From 2009.igem.org

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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
|
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
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

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!

10/19

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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