E. capsi Reducing phosphate pollution using engineered E. coli that - - PowerPoint PPT Presentation

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E. capsi Reducing phosphate pollution using engineered E. coli that - - PowerPoint PPT Presentation

Nov 08, 2022 342 likes •996 views

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

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  • E. capsi

Reducing 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
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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 billion Chinese 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

  • r

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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↑PO4

3- uptake

↑Poly-P

→

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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

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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

  • f

Poly-P Synthesis?

Bacterial Microcompartment

PPK1

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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?

eric

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

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Localize PPK1 into 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

Brainstorming

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Originally present in Eut MCP

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Strategy

  • E. Capsi

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Strategy – E. capsi

  • E. Capsi Model

MCP PPK PO4

3-

PO4

3-

PO4

3-

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 PstI

Tannerella forsythia ATCC 43037 Kingella oralis ATCC 51147 Escherichia coli K12 1 Helicobacter pylori J99 1 Mycobacterium smegmatis mc2155 1 1 Mycobacterium tuberculosis CDC1551 1 Neisseria lactamica ATCC 23970 1 Neisseria mucosa ATCC 25996 1 Porphyromonas gingivalis ATCC 33277 2 Porphyromonas gingivalis W83 2 Selenomonas sputigena ATCC 35185 2 Veillonella parvula ATCC 17745 1 1 Vibrio cholerae N16961 1 Vibrio parahaemolyticus 1 2 Zymomonas mobilis ATCC 29191 1

Organism EcoRI XbaI SpeI PstI

Tannerella forsythia ATCC 43037 Kingella oralis ATCC 51147 Escherichia coli K12 1 Helicobacter pylori J99 1 Mycobacterium smegmatis mc2155 1 1 Mycobacterium tuberculosis CDC1551 1 Neisseria lactamica ATCC 23970 1 Neisseria mucosa ATCC 25996 1 Porphyromonas gingivalis ATCC 33277 2 Porphyromonas gingivalis W83 2 Selenomonas sputigena ATCC 35185 2 Veillonella parvula ATCC 17745 1 1 Vibrio cholerae N16961 1 Vibrio parahaemolyticus 1 2 Zymomonas mobilis ATCC 29191 1

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Achievements

PPK1 Constructs

Kingella oralis (BBa_K1217003) Tanneralla forsythia (BBa_K1217000)

Kingella oralis Tanneralla forsythia

ppk1

Sequence 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

Expression

EutS = 11 kDa

Achievements

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PPK1 BBa_K1217003

PPK1 in native MCP

BBa_K1217008

PPK1 in simple MCP

BBa_K1217010

PPK1 BBa_K1217003

PPK1 in native MCP

BBa_K1217008

PPK1 in simple MCP

BBa_K1217010

Characterization 3 functional constructs

Achievements

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  • 1. Poly-P synthesis
  • 2. Phosphate uptake

medium phosphate level intracellular poly-P level

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Characterization

Procedure

Induce Expression Time: 0 hr Time: 3 hr Time: 6 hr PPK1 Wild type PPK1 in native MCP PPK1 in simple MCP blank

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

500 1000 1500 5 10 15 20

P45 (ng) A550nm

y = 0.0178x R2 = 0.994

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Characterization intracellular poly-P level

All 3 constructs have higher poly-P amount accumulated.

Achievements

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Poly-P synthesis

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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

PO4

3- standard

0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.1 0.2 0.3 0.4

PO4

3- (nmol)

A620nm

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Characterization Medium phosphate level

Achievements

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Medium phosphate level

2 4 6 8 2.0 2.2 2.4 2.6

Wild Type PPK1 PPK1 in Native MCP Medium Time (hr) phosphate (mM)

Basal PO4 uptake More PO4 removed!

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Quick Summary

Polyphosphate synthesis

PPK1 BBa_K1217003

PPK1 in native MCP

BBa_K1217008

PPK1 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!

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Human Practice !

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Nature Medicine. (2013). 19 (9).

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List List of

  • f Act

Activities ivities!

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

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Guest Guest Sp Speak eaker er in in Ca Captur pture e Sc Science ience Video Competition ideo Competition 201 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

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24th Aug 2013 HKU campus

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Talk alk to to Bi Bioc

  • chemist

hemist Fresh eshman man – Futu Future play e player ers in s in iGEM iGEM !

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Talk alk to to Bi Bioc

  • chemist

hemist Fresh eshman man – Futu Future play e player ers in s in iGEM ! 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

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5th- 8th Aug 2013 National Chiao Tung University, Taiwan

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Sharing Sharing with other with other Chin Chinese ese iGE iGEM Teams eams

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Sharing Sharing with other with other Chin Chinese ese iGE iGEM Teams eams

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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

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Pr Promot

  • motional V

ional Video ideo

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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 Our Jour

  • urney

ney thr through

  • ugh

From May to Now!

  • E. capsi

BBa_K1217003 BBa_K1217008 BBa_K1217010

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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

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Thank you!

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Questions?

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Future Directions

A promising metabolic engineering platform

MCP

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Future Directions

MCP

Exploited as a different tool? A Delivery vesicle?

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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

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EutS monomer is suitable for tagging at its N terminus

MPC as a target specific delivery system

Future Directions

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EutS monomer is suitable for tagging at its N terminus

MPC as a target specific delivery system

Future Directions

Interiror surface Cytosol

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  • 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

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  • 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)

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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 phosphate to 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]

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Modelling Description