day 1:

Day 2:Day 2:


2222

Achieving More Robust Achieving More Robust BistabilityBistabilityContinue playing with environmental parameters?

Try 31C? ….If a more complete ON state is achieved, perhaps we will see a difference between maintaining the OFF state and maintaining the ON state.

Alter the parameters of the underlying circuit?Put the switch circuit on a medium copy plasmid?Make sure that both the LacIts promoter and the CI promoter have canonical polymerase binding regions or stronger RBSs.

Is CI activity weak at 30C?Is state maintained in the Collins switch at 30C?

And now, the long-And now, the long-awaited …awaited …

T-Shirt DesignT-Shirt DesignOrr & Yin


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FrontFrontBBa_RTeKN0BBa_RTeKN0

PromoterPromoterPizza-InduciblePizza-Inducible

BBa_B2WTF!BBa_B2WTF!

RBSRBSReference NA; Reference NA; could possibly work … maybecould possibly work … maybe

BBa_C(rand # of choice)BBa_C(rand # of choice) BBa_B6D1N0BBa_B6D1N0

TerminatorTerminatorStrength 5.5; units NAStrength 5.5; units NA

CDSCDSBioWiredSketch-a-LatorBioWiredSketch-a-Lator

= …= …

++


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BackBack

KXKX

Oh DannyOh Danny

XOrrXOrr

ScarecrowScarecrow

Number 2Number 2

Crouching ConnieCrouching Connie

T. RialtoT. Rialto

Chickwich Lady Chickwich Lady

Glowing Green YinGlowing Green Yin

MacDaddyMacDaddy

Chris the DestroyerChris the Destroyer

Hidden HingHidden Hing

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Pam, Pam, GeorgeGeorge

Kit, RadhikaKit, RadhikaAlainAlain

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BioSketch The bacterial sketch pad.

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