Synthetic Biology Research Summer 2012

Ben Clarkson

Cell-Cell Communication

• Light– Fast, specific (wavelengths), sensitive

• How to use light to communicate between cells?

Proton Channels/Pumps

• Light-gated

• e-BO– 560 nm

pH sensitive promoters: cadA

cadB cadA

CadC

Cad1 Cad2

cadAP

pH sensitive promoters: cadA

cadB cadA

CadC

Cad1 Cad2

CadC

pH sensitive promoters: cadA

cadB cadA

CadC

Cad1 Cad2

H+

H+

H+ H+

H+

Cad1 Cad2Cad1 Cad2

pH sensitive promoters: cadA

cadB cadA

CadC

H+

H+

H+ H+

H+

Cad1 Cad2

pH sensitive promoters: cpxP

cpxP

CpxA CpxR

DegP

CpxP

pH sensitive promoters: cpxP

cpxP

DegP

CpxP

At high pH:1. DegP breaks down cpxP

pH sensitive promoters: cpxP

cpxP

CpxA CpxR

DegP

CpxP

At high pH:1. DegP breaks down cpxP2. CpxA (no longer inhibited by

cpxP) phosphorylates CpxR

pH sensitive promoters: cpxP

cpxP

CpxA CpxR

DegP

CpxP

At high pH:1. DegP breaks down cpxP2. CpxA (no longer inhibited by

cpxP) phosphorylates CpxR3. CpxR activates cpxP (as well as

DegP)

ScpxP

LcpxP

Assembling Promoters: Colony PCR

5'-GCATGAATTCGCGGCCGCTTCTAGAG-CTCAAGGCCGAGAACGCGATCAAGTTCACTGCCGCGCGCCGTCAACATAATGACAGGCGTCTGGTGTGTCTGGCGAAGTGCTTTTAATGTGTCGATACCATTTTTCTTCGGCATCATTACGTCAAGCAAAAGTAAATCAATGCTGTCGTCCAGAAGATCAAGCGCCTGTTCCCCATCGTGGGCAACAATCACGTTGAAGCCTTCCATCTCGAGCAGCTCCTTTAATAGGGAAGTCAGCTCTCGGTCATCATCAACTAACAGGATTTTATTCATTGTTTAAATACCTCCGAGGCAGAAATTACGTCATCAGACGTCGCTAATCCATGACTTTACGTTGTTTTACACCCCCTGACGCATGTTTGCAGCCTGAATCGTAAACTCTCTATCGTTGA-3'

3‘- GAGTTCCGGCTCTTGCGCTAGTTCAAGTGACGGCGCGCGGCAGTTGTATTACTGTCCGCAGACCACACAGACCGCTTCACGAAAATTACACAGCTATGGTAAAAAGAAGCCGTAGTAATGCAGTTCGTTTTCATTTAGTTACGACAGCAGGTCTTCTAGTTCGCGGACAAGGGGTAGCACCCGTTGTTAGTGCAACTTCGGAAGGTAGAGCTCGTCGAGGAAATTATCCCTTCAGTCGAGAGCCAGTAGTAGTTGATTGTCCTAAAATAAGTAACAAATTTATGGAGGCTCCGTCTTTAATGCAGTAGTCTGCAGCGATTAGGTACTGAAATGCAACAAAATGTGGGGGACTGCGTACAAACGTCGGACTTAGCATTTGAGAGATAGCAACT-ATGATCATCGCCGGCGACGTCTACG-'5

Forward Primer

Reverse Primer

Forward Primer

Reverse Primer

Forward Primer Reverse Primer

Promoter sequence

Colony PCR

Promoter

PromoterPromoter

Promoter

PromoterPromoter

PCR (template DNA=colony PCR products)

PromoterPromoter

PromoterPromoterPromoter

PromoterPromoter

PromoterPromoterPromoter

Ligation into Plasmid

Superfolder GFP with LVA (J119041)

pSB1A8

EcoRIXbaI SpeI

PstI

Superfolder GFP with LVA (J119041)

pSB1A8

EcoRIXbaI SpeI

PstI

EcoRI PstI

XbaI SpeI

Superfolder GFP with LVA (J119041)

pSB1A8

XbaI SpeI

Success!

EcoRI PstI

XbaI SpeI

Testing Promoters

RBS GFP

cadA (J100071)

pH 5.8 pH 6.5 pH 6.9 pH 7.5 pH 8 pH 8.50

5000

10000

15000

20000

25000

30000

35000

GFP Expression of J100081 at Various pH

pH

Fluo

resc

ence

/cel

l (RF

U)

LcpxP (J100072)

pH 5.8 pH 6.5 pH 6.9 pH 7.5 pH 8.0 pH 8.50

5000

10000

15000

20000

25000

30000

35000

40000 Level of GFP Expression of J100082 at Var-ious pH

pH

FLuo

resc

ence

/cel

l (RF

U)

ScpxP (J100073)

pH 5.8 pH 6.5 pH 6.9 pH 7.5 pH 8 pH 8.50

10000

20000

30000

40000

50000

60000

70000

80000

GFP Expression of J100087 at Various pH (90 gain)

pH

Fluo

resc

ence

/cel

l (RF

U)

Linking pH and Light

RBS LRE RBS Green luciferase

J100088: cadA promoterJ100089: LcpxP promoter

J100088 pH 5.8

J100088 pH 6.5

J100088 pH 6.9

J100088 pH 7.5

J100088 pH 8.0

J100088 pH 8.5

negati

ve co

ntrol

pBad E.

glowi

0.01000.02000.03000.04000.05000.06000.07000.08000.09000.0

10000.0

Luminescence of E. coli with J100088 at Various pH

Part, pH

Lum

ines

cenc

e/ce

ll (R

LU)

J100089 pH 5.8

J100089 pH 6.5

J100089 pH 6.9

J100089 pH 7.5

J100089 pH 8.0

J100089 pH 8.5

negati

ve co

ntrol

pBad E.

glowi

0.02000.04000.06000.08000.0

10000.012000.014000.016000.018000.020000.0

Luminescence of E. coli with J100089 at Various pH

Part, pH

Lum

ines

cenc

e/ce

ll (R

LU)

0 0.5 1 1.5 2 2.5 3 3.5 4 4.50

200400600800

1000120014001600

Luminescence of cells with J100088 and Luciferin at Different Time Points (Plate 1)

Luciferin added at t=0Luciferin added at t=1 hr.Luciferin added at t=2 hrs.Luciferin added at t=4 hrs.Negative control (no luciferin added)

Time of reading (hours)

Lum

ines

cenc

e/ce

ll (R

LU)

0 0.5 1 1.5 2 2.5 3 3.5 4 4.50.0

500.0

1000.0

1500.0

2000.0

2500.0

Luminescence of cells With J100089 and Luciferin at Different Time Points (Plate 1)

Luciferin added at t=0Luciferin added at t=1 hr.Luciferin added at t=2 hrs.Luciferin added at t=4 hrs.Negative control (no luciferin added)

Time of reading (hours)

Lum

ines

cenc

e/ce

ll (R

LU)

What next?

• Repeat experiment– Spacing– Different plates

• Other variables to test:– LysP represses CadC• Lysine=another variable?

– Luciferin– Retinal

Bacterioopsin vs. Bacteriorhodopsin: Moving Forward

• Adding retinal

• Cells with bacteriorhodopsin?

• Repeat experiment