Transcript Sticky Bacteria… Bacteri-adhesive… Cling-E. coli…

Sticky Bacteria…
Bacteri-adhesive…
Cling-E. coli…
A working title
Perry Tsai
June 25, 2007
The goals
Develop a system for random peptide
display on the bacterial surface, in order to
screen for peptides that bind to a target
with high affinity
 Use this system to identify consensus
peptide sequences that bind to targets of
interest

Lpp-OmpA membrane display
Surface peptide
extracellular
Lpp
(lipoprotein)
signal peptide
outer
membrane
periplasm
OmpA
(outer membrane protein A)
transmembrane domains
J. Francisco
C. Earhart
G. Georgiou
1992
The construct
NheI
PstI
LppOmpA1 or 2
in
pET29b+ vector
SpeI
BioBricks
PstI protein fusion
+
Random 30nt
oligo sequences
Digest/
ligation
Random peptide display and
screening
Transformation
+
MACS
(target with
magnetic bead)
Expression
FACS
(target with
fluorescent tag)
Sequence identification and
targeting generalization
… ctg gct ggt ttc …
=
L
A
G
F
Bacterial
targeting
Fluorescent
tagging
Affinity purification
Eukaryotic cell
targeting
Protein targeting
Using known peptide interactions
as positive controls
His6 tag + nickel beads
 Strep2 tag + streptavidin beads
 PDZ-binding tag + GST PDZ domains
 PDZ domains + PDZ-binding tag (GFP)

Digest and gel isolation
NheI
PstI
LppOmpA1 or 2
in
pET29b+ vector
LppOmpA1
NheI/PstI
LppOmpA2
NheI/PstI
Amplifying the random library:
PCR vs extension?
SpeI
PstI
5’ gcggctg ggag actagt nnb nnb nnb nnb nnb nnb nnb nnb nnb nnb gctagc tgataa ctgcag cccg 3’
PCR
Extension
Ligations: 2 hr vs. overnight?
Protein gel of LppOmpA1-His6 and
LppOmpA-Strep2
188kD
62
49
38
LppOmpA1
28
His6
18
14
6
3
-
LppOmpA2
Strep2
+
-
+
His6
Strep2
-
-
+
+
For the next week…
Figure out ways to increase ligation and
transformation efficiency
 Continue brainstorming

 Targets
 Signal

transduction systems
Testing MACS magnetic bead assay using
positive controls
iGEM Mafia


Bill, George, and Nick have
been killed by the Mafia
Perry and Alain were
sniffed out as members of
the Mafia and appropriately
lynched; one still remains.


Kevin was an innocent
man wrongly accused
and executed
Alex is God, and
therefore cannot be killed