Team:Todai-Tokyo/Notebook/bread

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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<BR>
 * mtlD primers with HAtag come<BR>

9/25

 * PCR of mtlD with new primer→failed<BR>

9/26

 * PCR of mtlD with new primer→successful<BR>

9/27

 * PCR of gpd1 promoter with Pfu Ultra<BR>

=October=

10/1~11

 * PCR of Glu1<BR>
 * TA cloning of Glu1<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

 * Sequencing of mtlD<BR>

10/14

 * cut mtlD and plate1 7D both by EcoRI and PstI<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

 * 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
(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>
 * Amplification of liner DNA (gpd1 deletion and mtlD expression) by Pfu Ultra II PCR<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> (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>
 * Amplification of liner DNA (gpd2 deletion and glu1 expression) by Pfu Ultra II PCR<BR>
 * made PYD plate<BR>