E. capsi Reducing phosphate pollution using engineered E. coli that harvests polyphosphate 1
1. Our Focus : Phosphate Pollution 2. Our Strategy : E. capsi 3. Our Achievement : Results and Future 4. Our General Public : Human Practice 2
Water Pollution in China • 1/3 of the industrial Phosphate Pollution waste water • 90 percent of household sewage Eutrophication in China is released into rivers and lakes without being Algal Bloom treated. • Accounts for half of the $69 billion “Dead Zone” Chinese economy loses every year. 3
Phosphate Removal Enhanced Biological Chemical Precipitation Phosphorous Removal (EBPR) Process PAOs Ca 2+ Fe 3+ Al 3+ Phosphate accumulating organisms 4
Chemical precipitation | EBPR process PAOs: Phosphate accumulating organisms 5
Synthetic Biology How to enhance phosphate uptake efficiencies? Natural existing PAOs or Engineer new microbes ? 6
Brainstorming Overview of Phosphate Uptake and Poly-P Synthesis 3- uptake ↑ PO 4 → ↑ Poly-P P transport system Poly-P Synthesis 7
Brainstorming How to increase Poly-P Synthesis? 1. Increase Poly-P synthesis rate and effectiveness 2. Decrease Poly-P degradation rate Polyphosphate Kinase 1 (PPK1) (EC 2.7.4.1) Poly-P n + ATP → Poly -P n+1 + ADP Increase PPK1 expression 8
Brainstorming How to increase Poly-P Synthesis? 2. Decrease Poly-P degradation rate “Enemies” against Poly -P Synthesis PPK Exopolyphosphatase PPX Poly-P n Poly-P n+1 (EC 3.6.1.11) Poly-P n + H 2 O → Poly -P n-1 + P i PPX Pi Compartmentalization of Poly-P Synthesis? Bacterial Microcompartment PPK1 Eukaryotic cell 9
Brainstorming A Nano-Bioreactor inside the Bacterial Cell Bacterial Microcompartment MCP Protein-based polyhedral microcompartment • 20-200 nm in diameter • Sequester functionally related enzymes • Regulate substrate, small metabolites access • 10
Brainstorming General Assembly of MCP 11
Brainstorming 3 Choices of Bacterial MCP Which One? eric 12
Brainstorming Eut MCP expression in E.coli • iGEM 2010 University of Minnesota Eut S Eut S, M, N, L, K • Eut Micrompartment genes from Salmonella enterica serovar Typhimurium LT2 • BBa_K311004 Discovered in 2012 Simple MCP Native MCP No Biobrick http://2010.igem.org/Team:Minnesota (2012) Engineered Protein Nano-Compartments for Targeted Enzyme Localization. PLoS ONE 7(3): e33342. doi:10.1371/journal.pone.0033342 13
Brainstorming Localize PPK1 into Eut MCP Originally present in Eut MCP Localization Signal peptide N 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 14
Strategy E. Capsi 15
Strategy – E. capsi E. Capsi Model PO 4 3- PO 4 3- A Sink for PO 4 PPX PPK Poly-P n Poly-P n+1 PO 4 ATP 3- ATP ADP MCP Native MCP/ Simple MCP 16
Achievements 17
Searching Organism Organism EcoRI EcoRI XbaI XbaI SpeI SpeI PstI PstI Tannerella forsythia ATCC 43037 Tannerella forsythia ATCC 43037 0 0 0 0 0 0 0 0 Kingella oralis ATCC 51147 Kingella oralis ATCC 51147 0 0 0 0 0 0 0 0 PPK1 gene Escherichia coli K12 Escherichia coli K12 1 1 0 0 0 0 0 0 Helicobacter pylori J99 Helicobacter pylori J99 1 1 0 0 0 0 0 0 ~ 80 kDa Mycobacterium smegmatis mc2155 Mycobacterium smegmatis mc2155 1 1 0 0 0 0 1 1 ~ 2100 bp Mycobacterium tuberculosis CDC1551 Mycobacterium tuberculosis CDC1551 0 0 0 0 0 0 1 1 Neisseria lactamica ATCC 23970 1 0 0 0 Neisseria lactamica ATCC 23970 1 0 0 0 Neisseria mucosa ATCC 25996 Neisseria mucosa ATCC 25996 1 1 0 0 0 0 0 0 compactible with assembly standard Porphyromonas gingivalis ATCC 33277 Porphyromonas gingivalis ATCC 33277 0 0 0 0 0 0 2 2 Porphyromonas gingivalis W83 0 0 0 2 Porphyromonas gingivalis W83 0 0 0 2 Selenomonas sputigena ATCC 35185 Selenomonas sputigena ATCC 35185 2 2 0 0 0 0 0 0 Avoid Veillonella parvula ATCC 17745 Veillonella parvula ATCC 17745 1 1 1 1 0 0 0 0 EcoRI , XbaI , SpeI , PstI sites Vibrio cholerae N16961 1 0 0 0 Vibrio cholerae N16961 1 0 0 0 Vibrio parahaemolyticus Vibrio parahaemolyticus 1 1 2 2 0 0 0 0 Zymomonas mobilis ATCC 29191 Zymomonas mobilis ATCC 29191 0 0 1 1 0 0 0 0 18
Achievements PPK1 Constructs Tanneralla forsythia Kingella oralis ppk1 Sequence Signal (BBa_K1217004) Kingella oralis (BBa_K1217003) (BBa_K1217005) Tanneralla forsythia (BBa_K1217000) 19
Achievements PPK1 Constructs Expression K. oralis ~ 77kDa T. forsythia ~ 78kDa Soluble Expression 20
Achievements PPK1 Constructs Expression K. oralis PPK1 higher expression Sequence has no mutations Chosen for further development 21
Achievements MCP Constructs Eut S, M, N, L, K Eut S Eut S Discovered in 2012 Native MCP Simple MCP BBa_K1217016 Bba_K311004 EutS = 11 kDa Expression 22
Achievements Characterization 3 functional constructs PPK1 PPK1 in native MCP PPK1 in simple MCP BBa_K1217003 BBa_K1217008 BBa_K1217010 1. Poly-P synthesis 2. Phosphate uptake PPK1 PPK1 in native MCP PPK1 in simple MCP BBa_K1217003 BBa_K1217008 BBa_K1217010 intracellular poly-P level medium phosphate level 23
Achievements Characterization Procedure PPK1 PPK1 Wild type PPK1 blank in simple MCP in native MCP Induce Expression Time: 0 hr Time: 3 hr Time: 6 hr 24
Achievements Characterization intracellular poly-P level Cell pellet PolyP Standard in DAPI Assay 20 Poly-P extraction 15 A 550nm y = 0.0178x 10 DAPI R 2 = 0.994 fluorescence 5 Fluorescent probe measurement interacts with DNA and poly-P 0 0 500 1000 1500 P45 (ng) Excitation: 415nm Emssion: 550nm 25
Achievements Characterization intracellular poly-P level All 3 constructs have higher poly-P amount accumulated. Poly-P synthesis 26
Achievements Characterization Phosphate level in the medium 3- standard PO 4 Culture medium 0.4 malachite Green molybdate 0.3 Malachite green free orthophosphate A 620nm colorimetric assay 0.2 y = 0.0075x R 2 = 0.9987 green complex 0.1 Absorbance: 620 nm 0.0 0.0 0.5 1.0 1.5 2.0 2.5 3- (nmol) PO 4 27
Achievements Characterization Medium phosphate level Medium phosphate level 2.6 Medium phosphate (mM) Wild Type 2.4 PPK1 Basal PO 4 uptake PPK1 in Native MCP 2.2 More PO 4 removed! 2.0 0 2 4 6 8 Time (hr) 28
Quick Summary PPK1 PPK1 in native MCP PPK1 in simple MCP BBa_K1217003 BBa_K1217008 BBa_K1217010 Polyphosphate synthesis Phosphate removal 29
Future Directions So stay tuned! More Experiments (of course…) Refine the system design: e.g. Phosphate-induced PPK1 expression Engineer suitable environmental strains Apply into waste water treatment plant 30
Human Practice ! Nature Medicine. (2013). 19 (9). 31
Human practice List List of of Act Activities ivities! Speaker in Capture Science Video Competition 2013 Talk to Biochemist Freshmen Guest Sharing with other Chinese iGEM Teams Promotional Video 32
Guest Guest Sp Speak eaker er in in Ca Captur pture e Sc Science ience Video Competition ideo Competition 201 2013 10 th Aug 2013 HKU campus 33
WOW~ I see the hope to improve our world. This is awesome!!! I have to re-consider the plan for my future studies . 34
Human practice Speaker in Capture Science Video Competition 2013 Talk to Biochemist Freshmen Guest Sharing with other Chinese iGEM Teams Promotional Video 35
Talk alk to to Bi Bioc ochemist hemist Fresh eshman man – Futu Future play e player ers in s in iGEM iGEM ! 24 th Aug 2013 HKU campus 36
Talk alk to to Bi Bioc ochemist hemist Fresh eshman man – Futu Future play e player ers in s in iGEM ! iGEM ! 37
Human practice Speaker in Capture Science Video Competition 2013 Talk to Biochemist Freshmen Guest Sharing with other Chinese iGEM Teams Promotional Video 38
Sharing with other Sharing with other Chin Chinese ese iGE iGEM Teams eams 5 th - 8 th Aug 2013 National Chiao Tung University, Taiwan 39
Sharing Sharing with other with other Chin Chinese ese iGE iGEM Teams eams 40
Human practice Speaker in Capture Science Video Competition 2013 Talk to Biochemist Freshmen Guest Sharing with other Chinese iGEM Teams Promotional Video 41
Pr Promot omotional V ional Video ideo 42
Human practice Speaker in Capture Science Video Competition 2013 Talk to Biochemist Freshmen Guest Sharing with other Chinese iGEM Teams Promotional Video 43
From May to Now! Our Our Jour ourney ney thr through ough E. capsi BBa_K1217003 BBa_K1217008 BBa_K1217010 44
Thanks To Faculty of Science, Faculty of Medicine, Department of Biochemistry Our fellow teammates, advisors and instructors Wei Ying Yen Dr Chan, Danny Ma Tsz Shan, Shannon Wang Linsheng, Samson Cho Charmaine Sze Man Dr Tanner, Julian Alexander Cheng Man Chung, Calvin Dr Watt, Rory Munro Lai Hei Ming Sy Kwun Hei, Samuel Chan Kwok Hei, Kenny Peng Fengjia, Fall Wong Nok Hei, Mickey Our Sponsors: 45
Thank you! 46
Questions? 47
Future Directions MCP A promising metabolic engineering platform
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