Team:Todai-Tokyo/Notebook/bread

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Revision as of 14:52, 21 October 2009

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the notebook

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