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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
Introduction
Harvard iGEM 2007
Potential Targets and Applications
Bind Proteins
Bind Toxins
Bind Viruses
Bind Tissue
Bind Other Cells
Introduction
Harvard iGEM 2007
Bind Surface
Bind DNA
Bacterial targeting
Quorum-sensing
Introduction
Harvard iGEM 2007
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
Bacterial Targeting
Harvard iGEM 2007
Membrane
Protein
Selecting/enriching for surface
engineered bacteria
• Direct Selection
– Direct magnetic beads
• Indirect selection
– MACS
– FACS
Bacterial Targeting
Harvard iGEM 2007
Figure here to illustrate
Direct selection and
indirect selection
Indirect Bead Assays
Cell Sorting with his and
strep2
Direct Bead Assays
Bacterial Targeting
Harvard iGEM 2007
After Separation
<NEW TITLE>
Test: Cell Sorting with
AIDA1 + sender
constructs (with his
and strep2)
Before Separation
his
Bacterial Targeting
Harvard iGEM 2007
Results: (MORE DESCRIPTIVE TITLE HERE)
Initial cultures
Enriched cultures
through selection
Successful selection of E.coli expressing His or Strep2 on surface
Bacterial Targeting
Harvard iGEM 2007
Bacterial targeting
Quorum-sensing
Quorum Sensing
Harvard iGEM 2007
Fec signal transduction
luxI/luxR Quorum Sensing
Reporter
R
Receiver
+
Sender
OHHL
Quorum Sensing
Harvard iGEM 2007
Cell-Cell Signaling Constructs
• Receivers (luxR + Reporter)
Receiver
– GFP Receivers (Bba_T9002)
– mRFP Receivers (Bba_F2620 + Bba_I13507)
– mCherry Receivers (Bba_F2620 + Bba_J06702)
• Senders (bicistronic luxI + Reporter)
– mRFP Sender
• tetR controlled (Bba_S03623 + Bba_I13507)
• lacI controlled (Bba_S03608 + Bba_I13507)
– GFP Sender
• tetR controlled (Bba_S03623 + Bba_E0240)
• lacI controlled (Bba_S03608 + Bba_E0240)
Sender
– mCherry Sender
• tetR controlled (Bba_S03623 + Bba_J06702)
• Single Cell
– Constitutive (Bba_J23039 + Bba_T9002)
– Quorum Controlled (Bba_R0062 + Bba_A340620 + Bba_C0261 + Bba_E0240)
• Construction Intermediates
Quorum Sensing
Harvard iGEM 2007
Switch-like Quorum
Response
Receiver
Sender
Quorum Sensing
Harvard iGEM 2007
Selection with Direct Magnetic
Beads
Control: no selection
Experimental:
Selection with beads
Quorum Sensing
Harvard iGEM 2007
60-fold Enrichment through Direct
Magnetic Beads
Control: no beads
Quorum Sensing
Harvard iGEM 2007
Selection with streptactin beads
The plate-drop experiment
Receiver
Quorum Sensing
Harvard iGEM 2007
Sender
Bacterial targeting
Quorum-sensing
Two Component System
Harvard iGEM 2007
Fec signal transduction
Motivation: Fec System
• Goal: Direct cell signaling
• Method: Re-engineer an existing signaling
pathway
• Fec system:
– well-characterized
– substrate specific
Two Component System
Harvard iGEM 2007
Overview of Fec System
Two Component System
Harvard iGEM 2007
Braun et al. “Gene Regulation by Transmembrane Signaling.”
Biometals 2006 Apr;19(2):103-13.
Fec: Motivation and Methods
• Re-engineer FecA: Mutate
loop 7 and/or loop 8
• Structural Evidence:
– L7 moves up to 11Å,
helix unwinds
– L8 moves up to 15Å
• Assume signaling will
occur with binding.
Two Component System
Harvard iGEM 2007
Loops
7&8
Ferguson AD et al. “Structural Basis of Gating by the Outer Membrane Transporter FecA.
Science 2002 Mar 1: 295(5560) 1715-9
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
Two Component System
Harvard iGEM 2007
Braun et al. “Gene Regulation by Transmembrane Signaling
Biometals 2006 Apr;19(2):103-13.
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)
Two Component System
Harvard iGEM 2007
9:36:00
12:00:00
14:24:00
CONCLUSION
• Targeting: AIDA
• Intercellular signaling: QS
• Intracellular signaling: Fec
Conclusion
Harvard iGEM 2007
Future Directions
• Targeting: AIDA
• Intercellular signaling: QS
• Intracellular signaling: Fec
Conclusion
Harvard iGEM 2007
ACKNOWLEDGEMENTS
Advisors
Teaching Fellows
George Church
Debra Auguste
Jagesh V. Shah
William Shih
Pamela Silver
Alain Viel
Tamara Brenner
Nicholas Guido
Bill Senapedis
Mike Strong
Harris Wang
Conclusion
Harvard iGEM 2007
Funding
HHMI
Harvard Provost
Harvard Life Sciences Division
Harvard School of Engineering and
Applied Sciences
N terminus modification of
AIDA1
Colony count
MACS Results
100
90
80
70
60
50
40
30
20
10
0
white
white
red
red
aida1+strep2
Cell Types
Bacterial Targeting
Harvard iGEM 2007
aida1+his
CSR by gene Design
• Fusion of tags & randomers to
extracellular portion of OmpA loop 1
(loop insertion)
– PCR product insertion (950 bps)
– Insertion of ds oligos
Bacterial Targeting
Harvard iGEM 2007
CSR by gene design
Bacterial Targeting
Harvard iGEM 2007
Bacterial Targeting:
Cell Surface Reengineering (CSR)
• CSR by PCR product digestion & ligation:
– Fusion of peptides to the C terminus of OmpA
– Fusion of peptides to the N terminus of AIDA1
• <OmpA AIDA1 structures>
• Fusion of tags & randomers to extracellular portion
of OmpA loop 1 (loop insertion)
– PCR product insertion (950 bps)
– Insertion of ds oligos
Bacterial Targeting
Harvard iGEM 2007