Team:Todai-Tokyo/Notebook/bread

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=='''Plan'''==
=='''Plan'''==
'''Aim:'''Create yeast that can be used to make sweet and low energy bread<BR>  
'''Aim:'''Create yeast that can be used to make sweet and low energy bread<BR>  
Line 6: 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>
-
→Replace gpd1 gene by mtlD and gpd2 gene by glu1<BR>
+
|<BR>
 +
v<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>
-
→Replace gpd1 gene by D-tagatose 3-epimerase gene and gpd2 gene by glu1. Transform a plasmid coding xylose isomerase gene.
+
|<BR>
 +
v<BR>
 +
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 17: Line 47:
=='''9/21'''==
=='''9/21'''==
-
*Miniprep of mtlD<BR>
+
*[[Team:Todai-Tokyo/Protocols/Miniprep|Miniprep]]  of mtlD<BR>
=='''9/22'''==
=='''9/22'''==
-
*read the sequence of mtlD→successful<BR>
+
*read the [[Team:Todai-Tokyo/Protocols/Sequencing|Sequencing]] of mtlD→successful<BR>
*mtlD primers with HAtag come<BR>
*mtlD primers with HAtag come<BR>
Line 32: Line 62:
*PCR of gpd1 promoter with Pfu Ultra<BR>
*PCR of gpd1 promoter with Pfu Ultra<BR>
-
=='''October'''==
+
='''October'''=
 +
=='''10/1~11'''==
*PCR of Glu1<BR>
*PCR of Glu1<BR>
*TA cloning of Glu1<BR>
*TA cloning of Glu1<BR>
*PCR of gpd1 promoter with ExTaq<BR>
*PCR of gpd1 promoter with ExTaq<BR>
 +
 +
== '''10/12''' ==
 +
*PCR of glu1 and gpd1<BR>
 +
*colony PCR of gal1
 +
*ligation of glu1 and pMD20-T vector
 +
*Cut mtlD with X and P
 +
 +
== '''10/13''' ==
 +
*[[Team:Todai-Tokyo/Protocols/Sequencing|Sequencing]] of mtlD<BR>
=='''10/14'''==
=='''10/14'''==
Line 41: Line 81:
*colony PCR of Glu1<BR>
*colony PCR of Glu1<BR>
 +
=='''10/15'''==
 +
*ligate mtlD and plate1 7D<BR>
 +
=='''10/17'''==
 +
*colony PCR of mtlD+plate1-7D<BR>
 +
=='''10/18'''==
 +
*[[Team:Todai-Tokyo/Protocols/Miniprep|Miniprep]]  of mtlD+plate1-7D<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'''==
 +
* Amplification of liner DNA (gpd1 deletion and mtlD expression) by Pfu Ultra II PCR<BR>
 +
(1) gpd1_5'_mtlD_5' primer<BR>
 +
T(ADH)_mtlD_3' primer<BR>
 +
Amplify mtlD gene(1149bp) by using the above 2 primers<BR><BR>
 +
mtlD plasmid（25 ng） 0.1 ul<BR>
 +
each primer（100 uM） 0.1 ul<BR>
 +
2.5mM dNTPs 2.0 ul<BR>
 +
10×PfuUltra II buffer 2.0 ul<BR>
 +
PfuUltra II 0.4 ul<BR>
 +
MilliQ 15.3 ul<BR>
 +
<BR>
 +
Program<BR>
 +
1 : 95 oC, 2min<BR>
 +
2 : 95 oC, 30sec<BR>
 +
3 : 55 oC, 30sec<BR>
 +
4 : 72 oC, 30sec<BR>
 +
5 : go to 2, 24times<BR>
 +
6 : 25 oC, for ever<BR>
 +
7 : End<BR>
 +
<BR>
 +
(2) T(ADH) primer<BR>
 +
gpd1_3'_T(TEF1) primer<BR>
 +
Amplify GFPKan4MX6 gene (1715bp) by using the above 2 primers<BR>
 +
<BR>
 +
pYM27 plasmid（30 ng） 0.1 ul<BR>
 +
each primer（100 uM） 0.1 ul<BR>
 +
2.5mM dNTPs 2.0 ul<BR>
 +
10×PfuUltra II buffer 2.0 ul<BR>
 +
PfuUltra II 0.4 ul<BR>
 +
MilliQ 15.3 ul<BR>
 +
<BR>
 +
Program<BR>
 +
1 : 95 oC, 2min<BR>
 +
2 : 95 oC, 30sec<BR>
 +
3 : 55 oC, 30sec<BR>
 +
4 : 72 oC, 30sec<BR>
 +
5 : go to 2, 24times<BR>
 +
6 : 25 oC, for ever<BR>
 +
7 : End<BR>
 +
<BR>
 +
Confirm those 2 PCR products by 1% agarose gel electrophoresis and purify them by Promega Gel Extract Kit.(Elute 30 ul MilliQ)<BR>
 +
<BR>
 +
(3)Overlap PCR<BR>
 +
(i) Amplify the full-length fragment by using 2 PCR products<BR>
 +
<BR>
 +
Overlap extension PCR<BR>
 +
5’ Fragment (99 ng)                 3.3 ul<BR>
 +
3’ Fragment (100 ng)       10 ul<BR>
 +
2.5mM dNTPs     2.0 ul<BR>
 +
10×PfuUltra II buffer         2.0 ul<BR>
 +
PfuUltra II 0.4 ul<BR>
 +
MilliQ         2.3 ul<BR>
 +
<BR>
 +
Program<BR>
 +
1 : 95 oC, 2min<BR>
 +
2 : 95 oC, 30sec<BR>
 +
3 : 55 oC, 30sec<BR>
 +
4 : 72 oC, 45sec<BR>
 +
5 : go to 2, 10times<BR>
 +
6 : 25 oC, for ever<BR>
 +
7 : End<BR>
 +
<BR>
 +
(ii) Amplify the full-length fragment by using 2 primers<BR>
 +
gpd1_5'_mtlD_5' primer<BR>
 +
gpd1_3'_T(TEF1) primer<BR>
 +
<BR>
 +
gpd1_5'_mtlD_5' primer（100 uM）   0.2 ul<BR>
 +
gpd1_3'_T(TEF1) primer（100 uM） 0.2 ul<BR>
 +
2.5mM dNTPs 2.0 ul<BR>
 +
10×PfuUltra II buffer 2.0 ul<BR>
 +
PfuUltra II 0.5 ul<BR>
 +
MilliQ 15.1 ul<BR>
 +
<BR>
 +
Add the above mixture to the former PCR reaction mixture<BR>
 +
<BR>
 +
Program<BR>
 +
1 : 95 oC, 2min<BR>
 +
2 : 95 oC, 30sec<BR>
 +
3 : 55 oC, 30sec<BR>
 +
4 : 72 oC, 45sec<BR>
 +
5 : go to 2, 24times<BR>
 +
6 : 25 oC, for ever<BR>
 +
7 : End<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>
 +
Amplify GFPHis3MX6 gene (約2500bp) by using the above 2 primers<BR>
 +
<BR>
 +
pFA6-GFP-His3MX6 plasmid 1 ul<BR>
 +
each primer（100 uM）      0.1 ul<BR>
 +
2.5mM dNTPs 2.0 ul<BR>
 +
10×PfuUltra II buffer 2.0 ul<BR>
 +
PfuUltra II 0.4 ul<BR>
 +
MilliQ 15.3 ul<BR>
 +
<BR>
 +
Program<BR>
 +
1 : 95 oC, 2min<BR>
 +
2 : 95 oC, 30sec<BR>
 +
3 : 55 oC, 30sec<BR>
 +
4 : 72 oC, 45sec<BR>
 +
5 : go to 2, 24times<BR>
 +
6 : 25 oC, for ever<BR>
 +
7 : End<BR>
 +
<BR>
 +
<BR>
 +
*made PYD plate<BR>

Latest revision as of 03:49, 22 October 2009

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the notebook

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

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

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




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