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Team:Todai-Tokyo/Notebook/bread
From 2009.igem.org
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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 Sequencing 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
10/1~11
- PCR of Glu1
- TA cloning of Glu1
- PCR of gpd1 promoter with ExTaq
10/12
- PCR of glu1 and gpd1
- colony PCR of gal1
- ligation of glu1 and pMD20-T vector
- Cut mtlD with X and P
10/13
- Sequencing of mtlD
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!
10/20
- 1. Amplification of liner DNA (gpd1 deletion and mtlD expression) by Pfu Ultra II PCR
(1) gpd1_5'_mtlD_5' primer T(ADH)_mtlD_3' primer Amplify mtlD gene(1149bp) by using the above 2 primers
mtlD plasmid（5-30 ng） 0.1 ul each primer（100 uM） 0.1 ul 2.5mM dNTPs 2.0 ul 10×PfuUltra II buffer 2.0 ul PfuUltra II 0.4 ul MilliQ 15.3 ul
Program 1 : 95 oC, 2min 2 : 95 oC, 30sec 3 : 55 oC, 30sec 4 : 72 oC, 30sec 5 : go to 2, 24times 6 : 25 oC, for ever 7 : End
(2) T(ADH) primer gpd1_3'_T(TEF1) primer Amplify GFPKan4MX6 gene (1715bp) by using the above 2 primers
pYM27 plasmid（5-30 ng） 0.1 ul each primer（100 uM） 0.1 ul 2.5mM dNTPs 2.0 ul 10×PfuUltra II buffer 2.0 ul PfuUltra II 0.4 ul MilliQ 15.3 ul
Program 1 : 95 oC, 2min 2 : 95 oC, 30sec 3 : 55 oC, 30sec 4 : 72 oC, 30sec 5 : go to 2, 24times 6 : 25 oC, for ever 7 : End
Confirm those 2 PCR products by 1% agarose gel electrophoresis and purify them by Promega Gel Extract Kit.(Elute 30 ul MilliQ)
(3)Overlap PCR (i) Amplify the full-length fragment by using 2 PCR products
Overlap extension PCR 5’ Fragment 100 ng 3.3 ul 3’ Fragment 100 ng 10 ul 2.5mM dNTPs 2.0 ul 10×PfuUltra II buffer 2.0 ul PfuUltra II 0.4 ul MilliQ 2.3 ul
Program 1 : 95 oC, 2min 2 : 95 oC, 30sec 3 : 55 oC, 30sec 4 : 72 oC, 45sec 5 : go to 2, 10times 6 : 25 oC, for ever 7 : End
(ii) Amplify the full-length fragment by using 2 primers gpd1_5'_mtlD_5' primer gpd1_3'_T(TEF1) primer
gpd1_5'_mtlD_5' primer（100 uM） 0.2 ul gpd1_3'_T(TEF1) primer（100 uM） 0.2 ul 2.5mM dNTPs 2.0 ul 10×PfuUltra II buffer 2.0 ul PfuUltra II 0.5 ul MilliQ 15.1 ul
Add the above mixture to the former PCR reaction mixture
Program 1 : 95 oC, 2min 2 : 95 oC, 30sec 3 : 55 oC, 30sec 4 : 72 oC, 45sec 5 : go to 2, 24times 6 : 25 oC, for ever 7 : End
2. Amplification of liner DNA (gpd2 deletion and glu1 expression) by Pfu Ultra II PCR
(2) F2 primer
gpd2_3'_R1 primer
Amplify GFPHis3MX6 gene (約2500bp) by using the above 2 primers
plasmid（5-30 ng） 1 ul
each primer（100 uM） 0.1 ul 2.5mM dNTPs 2.0 ul 10×PfuUltra II buffer 2.0 ul PfuUltra II 0.4 ul MilliQ 15.3 ul
Program 1 : 95 oC, 2min 2 : 95 oC, 30sec 3 : 55 oC, 30sec 4 : 72 oC, 45sec 5 : go to 2, 24times 6 : 25 oC, for ever 7 : End
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