E. coslim

WHU-China 2012 WHU-China 2012

Synthetic probiotics to control obesity

WHU-China 2012

Obesity

Why are you still

working?

Do you want to try it?

No,

Thanks.

We can help you !

How can you help me lose weight?

We can function in

your intestine In this way.

I can degrade extra fatty acid in your intestine.

I will turn glucose to cellulose

I am a tough guy in human intestine

How to sense fatty acid and glucose?

We have promoters ！

Outline

• Introduction

• Sensing

• Device I: Fatty Acids Degradation

• Device II: Cellulose synthesis

• Device III: Colonization

• Human Gut Microbiota Modeling

• Safety

• Human Practice

Sense Glucose and Fatty Acid

Low glucose concentration

Direct Regulatory Pathway

cAMP

CRP

cAMP-CRP

ATP

Direct Regulatory Pathway

Low glucose concentration

Direct Regulatory Pathway

cAMP-CRP Transcription off

High glucose concentration

Transcription On

Direct Regulatory Pathway

CRP without cAMP

Direct Regulatory Pathway

Fluorescent measurement

Fluorescence Image

Direct Regulatory Pathway

Single cell recognition: FANCY

Indirect Regulatory Pathway

A B

Intermediate

protein

activate

PfadR and PxylR

How to degrade fatty acid?

Quite easy!

Outline

• Introduction

• Sensing

• Device I: Fatty Acids Degradation

• Device II: Cellulose synthesis

• Device III: Colonization

• Human Gut Microbiota Modeling

• Safety

• Human Practice

Device I: Fatty Acid Degradation

--- Modified from Cho et al. Microbiology. 2006

32

Genes from E. coli and S. enterica were used

Placed downstream PfadR

Device I: Fatty Acid Degradation

33

Cupric Acetate Pyridine Method

--- A set of reaction

34

In Vivo Experiment

Overexpression of FadE and FadD had no significant changes

Overexpression of FadL showed about 1.3 degradation of fatty acid

In Vitro Experiment

How to convert glucose into cellulose?

A piece of cake!

Outline

• Introduction

• Sensing

• Device I: Fatty Acids Degradation

• Device II: Cellulose synthesis

• Device III: Colonization

• Human Gut Microbiota Modeling

• Safety

• Human Practice

39

Device II: Cellulose Synthesis

AdrA was taken into

consideration

Genes from E.coli

Placed downstream Pcar

40

Total Reducing Sugar Measurement

---Color change after the treatment of cellulase

Overexpression

Cooverexpression

Don’t forget

about me!

How to colonize in the intestine?

Let me show you!

Outline

• Introduction

• Sensing

• Device I: Fatty Acids Degradation

• Device II: Cellulose synthesis

• Device III: Colonization

• Human Gut Microbiota Modeling

• Safety

• Human Practice

Device III: Colonization

A magic molecule: c-di-GMP

Modified from Hengge, Nature review Microbiology, 2009

48

Semisolid Plate

Can you truly function in intestine?

51

Don’t worry !

This is E. coslim

Outline

• Introduction

• Sensing

• Device I: Fatty Acids Degradation

• Device II: Cellulose synthesis

• Device III: Colonization

• Human Gut Microbiota Modeling

• Safety

• Human Practice

Human Gut Microbiota

E.coslim

Human Gut

Microbiota

Huge quantity, diversity, complexity

Related to life activities Digestion, immunity, mental health

Forgotten organ

Gut microbiota is related to obesity

·Two major phyla of microbiota:

Firmicutes and Bacteriodetes

·Their proportion is related to

obesity.

·Firmicutes to Bacteriodetes:

High Obesity

Low Normal

Regulate the microbiota to make you slim!

Modeling on microbiota regulation

Simulation result in

obese people before

the colonization of

E.coslim

After colonization: different situations

Microbiota die out Fail to colonize

No significant change Colonization and regulation

Microbe proportion

Proportion of Firmicutes

decreases while that of

Bacteriodetes rises up

significantly after colonization

of E.coslim.

Is E.coslim safe?

Outline

• Introduction

• Sensing

• Device I: Fatty Acids Degradation

• Device II: Cellulose synthesis

• Device III: Colonization

• Human Gut Microbiota Modeling

• Safety

• Human Practice

Future Experiments to insure security

Future Experiments to insure security

Further security: Death Device

Prevent horizontal gene transfer

and overgrowth.

64

Double capsules design

Will people accept E.coslim if I want to promote it to the

public?

Outline

• Introduction

• Sensing

• Device I: Fatty Acids Degradation

• Device II: Cellulose synthesis

• Device III: Colonization

• Human Gut Microbiota Modeling

• Safety

• Human Practice

Survey

The majority of participants accepted bacterial

product.

Survey

Most of the students hold negative opinions toward

Genetically Modified Organisms (GMOs).

harmful safe

Survey

The public did not know why GMOs are potentially harmful.

59.29%

Publicizing

Achievements

• Two synthetic promoters PfadR and Pcar

• In vivo and in vitro experiments on fatty

acid degradation

• Cloning and testing the function of

cellulose synthetase genes

• Modeling on gut microbiota

• An open software Fancy

• 66 new biobrick parts

Acknowledgement

• We thank Professor Jianbo Wang, and Zhixiong Xie for leading us

• We thank Dr. Xiangdong Gao, Mengxiang Sun, Ling Zheng, Xiaofeng Tang, Tiangang Liu, Yulan Wang, Lijia Li, Hongbin Shu, Xiaodong Zhang, Wentao Ma, Lin Guo, Daisuke Kiga and John Cronan for advices and kind instruction

• We thank Dundee iGEM team and Tokyo tech iGEM team for providing and preparing us cDNA and plasmids

Acknowledgement

• We specially thank Professor Yan Zhou, Yan Wu and Xiangdong Chen for their kindness to provide research equipments and reagents.

• We thank Yan Long, Jing Che, and Xiaoyan Li for ordering reagents and equipment.

• We thank former WHU iGEMer Chao Zhang, Jing You, Zurong Wan, Fei Shu, Meng Zhang, Yun Yan for their advices and supports.

Thank you!

WHU- China iGEM 2012