Cling- E. coli : Bacteria on target
-
Upload
constance-mosley -
Category
Documents
-
view
27 -
download
0
description
Transcript of Cling- E. coli : Bacteria on target
![Page 1: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/1.jpg)
Cling-E. coli :Bacteria on target
Harvard iGEM 2007Ellenor BrownStephanie LoAlex PickettSammy Sambu
Kevin SheePerry TsaiShaunak VankudreGeorge Xu
![Page 2: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/2.jpg)
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
![Page 3: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/3.jpg)
The vision:Bacterial targeting
via membrane display
![Page 4: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/4.jpg)
The vision:Inter-cellular activationvia Lux quorum-sensing
![Page 5: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/5.jpg)
The vision:Intra-cellular activation
via Fec signal transduction
![Page 6: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/6.jpg)
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
![Page 7: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/7.jpg)
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
![Page 8: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/8.jpg)
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
![Page 9: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/9.jpg)
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
![Page 10: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/10.jpg)
After magneticselection
Direct Selection using Magnetic Beads
![Page 11: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/11.jpg)
Direct Selection using Magnetic Beads
![Page 12: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/12.jpg)
Fusion at C terminus: Bead Assay (His tag)
Beads
Pre-assay plates
![Page 13: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/13.jpg)
Loop Insertion
• PCR product digestion & ligation – Primer design– Digest-Ligate-Transform motif
• Gene design– Insertion points created for inserting synthetic
constructs
![Page 14: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/14.jpg)
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
![Page 15: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/15.jpg)
PCR productsLane1: Ladder
Lane2: 1st portion OmpA
Lane 3: strep2-OmpA portion 2
Lane 4: 6xHis-OmpA portion 2
![Page 16: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/16.jpg)
PCR: final plasmid as a template
Red line indicates the 1 kb band
![Page 17: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/17.jpg)
Gene Design
E PX SN
![Page 18: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/18.jpg)
Gene Design: Operations
N P
X PM
M
![Page 19: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/19.jpg)
Gene Design: Operations
N NX S
M
M
M
M
His/strep tags OR randomers
![Page 20: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/20.jpg)
PCR Results
Red line indicates the 1 kb line (7/8)
![Page 21: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/21.jpg)
Cell-Cell Signalling
Receiver
Sender
R
OHHL
Target(bead)
Reporter
luxI/luxR Quorum Sensing
+
![Page 22: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/22.jpg)
Cell-Cell Signaling:Constructs
SenderReceiver
Single Cell Construct – “JT”
Two Cell Construct
ReceiverSender
![Page 23: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/23.jpg)
Sharp increase in fluorescence indicates quorum activity
Amount of sender cells added
Fluorescence per cell
![Page 24: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/24.jpg)
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
![Page 26: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/26.jpg)
Plate Drop Experiment with Enriched Sender
![Page 27: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/27.jpg)
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.
![Page 28: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/28.jpg)
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.
![Page 29: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/29.jpg)
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
![Page 30: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/30.jpg)
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
![Page 31: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/31.jpg)
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
![Page 32: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/32.jpg)
• 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
![Page 33: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/33.jpg)
CONCLUSION
• To be added
![Page 34: Cling- E. coli : Bacteria on target](https://reader035.fdocuments.us/reader035/viewer/2022070400/568134ac550346895d9bc012/html5/thumbnails/34.jpg)
ACKNOWLEDGEMENTS
• Thank you.