HIT-Harbin iGEM 2011

Yogurts under normal temperature

………… A few days later

It is too acid

Why yogurt

Lactobacillus bulgaricus

Streptococcus thermophiles

Why yogurt

Yogurt is rich in nutrition.

More importantly, consumers who are moderately lactose-intolerant can consume yoghurt without unfavorable symptoms, especially for Chinese people.

Why yogurt

The distribution of people who are lactose-intolerant.

1.Post-acidification

2.Collagen

3.Food-grade vectors

4.Tests & results

1.Post-acidification

1. Post-acidification

Especially L. bulgaricus

1. Post-acidification

pH sensor

Cut off the pathway for lactic acid

1. Post-acidification

Some genes must be up-regulated or down-regulated in order to adapt to the acid environment.

Possible acid tolerance mechanisms of lactic acid bacteria.

rcfB promoter

H+

H+ H+

H+

H+

H+

H+ H+

pH=5.5 Up-regulate

In Lactococcus lactis IL1403

1. Post-acidification

1. Post-acidification

The metabolic pathway of lactose

1. Post-acidification

The lac operon in L. delbrueckii subsp. lactis

lacA OP

lacS lacZ lacR

lactose permease

Β-galactosidase

repressor

1. Post-acidification

lacA OP

lacS lacZ lacR

the lacR gene was inactivated by small nucleotides insertions and deletions in the sequence

The lac operon in L. bulgaricus

1. Post-acidification

H+

H+

H+

rcfB promoter

H+

H+ H+

H+

lacR RBS

lacA OP

LacR

T

lacS lacZ lacR

LacR

LacR

H+

H+ H+

H+

2.Collagen

2. Collagen

…………

2. Collagen

Zhou Qian, et al. Modern Food Science and Technology, 2008, 24(3):285-287.

2. Collagen

The parts for producing collagen

His T RBS blpC

p32 premotor

COLⅢα1

COLⅢα1 is part of the α1 chain of human collagen type Ⅲ.

2. Collagen

The sequence of collagen had been optimized for an efficient expression in S. thermophilus.

COLⅢα1 BlpC

COLⅢα1

BlpC is the signal peptide

COLⅢα1 BlpC

COLⅢα1

COLⅢα1

COLⅢα1

2. Collagen

BlpC

3.Food-grade vectors

3. Food-grade vector

The E.coli-Lactococcus lactis shuttle plasmid pMG36e

3. Food-grade vector

Change Emr to NisI

3. Food-grade vector

A long history of safe use in food production

Structure of Nisin

4.Tests & results

4. Tests & results

rcfB promoter

lacR RBS T

His T RBS blpC

p32 premotor

COLⅢα1

Our four biobricks.

E X S P

S P E X

nisI T RBS

nisI T RBS blpC

nisI for L. bulgaricus ——nisI(Ⅰ)

nisI for S.thermophilus——nisI (Ⅱ)

4. Tests & results

E X S P

E X S P

peptide

4. Tests & results

Ligation

rcfB promoter

lacR RBS T

S P

nisI T RBS

E X

peptide

S/X

His T RBS blpC

p32 premotor

COLⅢα1 nisI T RBS blpC

E X S P S/X

4. Tests & results

Ligate with the pMG36e

Ligate with the pMG36e

4. Tests & results

4. Tests & results

The two recombinant plasmids are transformd to L.bulgaricus and S.thermophiles separately by electroporation.

Further experiments are under way.

More needs to be done ……

• Whether the nisI genes work

• Knock out the erythromycin resistance gene

• Whether the collagen peptide is expressed

• Whether the pH sensor works

Acknowledgment:

Prof. Dechang Xu

Dr. Cuihong Dai

Prof. Steve Zeng

Sponsor:

Lec. Xue Han