JenniferBrophy SusanChen SherineCheung( UCSD ) EliciaFarrar - PowerPoint PPT Presentation

Jennifer
Brophy
 Susan
Chen
 Sherine
Cheung

( UCSD )
 Elicia
Farrar
 (Prairie
View
A&M )
 Gabriela
Guzman
 Patrick
Harrigan
 Tom
Huffaker
( Piedmont
HS )
 Joseph
Silo
( Contra
Costa
CC )
 MaDhew
Walters
 John
Wang


UC
Berkeley
iGEM
2009
 Cell
Surface
Display
in
Past
iGEM
Projects
 Kyoto 2008 Duke 2006 Titanium Binding Peptide Antibody Display Harvard 2006 Warsaw 2008 Antibody display “Bait and Prey” display

UC
Berkeley
iGEM
2009
 Cell
Surface
Display
Device
 Hag: an autotransporter-containing protein

UC
Berkeley
iGEM
2009
 Automated
Basic
Part
Synthesis
 PCR Oligos Basic Parts IDT 96 well Liquid Handling Basic Plate Robot Parts IDT 96 Order oligoDesigner Form

UC
Berkeley
iGEM
2009
 UC
Berkeley
iGEM
2009
 Automated
Assembly
 Basic Part Specialized Composite Part Entry Assembly Vector Vector Goal Parts Specialized Liquid Handling Robot CSV (Robot Instructions) Assembly Clotho Vector Assembly Tree

UC
Berkeley
iGEM
2009
 Success
Rates
 We
made
 >800
 parts!
 Success
Rates:
 
<7%
gave
no
colonies
 
12‐15%
colonies
are
co‐transformed
 
~10%
sequenced:

90%
were
correct


UC
Berkeley
iGEM
2009
 Cell
Surface
Display
Constructs
 azo1653 AtD OprF AtD cl02365 beta helix AG4 VtaA11 beta roll cel3A Hag AtD INP repeats cel5B Pcryo_1225AtD (gly-ser) 14 repeats cel6A Hia AtD GFP-LVA cel9 A upaG_short KILR CPG_L2 EILD CPG_L6 GS5-IILK espP(beta) needle scFv ehaB strep tag eCPX TshA VirG YuaQ AtD AIDA-I AtD Ag43_short

UC
Berkeley
iGEM
2009
 Passenger:
Ag4
pepQde
 Functional Assay: AgNO 3 reduction AgNO 3 incubate
 negative control Ag4 peptide + (DH10B) espP(beta) Ag Reddish color indicates the reduction of AgNO 3 to Ag Results:

 • Displayers+Ag4:
4
out
of
7
hits
(57%)


UC
Berkeley
iGEM
2009
 Passenger:
anQ‐type
III
Needle
scFv
 Functional Assay: dot blot wash
 lyse
 CPG_L2 espP(beta) AIDA-1 Negative Control (1363) Dot
blot
for
Needle
 Results:

 protein
 • Displayers
only:
0
out
of
14
hits
(0%)
 • Displayers
+
Needle
scFv:
3
out
of
14
 hits
(21%)


UC
Berkeley
iGEM
2009
 Passenger:
Leucine
zippers
 FuncOonal
Assay:
flocculaOon
in
soluOon
 EILD KILR EILD/KILR Results:
 • 
Displayer
+
leucine
zippers:
21
out
of
 28
hits
(75%)
 • 
Homodimers
and
heterodimers
both
 form


UC
Berkeley
iGEM
2009
 Passenger:
Cellulases

 FuncOonal
Assay:
Azo
CM
cellulose
degradaOon
 ~ ~ Add precipitation buffer Soluble low MW Azo CM Cel5b
+
cl02365
 Insoluble high MW Azo CM compared
to
 negaQve
control
 Results:
 (1363)
 • Only
Cel5b
showed
cellulase
acQvity
 • AcQvity
required
only
a
prepro
sequence


UC
Berkeley
iGEM
2009
 Passenger:
Strep
tag
 PosiQve
(CPX)
 FuncOonal
Assay:
streptavidin
binding
 NegaQve
 AIDA‐1
AtD
 Hia
AtD
 Hag
AtD
 Incubate
cells
 Pellet
cells
 Remove
 with
PE‐ unbound
PE‐ OprF
AtD
 streptavidin
 streptavidin,
 wash
 VtaA11
AtD
 Results:
 ehaB
 • 
Displayer
+
Strep
tag:
4
hits
out
of
 14
constructs
(29%)
 Pcryo_1225
AtD
 • 
Displayer
+
INP
spacers
+
Strep
tag:
 cl02365
AtD
 9
hits
out
of
14
constructs
(64%)
 Displayers
with
 INP
spacers


UC
Berkeley
iGEM
2009
 Passenger:
Mussel
Adhesive
Protein
Mgfp‐5
 FuncOonal
Assay:
BCA
total
protein
assay
 AIDA-1 AtD + Beta Roll + (+) mgfp5 Remove
 Supernatant
 (+) AIDA-1 AtD + mgfp5 Lyse
 (-) AIDA-1 AtD Add
BCA
 reagents
 espP(beta) + (+) INP spacers + mgfp5 Results:
 espP(beta) + • Displayer
+
Mgfp‐5:
7
hits
out
of
14
 (-) mgfp5 constructs
(50%)
 • 
Displayer
+
Spacer
+
Mgfp‐5:
70
 (-) espP(beta) hits
out
of
71
constructs
(99%)


UC
Berkeley
iGEM
2009
 The
Matrix


UC
Berkeley
iGEM
2009
 The
Math
 Displayer Activity <AIDA-1 AtD> 0.64 <VtaA11 AtD> 0.57 <Hia AtD> 0.47 <Hag AtD> 0.41 <espP(beta)> 0.38 <CPG_L2> 0.36 <azo1653 AtD> 0.36 <upaG_short> 0.34 <yuaQ AtD> 0.31 <eCPX> 0.23 <ehaB> 0.17 <cl02365 AtD> 0.11 <Pcryo_1225 AtD> 0.08 <OprF AtD> 0.05

UC
Berkeley
iGEM
2009
 Conclusions
 • FuncQonal
display
depends
on
the
nature
of
the
individual
systems
 • AutomaQon
facilitates
systemaQc
opQmizaQon
of
display
 • Models
help
establish
preliminary
principles
of
raQonal
design
 • Spacer
elements
tend
to
increase
rate
of
successful
display


UC
Berkeley
iGEM
2009
 Human
pracQces
 • Tested
UCB
sogware
team’s
Clotho
program
 • Gave
the
UCB
sogware
team
a
“boot
camp”
lab
experience
to
beker
 understand
the
needs
of
their
users
 • Ars
syntheQca
syntheQc
biology
tutorial


UC
Berkeley
iGEM
2009
 Acknowledgements