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

Titanium Binding Peptide

Duke 2006

Antibody Display

Harvard 2006

Antibody display

Warsaw 2008

“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

Basic Parts Liquid Handling Robot IDT 96 well Plate IDT 96 Order Form

• ligoDesigner

UC
Berkeley
iGEM
2009
 Automated
Assembly
 UC
Berkeley
iGEM
2009


Basic Part

Specialized Assembly Vector Entry Vector Composite Part Clotho

Assembly Tree Specialized Assembly Vector CSV (Robot Instructions) Liquid Handling Robot Goal Parts

UC
Berkeley
iGEM
2009
 Success
Rates
 Success
Rates:



<7%
gave
no
colonies
 
12‐15%
colonies
are
co‐transformed
 
~10%
sequenced:

90%
were
correct


We
made
>800
parts!


UC
Berkeley
iGEM
2009
 Cell
Surface
Display
Constructs


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

UC
Berkeley
iGEM
2009
 Passenger:
Ag4
pepQde


Results:



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


negative control (DH10B) Ag4 peptide + espP(beta) Reddish color indicates the reduction of AgNO3 to Ag Ag Functional Assay: AgNO3 reduction

incubate
 AgNO3

Passenger:
anQ‐type
III
Needle
scFv


Results:



• Displayers
only:
0
out
of
14
hits
(0%)

• Displayers
+
Needle
scFv:
3
out
of
14


hits
(21%)
 Dot
blot
for
Needle
 protein


wash
 lyse
 CPG_L2 espP(beta) AIDA-1 Negative Control (1363) Functional Assay: dot blot

UC
Berkeley
iGEM
2009


Results:


• Displayer
+
leucine
zippers:
21
out
of


28
hits
(75%)


• Homodimers
and
heterodimers
both


form
 FuncOonal
Assay:
flocculaOon
in
soluOon


EILD KILR EILD/KILR

Passenger:
Leucine
zippers
 UC
Berkeley
iGEM
2009


Results:


• Only
Cel5b
showed
cellulase
acQvity

• AcQvity
required
only
a
prepro
sequence


Passenger:
Cellulases



Cel5b
+
cl02365
 compared
to
 negaQve
control
 (1363)


~ ~

Add precipitation buffer Soluble low MW Azo CM Insoluble high MW Azo CM

UC
Berkeley
iGEM
2009


FuncOonal
Assay:
Azo
CM
cellulose
degradaOon


Displayers
with
 INP
spacers
 NegaQve
 PosiQve
(CPX)
 AIDA‐1
AtD
 Hia
AtD
 Hag
AtD
 OprF
AtD
 VtaA11
AtD
 ehaB
 Pcryo_1225
AtD
 cl02365
AtD


Results:


• Displayer
+
Strep
tag:
4
hits
out
of


14
constructs
(29%)


• Displayer
+
INP
spacers
+
Strep
tag:


9
hits
out
of
14
constructs
(64%)
 FuncOonal
Assay:
streptavidin
binding


Incubate
cells
 with
PE‐ streptavidin
 Pellet
cells
 Remove
 unbound
PE‐ streptavidin,
 wash


Passenger:
Strep
tag
 UC
Berkeley
iGEM
2009


Results:


• Displayer
+
Mgfp‐5:
7
hits
out
of
14


constructs
(50%)


• Displayer
+
Spacer
+
Mgfp‐5:
70


hits
out
of
71
constructs
(99%)


Passenger:
Mussel
Adhesive
Protein
Mgfp‐5


(+) (-)

espP(beta) + INP spacers + mgfp5 espP(beta) + mgfp5

(-)

espP(beta) AIDA-1 AtD + Beta Roll + mgfp5 AIDA-1 AtD + mgfp5

(+) (+)

AIDA-1 AtD

(-) Remove
 Supernatant
 Lyse


FuncOonal
Assay:
BCA
total
protein
assay


Add
BCA
 reagents


UC
Berkeley
iGEM
2009


The
Matrix
 UC
Berkeley
iGEM
2009


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

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


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


Acknowledgements
 UC
Berkeley
iGEM
2009