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Team:Todai-Tokyo/Notebook/bread
From 2009.igem.org
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*PCR of gpd1 promoter with Pfu Ultra<BR> | *PCR of gpd1 promoter with Pfu Ultra<BR> | ||
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*PCR of Glu1<BR> | *PCR of Glu1<BR> | ||
*TA cloning of Glu1<BR> | *TA cloning of Glu1<BR> | ||
Revision as of 12:26, 18 October 2009
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Contents |
Plan
Aim:Create yeast that can be used to make sweet and low energy bread
Methods:
1.Clone the glu1 (glucoamylase) from Saccharomycopsis fibuligera and insert it in the yeast chromosome by homologous recombination.
2-a Clone mtlD (mannitol synthase) from E.coli and insert it in the yeast chromosome by homologous recombination
→Replace gpd1 gene by mtlD and gpd2 gene by glu1
2-b Clone xylose isomerase gene and D-tagatose 3-epimerase gene from Mesorhizobium loti.
→Replace gpd1 gene by D-tagatose 3-epimerase gene and gpd2 gene by glu1. Transform a plasmid coding xylose isomerase gene.
~9/20
- PCR of mtlD
- PCR of gpd1 promoter
- TA cloning of mtlD
9/21
- Miniprep of mtlD
9/22
- read the sequence of mtlD→successful
- mtlD primers with HAtag come
9/25
- PCR of mtlD with new primer→failed
9/26
- PCR of mtlD with new primer→successful
9/27
- PCR of gpd1 promoter with Pfu Ultra
10/1~13
- PCR of Glu1
- TA cloning of Glu1
- PCR of gpd1 promoter with ExTaq
10/14
- cut mtlD and plate1 7D both by EcoRI and PstI
- colony PCR of Glu1
10/15
- ligate mtlD and plate1 7D
10/17
- colony PCR of mtlD+plate1-7D
10/18
- Miniprep of mtlD+plate1-7D
- MtlD made a debut as an iGEM part!
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