HIT-Harbin - 2011.igem.org2011.igem.org/files/presentation/HIT-Harbin.pdf · HIT-Harbin iGEM 2011 ....
Transcript of HIT-Harbin - 2011.igem.org2011.igem.org/files/presentation/HIT-Harbin.pdf · HIT-Harbin iGEM 2011 ....
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HIT-Harbin iGEM 2011
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Yogurts under normal temperature
………… A few days later
It is too acid
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Why yogurt
Lactobacillus bulgaricus
Streptococcus thermophiles
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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.
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Why yogurt
The distribution of people who are lactose-intolerant.
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1.Post-acidification
2.Collagen
3.Food-grade vectors
4.Tests & results
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1.Post-acidification
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1. Post-acidification
Especially L. bulgaricus
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1. Post-acidification
pH sensor
Cut off the pathway for lactic acid
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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.
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rcfB promoter
H+
H+ H+
H+
H+
H+
H+ H+
pH=5.5 Up-regulate
In Lactococcus lactis IL1403
1. Post-acidification
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1. Post-acidification
The metabolic pathway of lactose
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1. Post-acidification
The lac operon in L. delbrueckii subsp. lactis
lacA OP
lacS lacZ lacR
lactose permease
Β-galactosidase
repressor
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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
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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+
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2.Collagen
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2. Collagen
…………
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2. Collagen
Zhou Qian, et al. Modern Food Science and Technology, 2008, 24(3):285-287.
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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.
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2. Collagen
The sequence of collagen had been optimized for an efficient expression in S. thermophilus.
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COLⅢα1 BlpC
COLⅢα1
BlpC is the signal peptide
COLⅢα1 BlpC
COLⅢα1
COLⅢα1
COLⅢα1
2. Collagen
BlpC
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3.Food-grade vectors
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3. Food-grade vector
The E.coli-Lactococcus lactis shuttle plasmid pMG36e
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3. Food-grade vector
Change Emr to NisI
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3. Food-grade vector
A long history of safe use in food production
Structure of Nisin
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4.Tests & results
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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
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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
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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
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4. Tests & results
Ligate with the pMG36e
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Ligate with the pMG36e
4. Tests & results
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4. Tests & results
The two recombinant plasmids are transformd to L.bulgaricus and S.thermophiles separately by electroporation.
Further experiments are under way.
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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
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Acknowledgment:
Prof. Dechang Xu
Dr. Cuihong Dai
Prof. Steve Zeng
Sponsor:
Lec. Xue Han
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