E. capsi Reducing phosphate pollution using engineered E. coli that harvests polyphosphate 1
E. capsiReducing phosphate pollution using
engineered E. coli
that harvests polyphosphate
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1. Our Focus : Phosphate Pollution
2. Our Strategy : E. capsi
3. Our Achievement : Results and Future
4. Our General Public : Human Practice
Water Pollution in China
• 1/3 of the industrial waste water
• 90 percent of household sewage in China is released into rivers and lakes without being treated.
• Accounts for half of the $69 billionChinese economy loses every year.
Phosphate Pollution
Eutrophication
Algal Bloom
“Dead Zone”
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Chemical Precipitation
Enhanced Biological
Phosphorous Removal
(EBPR) Process
Phosphate Removal
Ca2+
Al3+
Fe3+
Phosphate accumulating organisms
PAOs
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Chemical precipitation | EBPR process
PAOs: Phosphate accumulating organisms
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How to enhance
phosphate uptake efficiencies?
Synthetic Biology
Natural existing PAOs
or Engineer new microbes ?
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Brainstorming
Overview of Phosphate Uptake and Poly-P Synthesis
P transport system
Poly-P Synthesis
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↑PO43- uptake↑Poly-P →
How to increase Poly-P Synthesis?
Brainstorming
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1. Increase Poly-P synthesis rate and effectiveness
2. Decrease Poly-P degradation rate
Poly-Pn + ATP → Poly-Pn+1 + ADP
Polyphosphate Kinase 1 (PPK1)
(EC 2.7.4.1)
Increase PPK1 expression
How to increase Poly-P Synthesis?
Brainstorming
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2. Decrease Poly-P degradation rate
“Enemies” against Poly-P Synthesis
Exopolyphosphatase PPX
(EC 3.6.1.11)
Poly-Pn + H2O → Poly-Pn-1 + Pi
Pi
Poly-Pn Poly-Pn+1
PPK
PPX
Eukaryotic cell
Compartmentalization
of
Poly-P Synthesis? BacterialMicrocompartment
PPK1
Bacterial Microcompartment MCP
A Nano-Bioreactor inside the Bacterial Cell
• Protein-based polyhedral microcompartment
• 20-200 nm in diameter
• Sequester functionally related enzymes
• Regulate substrate, small metabolites access
Brainstorming
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General Assembly of MCP
Brainstorming
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3 Choices of Bacterial MCP
Which One?
Enteric
Brainstorming
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Eut MCP expression in E.coli
• iGEM 2010 University of Minnesota
• Eut Micrompartment genes
from Salmonella enterica serovar Typhimurium LT2
• BBa_K311004
http://2010.igem.org/Team:Minnesota
Eut S, M, N, L, K
(2012) Engineered Protein Nano-Compartments for Targeted Enzyme Localization. PLoS ONE 7(3): e33342. doi:10.1371/journal.pone.0033342
Native MCP
Eut S
Discovered in 2012
Simple MCP
Brainstorming
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No Biobrick
Localize PPK1 into Eut MCP
Localization Signal peptideN terminal 19 aa of EutC enzyme:MDQKQIEEIVRSVMASMGQ
(2012) Engineered Protein Nano-Compartments for Targeted Enzyme Localization. PLoS ONE 7(3): e33342. doi:10.1371/journal.pone.0033342
Brainstorming
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Originally present in Eut MCP
Strategy
E. Capsi
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Strategy – E. capsi
E. Capsi Model
MCP
PPK
PO43- PO4
3-
PO43- ATP ATP
Poly-Pn Poly-Pn+1
ADP
PPX
A Sink for PO4
Native MCP/ Simple MCP
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Achievements
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Avoid
EcoRI, XbaI, SpeI, PstI sites
PPK1 gene~ 80 kDa
~ 2100 bp
compactible with assembly standard
Searching
Organism EcoRI XbaI SpeI PstITannerella forsythia ATCC 43037 0 0 0 0
Kingella oralis ATCC 51147 0 0 0 0
Escherichia coli K12 1 0 0 0
Helicobacter pylori J99 1 0 0 0
Mycobacterium smegmatis mc2155 1 0 0 1
Mycobacterium tuberculosis CDC1551 0 0 0 1
Neisseria lactamica ATCC 23970 1 0 0 0
Neisseria mucosa ATCC 25996 1 0 0 0
Porphyromonas gingivalis ATCC 33277 0 0 0 2
Porphyromonas gingivalis W83 0 0 0 2
Selenomonas sputigena ATCC 35185 2 0 0 0
Veillonella parvula ATCC 17745 1 1 0 0
Vibrio cholerae N16961 1 0 0 0
Vibrio parahaemolyticus 1 2 0 0
Zymomonas mobilis ATCC 29191 0 1 0 0
Organism EcoRI XbaI SpeI PstITannerella forsythia ATCC 43037 0 0 0 0
Kingella oralis ATCC 51147 0 0 0 0
Escherichia coli K12 1 0 0 0
Helicobacter pylori J99 1 0 0 0
Mycobacterium smegmatis mc2155 1 0 0 1
Mycobacterium tuberculosis CDC1551 0 0 0 1
Neisseria lactamica ATCC 23970 1 0 0 0
Neisseria mucosa ATCC 25996 1 0 0 0
Porphyromonas gingivalis ATCC 33277 0 0 0 2
Porphyromonas gingivalis W83 0 0 0 2
Selenomonas sputigena ATCC 35185 2 0 0 0
Veillonella parvula ATCC 17745 1 1 0 0
Vibrio cholerae N16961 1 0 0 0
Vibrio parahaemolyticus 1 2 0 0
Zymomonas mobilis ATCC 29191 0 1 0 0
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AchievementsPPK1 Constructs
Kingella oralis (BBa_K1217003)
Tanneralla forsythia (BBa_K1217000)
Kingella oralisTanneralla forsythia
ppk1Sequence
Signal
(BBa_K1217004)
(BBa_K1217005)
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PPK1 Constructs
Expression
K. oralis ~ 77kDa
T. forsythia ~ 78kDa
Soluble Expression
Achievements
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PPK1 Constructs
Expression
K. oralis PPK1 higher expression
Sequence has no mutations
Chosen for
further development
Achievements
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MCP Constructs
Eut S, M, N, L, K
Native MCP
Eut S
Discovered in 2012
Simple MCP
Bba_K311004
Eut S
BBa_K1217016
ExpressionEutS = 11 kDa
Achievements
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PPK1
BBa_K1217003PPK1 in native MCP
BBa_K1217008PPK1 in simple MCP
BBa_K1217010
PPK1
BBa_K1217003PPK1 in native MCP
BBa_K1217008PPK1 in simple MCP
BBa_K1217010
Characterization 3 functional constructsAchievements
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1. Poly-P synthesis 2. Phosphate uptake
medium phosphate levelintracellular poly-P level
Characterization Procedure
Induce
Expression
Time: 0 hr
Time: 3 hr
Time: 6 hr
PPK1Wild type PPK1
in native MCP
PPK1
in simple MCPblank
Achievements
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Characterization intracellular poly-P level
Cell pellet
Poly-P extraction
DAPI
fluorescence
measurement
Excitation: 415nm
Emssion: 550nm
Achievements
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Fluorescent probe
interacts with DNA and poly-P
PolyP Standard in DAPI Assay
0 500 1000 15000
5
10
15
20
P45 (ng)
A5
50
nm
y = 0.0178x
R2 = 0.994
Characterization intracellular poly-P level
All 3 constructs have
higher poly-P amount
accumulated.
Achievements
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Poly-P synthesis
Characterization Phosphate level in the medium
Culture medium
Malachite green
colorimetric assay
Absorbance: 620 nm
y = 0.0075x
R2 = 0.9987
Achievements
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malachite Green
molybdate
free orthophosphate
green complex
PO43- standard
0.0 0.5 1.0 1.5 2.0 2.50.0
0.1
0.2
0.3
0.4
PO43-
(nmol)
A6
20
nm
Characterization Medium phosphate level
Achievements
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Medium phosphate level
0 2 4 6 82.0
2.2
2.4
2.6
Wild Type
PPK1
PPK1 in Native MCP
Medium
Time (hr)
ph
osp
hate
(m
M)
Basal PO4 uptake
More PO4 removed!
Quick Summary
Polyphosphate synthesis
PPK1
BBa_K1217003PPK1 in native MCP
BBa_K1217008PPK1 in simple MCP
BBa_K1217010
Phosphate removal
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Future Directions
Engineer suitable environmental strains
Apply into waste water treatment plant
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More Experiments (of course…)
Refine the system design:
e.g. Phosphate-induced PPK1 expression
So stay tuned!
Human Practice !
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Nature Medicine. (2013). 19 (9).
List of Activities!
Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
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Human practice
Guest Speaker in Capture Science
Video Competition 2013
10th Aug 2013 HKU campus
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This is awesome!!!
I have to re-consider the plan for my future
studies.
WOW~ I see
the hope to
improve our
world.
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Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
Human practice
24th Aug 2013 HKU campus36
Talk to Biochemist Freshman –
Future players in iGEM !
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Talk to Biochemist Freshman –
Future players in iGEM !
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Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
Human practice
5th- 8th Aug 2013
National ChiaoTung University, Taiwan
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Sharing with other Chinese
iGEM Teams
Sharing with other Chinese
iGEM Teams
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Human practice
Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
Promotional Video
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Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
Human practice
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Our Journey through
From May to Now!
E. capsiBBa_K1217003
BBa_K1217008
BBa_K1217010
Faculty of Science,
Faculty of Medicine,
Department of Biochemistry
Thanks To
Our fellow teammates, advisors and instructors
Our Sponsors:
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Wei Ying Yen
Cho Charmaine Sze Man
Cheng Man Chung, Calvin
Chan Kwok Hei, Kenny
Dr Chan, Danny
Dr Tanner, Julian Alexander
Dr Watt, Rory Munro
Ma Tsz Shan, Shannon
Wang Linsheng, Samson
Lai Hei Ming
Sy Kwun Hei, Samuel
Peng Fengjia, Fall
Wong Nok Hei, Mickey
Thank you!
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Questions?
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Future Directions
A promising
metabolic engineering platform
MCP
Future Directions
MCP
Exploited as a different tool?
A Delivery vesicle?
Future Directions
MPC as a target specific delivery system
Protein cage as nano-container:
Viral capsids, ferritins & heat shock proteins
Specific Targeting >> Surface editable
28 nm 12 nm 12 nm
EutS monomer is suitable for tagging at its N terminus
MPC as a target specific delivery system
Future Directions
EutS monomer is suitable for tagging at its N terminus
MPC as a target specific delivery system
Future Directions
Interiror surface
Cytosol
• designed targeting peptide to the EutS N terminus
• presented on the exterior surface of the MCP• pose little steric effect on the MCP assembly
MPC as a target specific delivery system
Future Directions
we hypothesize
• 6 Histidine tag with flexible linker
MPC as a target specific delivery system
Future Directions
As a proof of principle
Eut His-tag S (BBa_K1217015)
Eut His-tag S, MNLK (BBa_K1217015)
MPC as a target specific delivery system
Future Directions
Eut His-tag S (BBa_K1217015)
Eut His-tag S, MNLK (BBa_K1217015)
Easier Purification of MCP
Easier Recovery for Polyphosphate
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PROOF OF PRINCIPLE
Cargo Loading + Surface Tagging
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From collecting phosphateto collecting something else
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• Convenient product purification by surface tagging• ↑ Reaction rate by compartmentalization• ↓ Side reactions and thus ↑ yield by controlled
substrate and enzyme entry
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One of our fantasies that we’re most excited about:
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Cytotoxic agents/Drugs
Surface tag
Surface tag binder
Connector
Target cell
Target cell-specific receptor
Receptor ligand
One of our fantasies that we’re most excited about:
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Cytotoxic agents/Drugs
Surface tag
Surface tag binder
Connector
Target cell
Target cell-specific receptor
Receptor ligand
One of our fantasies that we’re most excited about:
• Targeted drug delivery• Can be further decorated to reduce
immunogenicity/side effects• Cocktail of cytotoxic agents/drugs
possible• Intermediate connector can be switched
and easily synthesized (chemical/biological)
• Many combinations:• [cargo loaded] x [surface tag] x [tag binder] x
[ligand] x [receptor]
Modelling Description