Team:Todai-Tokyo/Notebook/bread

From 2009.igem.org

(Difference between revisions)
(10/20)
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*MtlD made a debut as an iGEM part!<BR>  
*MtlD made a debut as an iGEM part!<BR>  
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=='''10/20'''==
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=='''10/20,21'''==
*1.  Amplification of liner DNA (gpd1 deletion and mtlD expression) by Pfu Ultra II PCR<BR>
*1.  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>
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<BR>
<BR>
Overlap extension PCR<BR>
Overlap extension PCR<BR>
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5’ Fragment 100 ng         3.3 ul<BR>
+
5’ Fragment (100 ng)                 3.3 ul<BR>
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3’ Fragment 100 ng 10 ul<BR>
+
3’ Fragment (100 ng)       10 ul<BR>
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2.5mM dNTPs 2.0 ul<BR>
+
2.5mM dNTPs     2.0 ul<BR>
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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>

Revision as of 15:00, 21 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

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

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

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!

10/20,21

  • 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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