e. capsi - international genetically engineered...
TRANSCRIPT
![Page 1: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/1.jpg)
E. capsiReducing phosphate pollution using
engineered E. coli
that harvests polyphosphate
1
![Page 2: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/2.jpg)
2
1. Our Focus : Phosphate Pollution
2. Our Strategy : E. capsi
3. Our Achievement : Results and Future
4. Our General Public : Human Practice
![Page 3: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/3.jpg)
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”
3
![Page 4: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/4.jpg)
Chemical Precipitation
Enhanced Biological
Phosphorous Removal
(EBPR) Process
Phosphate Removal
Ca2+
Al3+
Fe3+
Phosphate accumulating organisms
PAOs
4
![Page 5: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/5.jpg)
Chemical precipitation | EBPR process
PAOs: Phosphate accumulating organisms
5
![Page 6: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/6.jpg)
How to enhance
phosphate uptake efficiencies?
Synthetic Biology
Natural existing PAOs
or Engineer new microbes ?
6
![Page 7: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/7.jpg)
Brainstorming
Overview of Phosphate Uptake and Poly-P Synthesis
P transport system
Poly-P Synthesis
7
↑PO43- uptake↑Poly-P →
![Page 8: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/8.jpg)
How to increase Poly-P Synthesis?
Brainstorming
8
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
![Page 9: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/9.jpg)
How to increase Poly-P Synthesis?
Brainstorming
9
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
![Page 10: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/10.jpg)
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
10
![Page 11: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/11.jpg)
General Assembly of MCP
Brainstorming
11
![Page 12: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/12.jpg)
3 Choices of Bacterial MCP
Which One?
Enteric
Brainstorming
12
![Page 13: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/13.jpg)
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
13
No Biobrick
![Page 14: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/14.jpg)
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
14
Originally present in Eut MCP
![Page 15: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/15.jpg)
Strategy
E. Capsi
15
![Page 16: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/16.jpg)
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
16
![Page 17: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/17.jpg)
Achievements
17
![Page 18: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/18.jpg)
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
18
![Page 19: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/19.jpg)
AchievementsPPK1 Constructs
Kingella oralis (BBa_K1217003)
Tanneralla forsythia (BBa_K1217000)
Kingella oralisTanneralla forsythia
ppk1Sequence
Signal
(BBa_K1217004)
(BBa_K1217005)
19
![Page 20: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/20.jpg)
PPK1 Constructs
Expression
K. oralis ~ 77kDa
T. forsythia ~ 78kDa
Soluble Expression
Achievements
20
![Page 21: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/21.jpg)
PPK1 Constructs
Expression
K. oralis PPK1 higher expression
Sequence has no mutations
Chosen for
further development
Achievements
21
![Page 22: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/22.jpg)
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
22
![Page 23: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/23.jpg)
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
23
1. Poly-P synthesis 2. Phosphate uptake
medium phosphate levelintracellular poly-P level
![Page 24: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/24.jpg)
Characterization Procedure
Induce
Expression
Time: 0 hr
Time: 3 hr
Time: 6 hr
PPK1Wild type PPK1
in native MCP
PPK1
in simple MCPblank
Achievements
24
![Page 25: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/25.jpg)
Characterization intracellular poly-P level
Cell pellet
Poly-P extraction
DAPI
fluorescence
measurement
Excitation: 415nm
Emssion: 550nm
Achievements
25
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
![Page 26: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/26.jpg)
Characterization intracellular poly-P level
All 3 constructs have
higher poly-P amount
accumulated.
Achievements
26
Poly-P synthesis
![Page 27: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/27.jpg)
Characterization Phosphate level in the medium
Culture medium
Malachite green
colorimetric assay
Absorbance: 620 nm
y = 0.0075x
R2 = 0.9987
Achievements
27
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
![Page 28: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/28.jpg)
Characterization Medium phosphate level
Achievements
28
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!
![Page 29: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/29.jpg)
Quick Summary
Polyphosphate synthesis
PPK1
BBa_K1217003PPK1 in native MCP
BBa_K1217008PPK1 in simple MCP
BBa_K1217010
Phosphate removal
29
![Page 30: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/30.jpg)
Future Directions
Engineer suitable environmental strains
Apply into waste water treatment plant
30
More Experiments (of course…)
Refine the system design:
e.g. Phosphate-induced PPK1 expression
So stay tuned!
![Page 31: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/31.jpg)
Human Practice !
31
Nature Medicine. (2013). 19 (9).
![Page 32: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/32.jpg)
List of Activities!
Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
32
Human practice
![Page 33: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/33.jpg)
Guest Speaker in Capture Science
Video Competition 2013
10th Aug 2013 HKU campus
33
![Page 34: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/34.jpg)
This is awesome!!!
I have to re-consider the plan for my future
studies.
WOW~ I see
the hope to
improve our
world.
34
![Page 35: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/35.jpg)
35
Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
Human practice
![Page 36: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/36.jpg)
24th Aug 2013 HKU campus36
Talk to Biochemist Freshman –
Future players in iGEM !
![Page 37: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/37.jpg)
37
Talk to Biochemist Freshman –
Future players in iGEM !
![Page 38: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/38.jpg)
38
Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
Human practice
![Page 39: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/39.jpg)
5th- 8th Aug 2013
National ChiaoTung University, Taiwan
39
Sharing with other Chinese
iGEM Teams
![Page 40: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/40.jpg)
Sharing with other Chinese
iGEM Teams
40
![Page 41: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/41.jpg)
41
Human practice
Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
![Page 42: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/42.jpg)
Promotional Video
42
![Page 43: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/43.jpg)
43
Speaker in Capture Science Video Competition 2013
Talk to Biochemist Freshmen
Guest Sharing with other Chinese iGEM Teams
Promotional Video
Human practice
![Page 44: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/44.jpg)
44
Our Journey through
From May to Now!
E. capsiBBa_K1217003
BBa_K1217008
BBa_K1217010
![Page 45: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/45.jpg)
Faculty of Science,
Faculty of Medicine,
Department of Biochemistry
Thanks To
Our fellow teammates, advisors and instructors
Our Sponsors:
45
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
![Page 46: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/46.jpg)
Thank you!
46
![Page 47: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/47.jpg)
Questions?
47
![Page 48: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/48.jpg)
Future Directions
A promising
metabolic engineering platform
MCP
![Page 49: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/49.jpg)
Future Directions
MCP
Exploited as a different tool?
A Delivery vesicle?
![Page 50: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/50.jpg)
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
![Page 51: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/51.jpg)
EutS monomer is suitable for tagging at its N terminus
MPC as a target specific delivery system
Future Directions
![Page 52: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/52.jpg)
EutS monomer is suitable for tagging at its N terminus
MPC as a target specific delivery system
Future Directions
Interiror surface
Cytosol
![Page 53: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/53.jpg)
• 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
![Page 54: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/54.jpg)
• 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)
![Page 55: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/55.jpg)
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
![Page 56: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/56.jpg)
![Page 57: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/57.jpg)
57
PROOF OF PRINCIPLE
Cargo Loading + Surface Tagging
![Page 58: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/58.jpg)
58
From collecting phosphateto collecting something else
![Page 59: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/59.jpg)
59
• Convenient product purification by surface tagging• ↑ Reaction rate by compartmentalization• ↓ Side reactions and thus ↑ yield by controlled
substrate and enzyme entry
![Page 60: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/60.jpg)
60
One of our fantasies that we’re most excited about:
![Page 61: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/61.jpg)
61
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:
![Page 62: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/62.jpg)
62
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]
![Page 63: E. capsi - International Genetically Engineered Machine2013.igem.org/files/presentation/Hong_Kong_HKU.pdf · 2 1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our](https://reader031.vdocuments.site/reader031/viewer/2022022708/5be65e3909d3f2857c8ddd7a/html5/thumbnails/63.jpg)
Modelling Description