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

E. capsiReducing phosphate pollution using

engineered E. coli

that harvests polyphosphate

1

2

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”

3

Chemical Precipitation

Enhanced Biological

Phosphorous Removal

(EBPR) Process

Phosphate Removal

Ca2+

Al3+

Fe3+

Phosphate accumulating organisms

PAOs

4

Chemical precipitation | EBPR process

PAOs: Phosphate accumulating organisms

5

How to enhance

phosphate uptake efficiencies?

Synthetic Biology

Natural existing PAOs

or Engineer new microbes ?

6

Brainstorming

Overview of Phosphate Uptake and Poly-P Synthesis

P transport system

Poly-P Synthesis

7

↑PO43- uptake↑Poly-P →

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

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

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

General Assembly of MCP

Brainstorming

11

3 Choices of Bacterial MCP

Which One?

Enteric

Brainstorming

12

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

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

Strategy

E. Capsi

15

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

Achievements

17

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

AchievementsPPK1 Constructs

Kingella oralis (BBa_K1217003)

Tanneralla forsythia (BBa_K1217000)

Kingella oralisTanneralla forsythia

ppk1Sequence

Signal

(BBa_K1217004)

(BBa_K1217005)

19

PPK1 Constructs

Expression

K. oralis ~ 77kDa

T. forsythia ~ 78kDa

Soluble Expression

Achievements

20

PPK1 Constructs

Expression

K. oralis PPK1 higher expression

Sequence has no mutations

Chosen for

further development

Achievements

21

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

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

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

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

Characterization intracellular poly-P level

All 3 constructs have

higher poly-P amount

accumulated.

Achievements

26

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

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

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!

Quick Summary

Polyphosphate synthesis

PPK1

BBa_K1217003PPK1 in native MCP

BBa_K1217008PPK1 in simple MCP

BBa_K1217010

Phosphate removal

29

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!

Human Practice !

31

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

32

Human practice

Guest Speaker in Capture Science

Video Competition 2013

10th Aug 2013 HKU campus

33

This is awesome!!!

I have to re-consider the plan for my future

studies.

WOW~ I see

the hope to

improve our

world.

34

35

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 !

37

Talk to Biochemist Freshman –

Future players in iGEM !

38

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

39

Sharing with other Chinese

iGEM Teams

Sharing with other Chinese

iGEM Teams

40

41

Human practice

Speaker in Capture Science Video Competition 2013

Talk to Biochemist Freshmen

Guest Sharing with other Chinese iGEM Teams

Promotional Video

Promotional Video

42

43

Speaker in Capture Science Video Competition 2013

Talk to Biochemist Freshmen

Guest Sharing with other Chinese iGEM Teams

Promotional Video

Human practice

44

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:

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

Thank you!

46

Questions?

47

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

57

PROOF OF PRINCIPLE

Cargo Loading + Surface Tagging

58

From collecting phosphateto collecting something else

59

• Convenient product purification by surface tagging• ↑ Reaction rate by compartmentalization• ↓ Side reactions and thus ↑ yield by controlled

substrate and enzyme entry

60

One of our fantasies that we’re most excited about:

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:

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]

Modelling Description