Transcript Bacteria on target

Cling-E. coli :
Bacteria on target
Harvard iGEM 2007
Ellenor Brown
Stephanie Lo
Alex Pickett
Sammy Sambu
Kevin Shee
Perry Tsai
Shaunak Vankudre
George Xu
The motivation
To develop a system for directing bacteria
to a target of interest and effecting
downstream activity
• Bacterial targeting is necessary for spatiallyspecific activity in the body or in nature
• Post-targeting activity and transmembrane
signalling are the next step in engineering genetic
circuits that interface extracellular and
intracellular environments
The vision:
Bacterial targeting
via membrane display
The vision:
Inter-cellular activation
via Lux quorum-sensing
The vision:
Intra-cellular activation
via Fec signal transduction
Surface Engineered Bacteria
Engineered to Bind and Signal
OmpA – C terminal insertion
Fusion Protein
OmpA-Loop1 insertion
AIDA-1 – N terminal insertion
FecA – loop insertion
Membrane
Protein
Surface Engineered Bacteria
Engineered to Bind and Signal
Positive Signal
AIDA-1 his
or
AIDA-1 strep2
Background
Sender LuxI RFP
Kan
Co-transform
Amp
Amp and Kan
Surface Engineered Bacteria
Engineered to Bind and Signal
Positive Signal
AIDA-1 his
or
AIDA-1 strep2
Background
Sender LuxI RFP
Kan
Amp and Kan
Amp
Co-transform
signal
2 Methods for selecting/enriching for surface engineered bacteria
Indirect Selection
Direct Selection
MACS Magnetic Beads
And Antibodies
Talon and Streptactin
Magnetic Beads
Streptactin Magnetic beads
-bind strep2 (strep binding peptide)
MACS microbeads
-binds Mouse IgG antibodies
or
Talon Magnetic beads
-bind polyhistidine tag
Y
Y
Anti-Strep2 tag Mouse IgG
antibody
Anti-his tag antibody
Labeled cells
retained
Unlabeled cells
Washed away
Y
Y
Direct Selection using Magnetic Beads
After magnetic
selection
Direct Selection using Magnetic Beads
Fusion at C terminus: Bead Assay (His tag)
Pre-assay plates
Beads
Loop Insertion
• PCR product digestion & ligation
– Primer design
– Digest-Ligate-Transform motif
• Gene design
– Insertion points created for inserting synthetic
constructs
Loop Insertion: PCR products
Pre-loop1 OmpA
E
S
X
P
E
M
OmpA Portion with
Modified loop1
P
Complete
Plasmid
E|P digested
vector
PCR products
Lane1: Ladder
Lane2: 1st portion OmpA
Lane 3: strep2-OmpA portion 2
Lane 4: 6xHis-OmpA portion 2
PCR: final plasmid as a template
Red line indicates the 1 kb band
Gene Design
E X
N
S P
Gene Design: Operations
N
M
X
M
P
P
Gene Design: Operations
His/strep tags OR randomers
X
S
N
M
M
MN
M
PCR Results
Red line indicates the 1 kb line (7/8)
Cell-Cell Signalling
luxI/luxR Quorum Sensing
Reporter
R
Target
(bead)
Receiver
+
Sender
OHHL
Cell-Cell Signaling:
Constructs
Sender
Single Cell Construct – “JT”
Receiver
Two Cell Construct
Receiver
Sender
Sharp increase in fluorescence indicates
quorum activity
Fluorescence per
cell
Amount of sender molecule added
Testing for self-induction:
Fluorescence over OD at various times
3000
0
20
40
60
80
100
120
140
160
180
200
220
240
After Overnight
Fluorescence over OD
2500
2000
1500
1000
500
0
Nondiluted
JT 1in200 dilution
T02 nondilute
Direct Magnetic Beads: Good Enrichment
Plate Drop Experiment with Enriched
Sender
OmpA C-Terminus Library
5uL
50uL
200uL
Vector 2
33
lots
10mer 14
149
lots
15mer 6
275
lots
uL transformant vs. colony count
5uL of ligation reaction
(4500 !! ng DNA) were
transformed
into
chemically competent
BL21 Gold DE3 Cells.
Direct Signaling from the Outer
Membrane: the Fec System
•
Advantages of Direct Signaling from
the Outer Membrane: Substrate
Specificity
•
The FecIRA system is the only wellcharacterized signaling scaffold in
Gram-negative bacteria
•
FecA is an iron transporter and signal
transducer on the outer membrane
of E. Coli K-12
•
When ferric citrate binds, FecA
activates periplasmic FecR, which
then activates the sigma factor FecI,
resulting in gene expression
•
The system is repressed by the Fur
repressor in iron-rich conditions
Braun et al. “Gene Regulation by Transmembrane Signaling
Biometals 2006 Apr;19(2):103-13.
Fec: Motivation and Methods
•
Structural information suggests possibility of
maintaining signaling with changed binding.
– L7 moves up to 11Å, helix unwinds
– L8 moves up to 15Å
•
Select binding targets by inserting random
library, controls known to bind nickel and
streptavidin into loops 7 and 8.
– Even if signaling cannot be maintained,
binding of controls proves that FecA can
be used as scaffold for surface
expression of peptides
•
Computational approach in collaboration
with the lab of Costas Maranas, Penn State
Dept of Chemical Engineering.
Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transport
Science 2002 Mar 1: 295(5560) 1715-9
Results
FecA Induction in Presence of Sodium Citrate in LB
•
•
Wild Type Induction of FecA with
Sodium Citrate and a GFP Reporter
shows approximately 2000 RFU
increase
MACS Results
Results from Nickel and His
Fluorescence Assays
2000
1500
RFUs
•
1000
500
0
0:00:00
2:24:00
4:48:00
7:12:00
Time(mins)
9:36:00
12:00:00
14:24:00
Biobricking the Fec System
Construct Features:
•Swappable FecA - FecA is
flanked by Nhe1 and AflII sites
to allow the easy mutagenesis
and replacement of FecA.
•Variable Promoters - each
component will be on a
separate constitutive promoter.
•The optimization of GFP
expression using promoters of
different strengths is planned.
Biobricking the Fec System
• Mutagenesis of Fec promoter
to weaken gene expression,
providing a range of sensitivity.
•Mutagenesis of the Fec
promoter to remove FUR
repressor binding site, allowing
easier assays.