Team:Todai-Tokyo/Notebook/bread

From 2009.igem.org

(Difference between revisions)
(10/20)
(10/20,21)
 
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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>
+
|<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>
+
|<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'''==
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*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>
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=='''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>
-
=='''10/20'''==
+
=='''10/19'''==
-
*1.  Amplification of liner DNA (gpd1 deletion and mtlD expression) by Pfu Ultra II PCR<BR>
+
*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>
(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 plasmid(5-30 ng) 0.1 ul<BR>
+
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>
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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 plasmid(5-30 ng) 0.1 ul<BR>
+
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 100 ng         3.3 ul<BR>
+
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>
-
10×PfuUltra II buffer 2.0 ul<BR>
+
10×PfuUltra II buffer         2.0 ul<BR>
PfuUltra II 0.4 ul<BR>
PfuUltra II 0.4 ul<BR>
MilliQ         2.3 ul<BR>
MilliQ         2.3 ul<BR>
Line 172: Line 180:
<BR>
<BR>
<BR>
<BR>
-
2.  Amplification of liner DNA (gpd2 deletion and glu1 expression) by Pfu Ultra II PCR<BR>
+
* Amplification of liner DNA (gpd2 deletion and glu1 expression) by Pfu Ultra II PCR<BR>
-
(2) F2 primer<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>
-
plasmid(5-30 ng) 1 ul<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>
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7 : End<BR>
7 : End<BR>
<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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