Transcript Media:Synthetic_Biology_Research_Summer_2012_presentation.pptx

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
cadAP
cadB
cadA
CadC
Cad1
Cad2
pH sensitive promoters: cadA
cadB
cadA
CadC
Cad1
Cad2
pH sensitive promoters: cadA
cadB
H+
H+
cadA
H+
H+
CadC
H+
Cad1
Cad2
pH sensitive promoters: cadA
cadB
H+
H+
cadA
H+
H+
CadC
H+
Cad1
Cad2
pH sensitive promoters: cpxP
cpxP
CpxP
CpxA
CpxR
DegP
pH sensitive promoters: cpxP
cpxP
CpxP
At high pH:
1. DegP breaks down cpxP
DegP
pH sensitive promoters: cpxP
cpxP
CpxP
CpxA
CpxR
At high pH:
1. DegP breaks down cpxP
2. CpxA (no longer inhibited by
cpxP) phosphorylates CpxR
DegP
pH sensitive promoters: cpxP
cpxP
CpxP
CpxA
CpxR
At high pH:
1. DegP breaks down cpxP
2. CpxA (no longer inhibited by
cpxP) phosphorylates CpxR
3. CpxR activates cpxP (as well as
DegP)
DegP
ScpxP
LcpxP
Assembling Promoters: Colony PCR
Forward Primer
5'-GCATGAATTCGCGGCCGCTTCTAGAGCTCAAGGCCGAGAACGCGATCAAGTTCACTGCCGCGCGCCGTCAACATAATGACAGGCGTCTGGTGTGTCTGGCGAAGTGCTTTTAATGT
GTCGATACCATTTTTCTTCGGCATCATTACGTCAAGCAAAAGTAAATCAATGCTGTCGTCCAGAAGATCAAGCGCCTGTTCCCCATCGTGGG
CAACAATCACGTTGAAGCCTTCCATCTCGAGCAGCTCCTTTAATAGGGAAGTCAGCTCTCGGTCATCATCAACTAACAGGATTTTATTCATTG
TTTAAATACCTCCGAGGCAGAAATTACGTCATCAGACGTCGCTAATCCATGACTTTACGTTGTTTTACACCCCCTGACGCATGTTTGCAGCCT
GAATCGTAAACTCTCTATCGTTGA-3'
3‘GAGTTCCGGCTCTTGCGCTAGTTCAAGTGACGGCGCGCGGCAGTTGTATTACTGTCCGCAGACCACACAGACCGCTTCACGAAAATTACAC
AGCTATGGTAAAAAGAAGCCGTAGTAATGCAGTTCGTTTTCATTTAGTTACGACAGCAGGTCTTCTAGTTCGCGGACAAGGGGTAGCACCC
GTTGTTAGTGCAACTTCGGAAGGTAGAGCTCGTCGAGGAAATTATCCCTTCAGTCGAGAGCCAGTAGTAGTTGATTGTCCTAAAATAAGTA
ACAAATTTATGGAGGCTCCGTCTTTAATGCAGTAGTCTGCAGCGATTAGGTACTGAAATGCAACAAAATGTGGGGGACTGCGTACAAACGT
CGGACTTAGCATTTGAGAGATAGCAACT-ATGATCATCGCCGGCGACGTCTACG-'5
Reverse Primer
Forward Primer
Reverse Primer
Forward Primer
Promoter sequence
Reverse Primer
Colony PCR
PCR (template DNA=colony PCR products)
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Promoter
Ligation into Plasmid
XbaI
EcoRI
SpeI
Superfolder GFP with
LVA (J119041)
pSB1A8
PstI
EcoRI
PstI
XbaI
SpeI
XbaI
EcoRI
SpeI
Superfolder GFP with
LVA (J119041)
pSB1A8
PstI
Success!
EcoRI XbaI
SpeIPstI
Superfolder GFP with
LVA (J119041)
XbaI
SpeI
pSB1A8
Testing Promoters
RBS
GFP
cadA (J100071)
GFP Expression of J100081 at Various pH
35000
30000
Fluorescence/cell (RFU)
25000
20000
15000
10000
5000
0
pH 5.8
pH 6.5
pH 6.9
pH 7.5
pH
pH 8
pH 8.5
LcpxP (J100072)
40000
35000
Level of GFP Expression of J100082 at Various
pH
FLuorescence/cell (RFU)
30000
25000
20000
15000
10000
5000
0
pH 5.8
pH 6.5
pH 6.9
pH 7.5
pH
pH 8.0
pH 8.5
ScpxP (J100073)
GFP Expression of J100087 at Various pH (90
gain)
80000
70000
Fluorescence/cell (RFU)
60000
50000
40000
30000
20000
10000
0
pH 5.8
pH 6.5
pH 6.9
pH 7.5
pH
pH 8
pH 8.5
Linking pH and Light
LRE
J100088: cadA promoter
J100089: LcpxP promoter
10000.0
9000.0
8000.0
7000.0
6000.0
5000.0
4000.0
3000.0
2000.0
1000.0
0.0
J100088 pH 5.8 J100088 pH 6.5 J100088 pH 6.9 J100088 pH 7.5 J100088 pH 8.0 J100088 pH 8.5
negative
control
pBad E. glowi
Part, pH
Luminescence of E. coli with J100089 at Various pH
20000.0
18000.0
Luminescence/cell (RLU)
Luminescence/cell (RLU)
Luminescence of E. coli with J100088 at Various pH
16000.0
14000.0
12000.0
10000.0
8000.0
6000.0
4000.0
2000.0
0.0
J100089 pH 5.8J100089 pH 6.5J100089 pH 6.9J100089 pH 7.5J100089 pH 8.0J100089 pH 8.5
Part, pH
negative
control
pBad E. glowi
1600
1400
1200
1000
800
600
400
200
0
Luciferin added at t=0
Luciferin added at t=1 hr.
Luciferin added at t=2 hrs.
Luciferin added at t=4 hrs.
Negative control (no luciferin added)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Time of reading (hours)
Luminescence of cells With J100089 and Luciferin at Different
Time Points (Plate 1)
2500.0
Luminescence/cell (RLU)
Luminescence/cell (RLU)
Luminescence of cells with J100088 and Luciferin at Different
Time Points (Plate 1)
2000.0
Luciferin added at t=0
1500.0
Luciferin added at t=1 hr.
1000.0
Luciferin added at t=2 hrs.
Luciferin added at t=4 hrs.
500.0
Negative control (no luciferin added)
0.0
0
0.5
1
1.5
2
2.5
Time of reading (hours)
3
3.5
4
4.5
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