"
Team:Todai-Tokyo/Notebook/bread
From 2009.igem.org
(→10/20) |
(→10/20,21) |
||
| (10 intermediate revisions not shown) | |||
| Line 31: | Line 31: | ||
1.Clone the glu1 (glucoamylase) from Saccharomycopsis fibuligera and insert it in the yeast chromosome by homologous recombination.<BR> | 1.Clone the glu1 (glucoamylase) from Saccharomycopsis fibuligera and insert it in the yeast chromosome by homologous recombination.<BR> | ||
2-a Clone mtlD (mannitol synthase) from E.coli and insert it in the yeast chromosome by homologous recombination<BR> | 2-a Clone mtlD (mannitol synthase) from E.coli and insert it in the yeast chromosome by homologous recombination<BR> | ||
| - | + | |<BR> | |
| + | v<BR> | ||
Replace gpd1 gene by mtlD and gpd2 gene by glu1<BR> | Replace gpd1 gene by mtlD and gpd2 gene by glu1<BR> | ||
2-b Clone xylose isomerase gene and D-tagatose 3-epimerase gene from Mesorhizobium loti.<BR> | 2-b Clone xylose isomerase gene and D-tagatose 3-epimerase gene from Mesorhizobium loti.<BR> | ||
| - | + | |<BR> | |
| + | v<BR> | ||
Replace gpd1 gene by D-tagatose 3-epimerase gene and gpd2 gene by glu1. Transform a plasmid coding xylose isomerase gene. | Replace gpd1 gene by D-tagatose 3-epimerase gene and gpd2 gene by glu1. Transform a plasmid coding xylose isomerase gene. | ||
| + | ='''September'''= | ||
=='''~9/20'''== | =='''~9/20'''== | ||
| Line 59: | Line 62: | ||
*PCR of gpd1 promoter with Pfu Ultra<BR> | *PCR of gpd1 promoter with Pfu Ultra<BR> | ||
| + | ='''October'''= | ||
=='''10/1~11'''== | =='''10/1~11'''== | ||
*PCR of Glu1<BR> | *PCR of Glu1<BR> | ||
| Line 83: | Line 87: | ||
=='''10/18'''== | =='''10/18'''== | ||
*[[Team:Todai-Tokyo/Protocols/Miniprep|Miniprep]] of mtlD+plate1-7D<BR> | *[[Team:Todai-Tokyo/Protocols/Miniprep|Miniprep]] of mtlD+plate1-7D<BR> | ||
| - | *MtlD made a debut as an iGEM part!<BR> | + | *MtlD made a debut as an iGEM part!<BR> |
| + | |||
| + | =='''10/19'''== | ||
| + | *read the sequence of mtlD | ||
| + | *PCR of mtlD and gpdI | ||
=='''10/20,21'''== | =='''10/20,21'''== | ||
| - | * | + | * Amplification of liner DNA (gpd1 deletion and mtlD expression) by Pfu Ultra II PCR<BR> |
(1) gpd1_5'_mtlD_5' primer<BR> | (1) gpd1_5'_mtlD_5' primer<BR> | ||
T(ADH)_mtlD_3' primer<BR> | T(ADH)_mtlD_3' primer<BR> | ||
Amplify mtlD gene(1149bp) by using the above 2 primers<BR><BR> | Amplify mtlD gene(1149bp) by using the above 2 primers<BR><BR> | ||
| - | mtlD | + | mtlD plasmid（25 ng） 0.1 ul<BR> |
each primer（100 uM） 0.1 ul<BR> | each primer（100 uM） 0.1 ul<BR> | ||
2.5mM dNTPs 2.0 ul<BR> | 2.5mM dNTPs 2.0 ul<BR> | ||
| Line 111: | Line 119: | ||
Amplify GFPKan4MX6 gene (1715bp) by using the above 2 primers<BR> | Amplify GFPKan4MX6 gene (1715bp) by using the above 2 primers<BR> | ||
<BR> | <BR> | ||
| - | pYM27 | + | pYM27 plasmid（30 ng） 0.1 ul<BR> |
each primer（100 uM） 0.1 ul<BR> | each primer（100 uM） 0.1 ul<BR> | ||
2.5mM dNTPs 2.0 ul<BR> | 2.5mM dNTPs 2.0 ul<BR> | ||
| Line 133: | Line 141: | ||
<BR> | <BR> | ||
Overlap extension PCR<BR> | Overlap extension PCR<BR> | ||
| - | 5’ Fragment ( | + | 5’ Fragment (99 ng) 3.3 ul<BR> |
3’ Fragment (100 ng) 10 ul<BR> | 3’ Fragment (100 ng) 10 ul<BR> | ||
2.5mM dNTPs 2.0 ul<BR> | 2.5mM dNTPs 2.0 ul<BR> | ||
| Line 172: | Line 180: | ||
<BR> | <BR> | ||
<BR> | <BR> | ||
| - | + | * Amplification of liner DNA (gpd2 deletion and glu1 expression) by Pfu Ultra II PCR<BR> | |
| - | ( | + | (1) F2 primer<BR> |
gpd2_3'_R1 primer<BR> | gpd2_3'_R1 primer<BR> | ||
Amplify GFPHis3MX6 gene (約2500bp) by using the above 2 primers<BR> | Amplify GFPHis3MX6 gene (約2500bp) by using the above 2 primers<BR> | ||
<BR> | <BR> | ||
| - | + | pFA6-GFP-His3MX6 plasmid 1 ul<BR> | |
each primer（100 uM） 0.1 ul<BR> | each primer（100 uM） 0.1 ul<BR> | ||
2.5mM dNTPs 2.0 ul<BR> | 2.5mM dNTPs 2.0 ul<BR> | ||
| Line 193: | Line 201: | ||
7 : End<BR> | 7 : End<BR> | ||
<BR> | <BR> | ||
| - | + | <BR> | |
| + | *made PYD plate<BR> | ||
Latest revision as of 03:49, 22 October 2009
| Home | The Team | The Project | Parts Submitted to the Registry | Modeling | Notebook | Protocols | Ethics |
|---|
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
|
v
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.
|
v
Replace gpd1 gene by D-tagatose 3-epimerase gene and gpd2 gene by glu1. Transform a plasmid coding xylose isomerase gene.
September
~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
October
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!
10/19
- read the sequence of mtlD
- PCR of mtlD and gpdI
10/20,21
- 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（25 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（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 (99 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
- Amplification of liner DNA (gpd2 deletion and glu1 expression) by Pfu Ultra II PCR
(1) F2 primer
gpd2_3'_R1 primer
Amplify GFPHis3MX6 gene (約2500bp) by using the above 2 primers
pFA6-GFP-His3MX6 plasmid 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
- made PYD plate
| Home | The Team | The Project | Parts Submitted to the Registry | Modeling | Notebook | Protocols | Ethics |
|---|
