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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. - PowerPoint PPT Presentation
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Cling-E. coli :Bacteria on target
Harvard iGEM 2007Ellenor BrownStephanie LoAlex PickettSammy Sambu
Kevin SheePerry TsaiShaunak VankudreGeorge Xu
The motivation
• Bacterial targeting is necessary for spatially-specific 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
To develop a system for directing bacteria to a target of interest and effecting
downstream activity
The vision:Bacterial targeting
via membrane display
The vision:Inter-cellular activationvia Lux quorum-sensing
The vision:Intra-cellular activation
via Fec signal transduction
Surface Engineered BacteriaEngineered to Bind and Signal
Fusion Protein
Membrane Protein
OmpA – C terminal insertion
OmpA-Loop1 insertion
AIDA-1 – N terminal insertion
FecA – loop insertion
AIDA-1 hisorAIDA-1 strep2
Surface Engineered BacteriaEngineered to Bind and Signal
Kan
Sender LuxI RFP
Amp Amp and Kan
Positive Signal Background
Co-transform
AIDA-1 hisorAIDA-1 strep2
Surface Engineered BacteriaEngineered to Bind and Signal
Kan
Sender LuxI RFP
Amp Amp and Kan
Positive Signal Background
Co-transform
signal
Direct Selection Talon and StreptactinMagnetic Beads
Indirect SelectionMACS Magnetic Beads And Antibodies
Streptactin Magnetic beads-bind strep2 (strep binding peptide)
or
Talon Magnetic beads-bind polyhistidine tag
2 Methods for selecting/enriching for surface engineered bacteria
MACS microbeads-binds Mouse IgG antibodies
Y Anti-Strep2 tag Mouse IgG antibody
Anti-his tag antibodyY
YY
YY
Labeled cellsretained
Unlabeled cellsWashed away
After magneticselection
Direct Selection using Magnetic Beads
Direct Selection using Magnetic Beads
Fusion at C terminus: Bead Assay (His tag)
Beads
Pre-assay plates
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
E S
Pre-loop1 OmpA
OmpA Portion with Modified loop1
E P
E|P digested vector
X P
Complete Plasmid
M
PCR productsLane1: 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 PX SN
Gene Design: Operations
N P
X PM
M
Gene Design: Operations
N NX S
M
M
M
M
His/strep tags OR randomers
PCR Results
Red line indicates the 1 kb line (7/8)
Cell-Cell Signalling
Receiver
Sender
R
OHHL
Target(bead)
Reporter
luxI/luxR Quorum Sensing
+
Cell-Cell Signaling:Constructs
SenderReceiver
Single Cell Construct – “JT”
Two Cell Construct
ReceiverSender
Sharp increase in fluorescence indicates quorum activity
Amount of sender cells added
Fluorescence per cell
Testing for self-induction: Fluorescence over OD at various times
0
500
1000
1500
2000
2500
3000
Nondiluted JT 1in200 dilution T02 nondilute
Flu
ore
scen
ce o
ver
OD
0
20
40
60
80
100
120
140
160
180
200
220
240
After Overnight
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 well-characterized 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 Transporter FecA. Science 2002 Mar 1: 295(5560) 1715-9
Results• 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
FecA Induction in Presence of Sodium Citrate in LB
0
500
1000
1500
2000
0:00:00 2:24:00 4:48:00 7:12:00 9:36:00 12:00:00 14:24:00
Time(mins)
RFU
s
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.
Biobricking the Fec System
CONCLUSION
• To be added
ACKNOWLEDGEMENTS
• Thank you.