Team:NCTU Formosatest

From 2014.igem.org

Contents

Note

  • April
    2014
  • May
    2014
  • June
    2014
  • July
    2014
  • August
    2014
  • September
    2014
  • October
    2014

April 2014

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None

4/11

Transformation of [PSB1K3] Kr, [PSB1A3] Ar, [PSB1C3] Cr (G2)

Transformation of [Plac+PSB1A3] Ar, [mGFP+PSB1A3] Ar (G3)

Transformation of [PSB1K3] Kr,[PSB1A3] Ar, [PSB1C3] Cr (G1)

4/12

Cultivation of [PSB1K3] Kr, [PSB1C3] Cr (G2)

Cultivation of [PSB1A3] Ar, [PSB1C3] Cr (G1)

Cultivation of [mGFP] Ar (G3)


4/13

Mini-prep of [mGFP] Ar, [PSB1K3] Kr (G3)

Mini-prep of [PSB1K3] Kr, [PSB1C3] Cr (G2)

Mini-prep of [PSB1A3] Ar, [PSB1C3] Cr (G1)

4/17

Transformation of [PSB1K3] Kr (G1)

4/18

Cultivation of [PSB1K3] Kr (G1)

4/19

Transformation of [Plac+PSB1A3] Ar, [PSB1K3] Kr, [PSB1A3] Ar, [PSB1C3] Cr (G3)

Mini-prep of [PSB1K3] Kr (G1)


4/20

Transformation of [PSB1A3] Ar (G2)

Electrophoresis of [PSB1K3] Kr, [PSB1C3] Cr plasmid (G2)

4/21

Cultivation of [PSB1K3] Kr, [PSB1A3] Ar, [PSB1C3] Cr (G2)

4/22

Mini-prep of [PSB1K3] Kr, [PSB1A3] Ar, [PSB1C3] Cr (G2)


4/28

Electrophoresis of digested [PSB1K3] Kr(EP), [PSB1C3] Cr(EP),[PSB1A3] Ar(EP) (G1)

Cultivation of [PSB1C3] Cr (G1)

4/29

Mini-prep of [PSB1C3] Cr (G1)

Mini-prep of [PSB1C3] Cr (G2)

Transformation of [B0032] Ar, and [BFP] Cr (G2)

Cultivation of [PSB1K3] Kr, [PSB1A3] Ar (G1)

Cultivation of [B0032] Ar, and [BFP] Cr (G2)

4/30

Mini-prep of [PSB1A3] Ar (G1)

Mini-prep of [B0032] Ar, and [BFP] Cr (G2)

Transformation of [B0032] Ar, and [BFP] Cr, [T7promoter] Ar Kr and [T7 polymerase] Cr (G2)

Transformation of [J23101] Ar,[K592100] Cr,[B0032] Ar,[B0015] Ar (G1)

Digestion of [PSBIC3] Cr(EP), [B0032] Ar(ES), [BFP] Cr(XP) mini (G2)

Electrophoresis of plasmid and digested prouct of[PSBIC3] Cr(EP), [B0032] Ar(ES), [BFP] Cr(XP) mini (G2)


May 2014

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5/1

Cultivation of [PSB1C3]Cr, [BFP]Cr, [T7promoter]ArKr, [T7 polymerase]Cr and [B0015]Ar (G2)

Transformation of [T7 polymerase]Cr (G2)

5/2

Cultivation of [J23101] Ar,[K592100] Cr,[B0032] Ar,[B0015] Ar (G1)

Mini-prep of [PSB1C3]Cr, [BFP]Cr, [T7promoter]ArKr, [T7 polymerase]Cr and [B0015]Ar (G2)

Digestion of [PSB1C3]Cr(EP), [BFP]Cr(XP), [T7 polymerase]Cr(ES), [T7promoter]ArKr(ES) and [B0015]Ar*2(ES & XP) (G2)

5/3

Mini-prep of [J23101] Ar,[K592100] Cr,[B0032] Ar,[B0015] Ar (G1)

Mini-pep of [B0032] Ar, [B0015] Ar,[BFP] Cr (G3)

Digestion of [J23101] Ar (ES),[K592100]Cr(ES),[B0032] Ar(XP),[B0015] Ar(XP),[PSB1A3] Ar(EP),[PSB1C3] Cr(EP),[PSB1K3] Kr(EP) mini (G1)

Electrophoresis of plasmid and digested product of [PSB1C3]Cr(EP), [BFP]Cr(XP), [T7 polymerase]Cr(ES), [T7promoter]ArKr(ES) and B0015*2(ES & XP) (G2)

Electrophoresis of digested product of [J23101] Ar (ES),[K592100] Cr(ES),[B0032] Ar(XP),[B0015] Ar(XP),[PSB1A3] Ar(EP),[PSB1C3]Cr(EP),[PSB1K3] Kr(EP) (G1)

Cultivation of [B0015] Ar, [PSB1A3] Ar, [PSB1K3] Kr (G1)

Ligation of [BFP+B0032+PSB1K3]Kr (G2)

Ligation of [J23101+B0032+PSB1C3] Cr (G1)

Transformation of ligation product [BFP+B0032+PSB1K3]Kr (G2)

Transformation of [T7polymerase]Cr and [B0015]Ar (G2)

5/4

Mini-prep of [B0015] Ar, [PSB1A3] Ar, [PSB1K3] Kr (G1)

Mini-prep of [B0015*3] Ar and [T7polymerase] Cr (G2)

Digestion of [B0015] Ar(XP), [PSB1A3] Ar(EP), [PSB1K3] Kr (EP)(G1)

Digestion of [B0015*3] Ar and [T7polymerase] Cr (G2)

Digestion of [B0015] Ar(XP), [BFP] Cr(XP), [PSB1A3] Ar(EP), [PSB1C3] Cr(EP), [B0032] Ar (XP) (G3)

Transformation of [Pcons promoter] Ar, [RBS+BFP] Kr and [T7 promoter] Kr (G2)

Transformation of [LacI] Ar (G3)

Electrophoresis of plasmid and digested product of [B0015*3] Ar and [T7polymerase]Cr (G2)

Electrophoresis of digested product of [B0015] Ar(XP), [PSB1A3] Ar(EP), [PSB1K3] Kr (EP), [K592100] Cr (ES)(G1)

Cultivation of [B0015] Ar, [PSB1A3] Ar(G1)

Cultivation of [Plac] Ar, [PSB1C3] Cr, [mGFP] Ar (G3)

5/5

Mini-prep of [PSB1A3] Ar,[B0015]Ar (G1)

Mini-prep of [Plac] Ar, [PSB1C3] Cr, [mGFP] Ar (G3)

Digestion of [PSB1A3] Ar(EP),[B0015]Ar(XP)(G1)

Electrophoresis of digested product of [PSB1A3] Ar(EP),[B0015]Ar(XP),[PSB1K3]Kr(EP) (G1)

5/6

Mini-prep of [LacI] Ar (G3)

5/7

Transformation of [PT7polymerase]Cr and [J61048]Ar (G2)

5/8

Transformation of [B0015*3] Ar and [J61048*2] Ar (G2)

Transformation of [B0015] Ar and [J61048] Ar (G3)

Transformation of [B0015]Ar,[J61048]Ar,[J23101]Ar (G1)

Mini-prep of [PT7polymerase]Cr, [T7polymerase*2]Cr and [B0015*2]Ar (G2)

Digestion of [T7polymerase] Kr (EP) and [B0015] Ar (ES) (G2)

Digestion of [Plac] Ar (ES), [mGFP] Ar (ES), [LacI] Ar (ES), [PSB1C3] Cr (EP) (G3)

Electrophoresis of digested product of [Plac] Ar (ES), [mGFP] Ar (ES), [LacI] Ar (ES), [PSB1C3] Cr (EP) (G3)

5/9

Cultivation of [B0015] Ar, [J61048] Ar [LacI] Ar (G3)

5/10

Mini-prep of [LacI] Ar (G3)


5/11

Cultivation of [J61048] Ar (G2) (ES)

Electrophoresis of plasmid and digested product of [B0015]Ar(ES), [T7polymerase] Kr(only mini) and [T7promoter] Kr (EP)

Transformation of [T7 promoter] Kr (G2)

5/12

Digestion of [T7polymerase] Kr (ES) and [J61048] Ar (ES) (G2)

5/13

Transformation of [T7 promoter] Kr (G2)

5/15

Electrophoresis of plasmid and digested product of [J61048]Ar (ES), [T7polymerase*3]Kr(ES & XP) and [PSBIK3]Kr(EP) (G2)

Cultivation of [B0015]Ar, [J61048]Ar, [J23101]Ar, [PSB1K3]Kr (G1)

Mini-prep of [PSB1K3]Kr (G1)

Digestion of [PSB1K3]Kr (EP) (G1)


5/19

Cultivation of [J23101]Ar,[B0015]Ar,[J61048]Ar (G1)

5/20

Transformation of [T7polymerase] Kr and [PSB1K3] Kr (G2)

Electrophoresis of plasmid and digested product of [T7polymerase*6] Kr and [PSB1K3*4]Kr (G2)

Mini-prep of [J23101]Ar, [B0015]Ar, [J61048]Ar (G1)

Digestion of [J23101]Ar (ES), [B0015]Ar (XP), [J61048]Ar (XP) (G1)

5/21

Digestion of [T7polymerase*6] Kr and [PSB1K3*2] Kr (G2)

Mini-prep of [T7polymerase] Kr and [PSB1K3] Kr (G2)

Electrophoresis of plasmid and digested product of [J23101]Ar(ES), [B0015]Ar(XP), [J61048]Ar(XP) and digested product [PSB1K3]Kr(EP) (G1)

Ligation of [K592100+J61048+PSB1K3] Kr (G1)

5/22

Electrophoresis of plasmid and digested product of [T7polymerase] Kr (ES) and [PSB1K3]Kr (EP) (G2)

5/23

Transformation of ligation product [K592100+J61048+PSB1K3]Kr (G1)

5/24

Digestion of [J61048] Ar (XP) (G2)

PCR of [BFP+B0032*4] Kr and [T7 promoter*1] Ar Kr (G2)

Electrophoresis of PCR products [BFP+B0032*4] Kr and [T7 promoter*1] Ar Kr, [J61048] Ar (G2)


5/27

Electrophoresis of digested product of [mGFP] Ar (ES), [J61048] Ar (XP) ,[B0015] Ar (XP) (G3)

Digestion of [J61048] Ar (XP) and [T7polymerase] Kr (ES) (G2)

Cultivation of [PSB1K3*3] Kr(G2)

5/28

PCR of [PSBIK3*5]Kr , [PSBIA3*5]Ar , [PSBIC3*5]Cr ,[J61048*5]Ar ,[B0015*5]Ar and [BFP*5](G1)

Mini-prep of [PSB1K3*3] Kr(G2)

Digestion of [PSB1K3*3] Kr (EP) (G2)

Electrophoresis of plasmid and digested product of [PSB1K3] Kr (EP), [T7polymerase] Kr (ES), [J61048] Ar (XP) (G2)

5/29

Digestion of [mGFP] Ar (ES), [J61048] Ar (XP) ,[B0015] Ar (XP) (G3)

Transformation of [J23101] Ar (G1)

Transformation of [T7 promoter] Ar Kr (G2)

Electrophoresis of plasmid and digested product of [J61048] Ar (ES), [T7polymerase] Kr (ES) (G2)

Ligation of [J61048+J61048] Cr, [T7polymerase+J61048] Kr, [RBS+BFP] Kr (G2)

Ligation of [Plac+B0032+PSB1K3] Kr (G3)

5/30

Transformation of ligation product (G2)

Transformation of ligation product [Plac+B0032+PSB1K3] Kr (G3)


June 2014

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6/2

PCR of [T7 promoter*4] Ar Kr, [J61048+J61048*3] Cr, [T7polymerase+J61048*3] Kr and [B0032+BFP*3] Kr (G2)

Cultivation of [T7 promoter*4] Ar Kr, [J61048+J61048*3] Cr, [T7polymerase+J61048*3] Kr and [B0032+BFP*3] Kr (G2)

Electrophoresis of [T7 promoter*4] Ar Kr, [J61048+J61048*3] Cr, [T7polymerase+J61048*3]Kr and [B0032+BFP*3] Kr plasmid (G2)

6/3

PCR of [T7 promoter*5] Ar Kr, [B0032+BFP*5] Kr , [J61048+J61048*5] Cr and [T7polymerase+J61048*5] Kr (G2)

Electrophoresis of PCR products (G2)

Cultivation of [B0032+BFP*10] Kr, [J61048+J61048*10] Cr and [T7polymerase+J61048*5] Kr (G2)

6/5

Transformation of ligation product [K592100+J61048+PSBIK3] Kr (G1)

Ligation of [BFP+B0032] Kr and [T7polymerase+J61048] Kr (G2)

Ligation of [mGFP+B0015+PSB1K3] Kr and [mGFP+J61048+PSB1K3] Kr (G3)

6/6

Mini-prep of [J61048+J61048*3] Cr and [T7 promoter*3]Ar Kr (G2)

Digestion of [J61048+J61048*3] Cr (XP) and [T7 promoter*3]Ar Kr(ES) (G2)

Transformation of [B0032+BFP] Kr and [T7polymerase+J61048] Kr (G2)

Transformation of ligation product [mGFP+B0015+PSB1K3] Kr and [mGFP+J61048+PSB1K3] Kr (G3)

6/7

PCR of [PSB1K3*6] Kr, [K592100+J61048+PSB1K3*10] Kr,[J23101*8] Ar ,[B0032*8] Ar (G1)

PCR of [mGFP+B0015+PSB1K3*6] Kr and [mGFP+J61048+PSB1K3*6] Kr (G3)


6/8

Electrophoresis of plasmid and digestion product of [J61048+J61048](XP), [T7 promoter](ES)

PCR of [B0032+BFP*5] Kr and [T7polymerase+J61048*5] Kr (G2)

Cultivation of [B0032+BFP*5] Kr and [T7polymerase+J61048*5] Kr (G2)

Electrophoresis of [B0032+BFP*5] Kr and [T7polymerase+J61048*5] Kr (G2)

6/9

Transformation of [B0023*2 ] Ar ,[J23101*2] Ar, [K592100+J61048*2] Kr and [PSB1K3*2] Kr (G1)

Electrophoresis of plasmid and digestion product of [RBS+BFP]Kr (ES),[T7 polymerase+J61048]Kr(XP),[T7 promoter]Kr Cr (ES),[J61048+J61048]Cr(XP) (G2)

Mini-prep of the plasmid and then digested the plasmid of [T7 polymerase+J61048]Kr(XP),[RBS+BFP]Kr(ES) (G2)

6/10

Electrophoresis of plasmid and digestion product of [K592100+J61048*2] Kr (XP), [PSB1K3*2] Kr (EP),[J23101*2] Ar (SP) and [B0023*2 ] Ar (SP) (G1)

6/11

Digestion of [mGFP] Ar (ES), [J61048] Ar (SP) ,[B0015] Ar (SP) (G3)

6/12

Ligation of [mGFP+B0015+PSB1K3] Kr and [mGFP+J61048+PSB1K3] Kr (G3)

PCR of [Plac+B0032+PSB1K3*12] Kr (G3)

6/13

Transformation of ligation product [mGFP+B0015+PSB1K3] Kr and [mGFP+J61048+PSB1K3] Kr (G3)

Cultivation of [Plac+B0032+PSB1K3*4] Kr (G3)

6/14

Mini-prep of [Plac+B0032+PSB1K3*2] Kr (G3)

Cultivation of [Plac+B0032+PSB1K3*4] Kr (G3)

PCR of [mGFP+B0015+PSB1K3*6] Kr


6/15

Mini-prep of [Plac+B0032+PSB1K3*4] Kr (G3)

Digestion of [Plac+B0032+PSB1K3*4] Kr (ES) (G3)


6/24

Digestion of [J23101] Ar (ES) and [B0032] Ar (XP) (G1)

Ligation of [B0032+K592100+J61048] Ar (G1)

Electrophoresis of plasmid and digestion product of [K592100+J61048] Kr (XP), [J23101] Ar (SP) and [B0032] Ar (SP) (G1)

Digestion of [T7 polymerase +J61048]Kr(XP),[J61048+J61048]Cr(XP) (G2)

6/27

Digestion of [J61048+J61048] Cr (XP) (G2)

Electrophoresis of [RBS+BFP]kr(ES)(mini),[T7polymerase](ES)(mini),[T7 promoter](mini)

6/28

Transformation of mini [B0032+BFP+J61048] Cr (G2)


6/29

PCR of [B0032+BFP+J61048]Cr

Electrophoresis of the PCR products (G2)


July 2014

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7/1

Ligation of [J23101+B0032+PSB1C3] Cr (G1)

Digestion of [B0032] Ar

Electrophoresis of plasmid and digestion products of[B0032+BFP+J61048]Cr (XP) (G2)

7/2

Transformation of ligation product [B0032+K592100+J61048] Ar and [J23101+B0032] Cr (G1)

Cultivation of [B0032+BFP+J61048] Cr in LB broth (G2)

7/3

Mini-prep of [B0032+BFP+J61048] and then digestion: (XP)

Electrophoresis of plasmid and digestion products of[B0032+BFP+J61048]Cr (XP) (G2)

7/4

Digestion of [T7 promoter](SP) (G2)

Cultivation of [J23101] Ar (G3)

7/5

PCR of [B0032+K592100+J61048*11] Ar (G1)

Electrophoresis of PCR products of(G1)

Ligation of [J23101+B0032+PSB1C3*3] Cr (G1)

Electrophoresis of plasmid and digestion products of[T7promoter](SP),[B0031+BFP+J61048](XP)

Ligation of [T7 promoter])(SP)+[B0032+BFP+J61048](XP) (G2)


7/6

Transformation of the ligation products made at 7/5 (G2)

7/7

Transformation of [J23101+B0032*5] Cr (G1)

Cultivation of [B0032+K592100+J61048*2] Ar (G1)

Cultivation of [J23101] Ar (G3)

Digestion of [B0015] Ar (XP), [J61048] Ar (XP) (G3)

7/8

Mini-prep of [B0032+K592100+J61048*2] Ar and then digest of only one of two: (XP) (G1)

PCR of [T7+B0032+BFP+J61048] (G2)

Digestion of [J23101] Ar (ES) (G3)

7/9

Cultivation of [B0032+BFP+J61048]Cr and [T7+B0032+BFP+J61048]Kr(G2)

7/10

Mini-prep of [B0032+BFP+J61048]Cr

Digestion of [B0032+FP+J61048]Cr( XP)

Electrophoresis of plasmid and digestion products of[B0032+BFP+J61048]Cr (XP) (G2)

7/12

Digestion of [B0032+BFP+J612048](XP), [T7+B0032+BFP+J61048]Kr (XP) (G2)


7/14

Electrophoresis of [B0032+BFP+J61048]Cr,[T7+B0032+BFP+J61048]Kr (G2)

7/15

Cultivation of [B0032+BFP+J61048]Cr,[PT7+B0032+BFP+J61048]Kr E.coli in LB broth (G2)

7/16

Mini-prep of [B0032+BFP+J61048],[PT7+B0032+BFP+J61048]

Digestion of [B0032+BFP+J61048]Cr(XP),[PT7+B032+BFP+J61048]Kr(ES) (G2)

7/17

Electrophoresis of the plasmid and digestion products made yesterday (G2)

7/19

PCR of [J23101+B0032*12] Cr (G1)

Electrophoresis of PCR products of [B0032+K592100+J61048*2] Ar ,digestion product of [B0032+K592100+J61048] Ar (XP) and [J23101] Ar (SP) (G1)


7/20

Digestion of [B0032+K592100+J61048] Ar (EP) (G1)

Digestion of [J23101] Ar (ES) (G3)

Ligation of [B0032+K592100+J61048] Cr (G1)

Ligation of [J23101+B0032+PSB1K3] Kr (G3)

PCR of [J23101+B0032*4] Cr (G1)

Cultivation of [J61048+B0032*4]Cr E.coli in LB broth (G1)

Electrophoresis of the PCR products and digestion products of [J23101] Ar (SP) and [B0032+K592100+J61048] Ar (EP) (G1)

7/21

Transformation of ligation product made yesterday (G1)

Transformation of ligation product of [J23101+B0032+PSB1K3] Kr (G3)

Ligation of [mGFP+J61048+PSB1K3] Kr,[mGFP+B0015+PSB1K3] Kr (G3)

7/23

Cultivation of [B0032+K592100+J61048] Cr E.coli in LB broth (G1)

Digestion of [J23101+B0032*3] Cr (EP) (G1)

Digestion of [B0032+K592100+J61048] Ar (EP) (G1)

Ligation of [J23101+B0032] Cr (G1)

Mini-prep of [B0032+K592100+J61048] Cr

Transformation of [B0032+K592100+J61048] Cr and [J23101+B0032] Ar (G1)

7/24

PCR of [B0032+K592100+J61048*12] Cr (G1)

Electrophoresis of digestion products of[J23101+B0032*3] Cr (EP) and the PCR products (G1)

Transformation of [J23101+B0032] Cr (G1)

Digestion of [BFP] Cr (ES), [B0015] Ar (XP), [J61048] Ar (XP), [B0032] Ar (XP), [BFP+B00315*2] Kr (XP) (G3)

7/26

Cultivation of [J23101+B0032*4] Cr E.coli in LB broth (G1)

Digestion of [J23101] Ar (SP), [B0032] Ar (XP) (G3)


7/27

Ligation of [J23101+B0032+PSB1C3] Cr (G3)

7/28

Digestion of [B0032+K592100+J61048*2] Cr (XP) (G1)

Transformation of [J23101] Ar (G3)

PCR of [J23101+B0032+PSB1C3*6] Cr (G3)

7/29

Electrophoresis of the digestion products of[B0032+K592100+J61048*2] Cr (XP) , [PSB1K3*2] Kr (EP) , [PSB1A3*3] Ar (EP) , [PSB1C3] Cr (EP) , [J23101] Ar (ES) , [B0032] Ar (SP) , [B0032] Ar (XP) , [B0032+K592100+J61048] Ar (XP) and [J23101] Ar (SP) (G1)

Electrophoresis of the digestion product of [B0032] Ar (EP),[PSB1C3] Cr (EP), [PSB1K3] Kr (EP), [PSB1A3] Ar (EP) and PCR product [J23101*6] Ar (G3)

Digestion of [B0032+K592100+J61048*2] Cr (XP) (G1)

Digestion of [B0032] Ar (EP), [PSB1C3] Cr (EP), [PSB1K3] Kr (EP), [PSB1A3] Ar (EP) (G3)

Mini-prep of [PT7+B0032+BFP+J61048] (G2)

PCR of [J23101*6] Ar (G3)

7/30

Electrophoresis of the digestion products of [B0032+K592100+J61048*2] Cr (XP) , [PSB1K3*2] Kr (EP) , [PSB1A3*3] Ar (EP) , [PSB1C3] Cr (EP) , [J23101] Ar (ES) , [B0032] Ar (SP) , [B0032] Ar (XP) , [B0032+K592100+J61048] Ar (XP) and [J23101] Ar (SP) (G1)

Electrophoresis of the plasmid and digestion products of [RBS],[B0032+BFP+J61048](XP),[PT7+B0032+BFP+J61048](XP) (G2)

7/31

Digestion of [B0032+K592100+J61048*2] Cr (XP) and [J23101] Ar (EP) (G1)

Ligation of [J23101+B0032+PSB1C3] Cr (G1)

Digestion of [B0032+K592100+J61048*2] Cr (XP) (G1)

Electrophoresis of the plasmid and digestion products of [B0032+K592100+J61048*2] Cr (XP) ,digestion products made in 7/7 and 7/30[B0032+K592100+J61048*2] Cr (XP) ; digestion product made in 7/31[B0032+K592100+J61048*2] Ar (XP) (G1)


August 2014

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None

None

8/11

Transformation of NCTU1 (G1)

Transformation of PBAN (G2)

8/12

Cultivation of [PBAN-4]Kr,[PBAN-5]Krand [PBAN-6]Kr E.coli in LB broth (G1)

Cultivation of [RBS+BFP+J61048]Cr ,[T7+RBS+BFP+J61048]Ar Kr

Mini-prep of [Pcons]Ar

Digestion of [Pcons]Ar (ES) (G2)

8/13

Mini-prep of [PBAN-4] Kr,[PBAN-5] Krand [PBAN-6] Kr (G1)

Digestion of [PBAN-4]Kr(EP),[PBAN-5]Kr(EP)and [PBAN-6]Kr(EP) (G1)

Electrophoresis of [Pcons](mini)&(dig ES),digestion of [Pcons](XP)

Ligation of [Pcons]+[RBS+BFP+J61048] (G2)

8/14

Electrophoresis of plasmid and digestion products of [PBAN-4] Kr(EP),[PBAN-5] Kr(EP)and [PBAN-6] Kr(EP) (G1)

Purification from electrophoresis of [PBAN]No.4&5&6(G1)

Transformation of the ligation product made yesterday

Electrophoresis of plasmid and digestion products of [PBAN]No.4&5&6,[Pcons],[Pcons+RBS+BFP+J61048] (G2)

8/15

Digestion of purification products yesterday:[PBAN]No.4&5&6 (EP) (G1)

Purification from electrophoresis of [PBAN]No.4&5&6

Transformation of [B0034]Ar (G2)

8/16

Electrophoresis of the purification products from electrophoresis,digestion products of[PBAN]No.4&5&6 (EP) (G1)

PCR of [B0034]

Electrophoresis of [PSB1K3](mini)&(dig E),[Pcons](dig SP),PCR products of B0034

Mini-prep of [B0034]Ar E.coli

Ligation of [Pcons]+[B0034] +PSB1K3

Digestion of [BFP](ES)

Electrophoresis of plasmid and digestion products of [PSB1K3],[PSB1C3],5/12[J61048],[Pcons])


8/17

Electrophoresis of the digestion products of[PBAN]No.4&5&6 (EP) (G1)


None

None

September 2014

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9/4

Cultivation of

Pcons+RBS+AseI PCR product-1,2,3,4,7,8 Ar

XhoI+PSB1K3 PCR product-1,2 Kr

PSB1A3 Ar

Cultivation plate test

Ar plate and Kr plate

Digestion of

[PBAN-HindIII] Kr*2 (EP)

[PBAN-NheI]Kr*2 (EP)

[PBAN-KpnI-HF]Kr*2 (EP)

[23101+B0034+PBAN (HindIII)]Ar(ES)

[23101+B0034+PBAN (NheI)]Ar(ES)

PCR of

[J23101+B0034+KpnI-HF] Ar*3

Ligation of

[J23101+B0034+KpnI-HF+PSB1C3]

Electrophoresis of

[PBAN-HindIII] Kr*2 (EP)

[PBAN-NheI]Kr*2 (EP)

[PBAN-KpnI-HF]Kr*2 (EP)

[23101+B0034+PBAN (HindIII)]Ar(ES)

[23101+B0034+PBAN (NheI)]Ar(ES)

[23101+B0034+PBAN (HindIII)]Ar(EP)*2

[23101+B0034+PBAN (NheI)]Ar(EP)

[23101+B0034+PBAN (KpnI-HF)]Ar(EP)*2

[23101+B0034+PBAN (KpnI-HF)]Ar PCR*3

9/5

Electrophoresis of

Well 1 Pcons+RBS+AseI--1 (EP) Ar

Well 2 Pcons+RBS+AseI--2 (EP) Ar

Well 3 Pcons+RBS+AseI--3 (EP) Ar

Well 4 Pcons+RBS+AseI--4 (EP) Ar

Well 5 Pcons+RBS+AseI--7 (EP) Ar （DNA sequencing）

Well 6 Pcons+RBS+AseI--8 (EP) Ar

Well 7 Pcons+RBS+NcoI--2 (EP) Cr （DNA sequencing）

Well 8 Pcons+RBS+Ndel--2 (EP) Cr （DNA sequencing）

Well 9 1 Kb marker

Well 10 Pcons+RBS+Ndel--13 (EP) Cr

Well 11 SacI--3 (XP) Kr

Well 12 SacI--4.1 (XP) Kr

Well 13 SacI--4.2 (XP) Kr

Well 14 XhoI--7 (EP) Kr （DNA sequencing）

Well 15 XhoI--8 (EP) Kr（DNA sequencing）

Well 16 SalI--3 (XP) Kr

Mini-prep of

Pcons+RBS+KpnI-HF-7

Pcons+RBS+KpnI-HF-8

Pcons+RBS+KpnI-HF-9

Digestion of

Pcons+RBS+KpnI-HF-7 (EP)

Pcons+RBS+KpnI-HF-8 (EP)

Pcons+RBS+KpnI-HF-9 (EP)

Cultivation of

Pcons+RBS+KpnI-HF Ar PCR product-7,8,9 Ar

Ligation of

[J23101+B0034+HindIII+B0034+PSB1K3] Kr

[J23101+B0034+HindIII+J61048+PSB1K3] Kr

[J23101+B0034+NheI+B0034+PSB1K3] Kr

[J23101+B0034+NheI+J61048+PSB1K3] Kr

Electrophoresis of digested product of

[Pcons+RBS+KpnI-HF]Ar (EP)*3


9/9

Cultivation of

Pcons+RBS+SacI PCR product-16 Ar

Pcons+RBS+SaII PCR product-17 Ar

RBS+BFP+Ter PCR product-16,17,29,30 Ar

Digestion of

XhoI+PSB1K3 (EP)

9/10

Mini-prep of

RBS+BFP+Ter-16 Ar

RBS+BFP+Ter-17 Ar

RBS+BFP+Ter-29 Ar

RBS+BFP+Ter-30 Ar

Pcons+RBS+Sac-16 Ar

Pcons+RBS+Sal-17 Ar

Digestion of

RBS+BFP+Ter-16 Ar(EP)

RBS+BFP+Ter-17 Ar(EP)

RBS+BFP+Ter-29 Ar(EP)

RBS+BFP+Ter-30 Ar(EP)

Pcons+RBS+SacI-16 Ar(EP)

Pcons+RBS+SalI-17 Ar(EP)

Electrophoresis of

Well 1 Marker

Well 2 Pcons+RBS+SacI-16 dig(EP)

Well 3 Pcons+RBS+SalI-17 dig(EP)

Well 4 RBS+BFP+Ter-16 dig(EP)

Well 5 RBS+BFP+Ter-17 dig(EP)

Well 6 RBS+BFP+Ter-29 dig(EP)

Well 7 RBS+BFP+Ter-30 dig(EP)

Well 8 XhoI-7 dig(XP) Kr

9/11

Digestion of

Pcons+RBS+PBAN(ES)(8 kind except of XhoI)

RBS+BFP+Ter(XP)

Ligation of

Pcons+RBS+XhoI-7 Ar

Cultivation of

Pcons+RBS+AseI-7 Ar

Pcons+RBS+SacI-16 Ar

Pcons+RBS+SalI-17 Ar

Pcons+RBS+NcoI-2 Ar

Pcons+RBS+NdeI-2 Ar

RBS+BFP+Ter-2 Ar

RBS+BFP+Ter-2 Ar

SalI-3 Kr

Ligation of

Pcons+RBS+XhoI-7 Ar

DNA sequencing of

Pcons+RBS+SaeI Ar 8ul VF2

Pcons+RBS+SalI Ar 8ul VF2

RBS+BFP+Ter-29 Ar 8ul VF2 + 8ul VR

RBS+BFP+Ter-30 Ar 5ul VF2 + 5ul VR

Digestion of

Pcons+RBS+HindIII dig(EP)

Pcons+RBS+NheI dig(EP)

Pcons+RBS+NcoI dig(EP)-->9/11 mini

Pcons+RBS+NdeI dig(EP)

Pcons+RBS+SacI dig(EP)

Pcons+RBS+SalI dig(EP)

Pcons+RBS+AseI dig(EP)

Pcons+RBS+KpnI dig(EP)

Ligation of

Pcons+RBS+BFP+Ter-30 Kr

9/12

Transformation of

Pcons+RBS+NheI+RBS+BFP+Ter Kr

Pcons+RBS+NdeI+RBS+BFP+Ter Kr

Pcons+RBS+HindIII+RBS+BFP+Ter Kr

Pcons+RBS+KpnI-HF+RBS+BFP+Ter Kr

Pcons+RBS+AseI+RBS+BFP+Ter Kr

Pcons+RBS+SacI+RBS+BFP+Ter Kr

Pcons+RBS+NcoI+RBS+BFP+Ter Kr

Pcons+RBS+SalI+RBS+BFP+Ter Kr

Pcons+RBS+BFP+Ter Kr

Pcons+RBS+XhoI Ar

Electrophoresis of

Well 1 Marker

Well 2 Pcons+RBS+NdeI dig(EP) Cr

Well 3 Pcons+RBS+NcoI dig(EP) Cr

Well 4 Pcons+RBS+SacI dig(EP) Ar

Well 5 Pcons+RBS+SalI dig(EP) Ar

Well 6 Pcons+RBS+AseI dig(EP) Ar

Well 7 Pcons+RBS+HindIII dig(EP) Ar

Well 8 Pcons+RBS+NheI dig(EP) Ar

Well 9 Pcons+RBS+KpnI dig(EP) Ar

9/13

Ligation of

Pcons+RBS+AseI Kr

Pcons+RBS+NdeI Kr

Pcons+RBS+SalI Kr

Pcons+RBS+SacI Kr

PCR of

Pcons+RBS+HindIII+RBS+BFP+Ter Kr x8

Pcons+RBS+NheI+RBS+BFP+Ter Kr x8

Pcons+RBS+NdeI+RBS+BFP+Ter Kr x8

Pcons+RBS+NcoI+RBS+BFP+Ter Kr x8

Pcons+RBS+KpnI-HF+RBS+BFP+Ter Kr x8

Pcons+RBS+AseI+RBS+BFP+Ter Kr x8

Pcons+RBS+Xhol Kr x8

Pcons+RBS+SalI+RBS+BFP+Ter Kr x16

Pcons+RBS+SacI+RBS+BFP+Ter Kr x16

Electrophoresis of

Gel 1

Pcons+RBS+SalI+RBS+BFP+Ter Kr x16

Gel 2

Pcons+RBS+SacI+RBS+BFP+Ter Kr x16

Gel 3

Pcons+RBS+HindIII+RBS+BFP+Ter Kr x8

Pcons+RBS+NheI+RBS+BFP+Ter Kr x8

Gel 4

Pcons+RBS+NdeI+RBS+BFP+Ter Kr x8

Pcons+RBS+NcoI+RBS+BFP+Ter Kr x8

Gel 5

Pcons+RBS+KpnI-HF+RBS+BFP+Ter Kr x8

Pcons+RBS+AseI+RBS+BFP+Ter Kr x8

Gel 6

Pcons+RBS+Xhol Kr x8

psb1K3 dig(EP)


9/16

Transformation of

psb3K3 *2

psb3C3

Digestion of

psb3K3 dig(EP)

psb1C3 dig(EP)

Cultivation of

Pcons+RBS+PBAN+RBS+BFP+Ter x8

9/17

Mini-prep of

PBAN：XhoI

SalI

SacI

KpnI

Ndel

AseI

NcoI

NheI

HindIII (fail)

Pcons+RBS+PBAN+RBS+BFP+Ter：AseI-4

Ndel-8

HindIII-4 (fail)

SacI-9(fail)

KpnI-1

NcoI-2

NheI-1

Digestion of

PBAN：XhoI

SalI

SacI

KpnI

Ndel

AseI

NcoI

AseI

Pcons+RBS+PBAN+RBS+BFP+Ter：AseI-4

Ndel-8

KpnI-1

NcoI-2

NheI-1

Electrophoresis of

Pcons+RBS+PBAN+RBS+BFP+Ter (Ndel)

Pcons+RBS+PBAN+RBS+BFP+Ter (NcoI)

Pcons+RBS+PBAN+RBS+BFP+Ter (SacI)

Pcons+RBS+PBAN+RBS+BFP+Ter (HindIII)

Pcons+RBS+PBAN+RBS+BFP+Ter (SalI)

Pcons+RBS+PBAN+RBS+BFP+Ter (NheI)

Pcons+RBS+PBAN+RBS+BFP+Ter (KpnI)

Pcons+RBS+PBAN+RBS+BFP+Ter (AseI)

PBAN AseI

PBAN Ndel

PBAN KpnI

PBAN SacI

PBAN SalI

PBAN XhoI

Pcons+RBS+XhoI-1 dig(EP)

Pcons+RBS+Xhol-1 dig(ES)

psb1C3 dig(ES)

Cultivation of

Pcons+RBS+PBAN+RBS+BFP+Ter (SacI)-4.12

Pcons+RBS+PBAN+RBS+BFP+Ter (HindIII)-6

Pcons+RBS+PBAN+RBS+BFP+Ter (SalI)-9

Digestion of

B0034 Kr dig(ES)

Pcons+RBS+AseI+RBS+BFP+Ter Kr dig(EP)

Pcons+RBS+NdeI+RBS+BFP+Ter Kr dig(EP)

NcoI Kr dig(EP)

Ndel Kr dig(EP)

SalI Kr dig(EP)

9/18

Electrophoresis of

Well 1 Marker

Well 2 B0034 Kr dig(ES)

Well 3 Pcons+RBS+AseI+RBS+BFP+Ter Kr dig(EP)

Well 4 Pcons+RBS+NdeI+RBS+BFP+Ter Kr dig(EP)

Well 5 NcoI Kr dig(EP)

Well 6 Ndel Kr dig(EP)

Well 7 SalI Kr dig(EP)

Electrophoresis of

Well 1 Marker

Well 2 B0034 Kr dig(ES)

Well 3 Pcons+RBS+AseI+RBS+BFP+Ter Kr dig(EP)

Well 4 Pcons+RBS+NdeI+RBS+BFP+Ter Kr dig(EP)

Well 5 NcoI Kr dig(EP)

Well 6 Ndel Kr dig(EP)

Well 7 SalI Kr dig(EP)

Mini-prep of

Pcons+RBS+HindIII+RBS+BFP+Ter-6

Pcons+RBS+SalI+RBS+BFP+Ter-9

Pcons+RBS+SacI+RBS+BFP+Ter-4

Pcons+RBS+SacI+RBS+BFP+Ter-11

Ligation of

Pcons+RBS+AseI+RBS+BFP+Ter Cr

Pcons+RBS+NdeI+RBS+BFP+Ter Cr

NheI Cr

NcoI Cr

9/20

Transformation of

AseI mini Cr

Electrophoresis of

Well 1 Marker

Well 2 HindIII-2 Kr dig(EP)

Well 3 SacI Kr dig(EP)

Well 4 Pcons+RBS+HindIII-6+RBS+BFP+Ter Kr dig(EP)

Well 5 Pcons+RBS+SacI-11+RBS+BFP+Ter Kr dig(EP)

Well 6 Pcons+RBS+SacI-9+RBS+BFP+Ter Kr dig(EP)

Well 7 RBS+BFP+Ter Ar dig(XP)

Well 8 Pcons+RBS+NheI+RBS+BFP+Ter Kr dig(EP)


9/21

Electrophoresis of PCR products of

Gel 1

Well 1 Marker

Well 2 3H1

Well 3 3H2

Well 4 3H3

Well 5 3H4

Well 6 3H5

Well 7 3H6

Well 8 Marker

Well 9 6K1

Well 10 6K2

Well 11 6K3

Well 12 6K4

Well 13 6K5

Well 14 6K6

Well 15 3Sl1

Well 16 3Sl2

Well 17 3Sl3

Gel 2

Well 1 Marker

Well 2 6Sl4

Well 3 6Sl5

Well 4 3Nh1

Well 5 3Nh2

Well 6 3Nh3

Well 7 3Nh4

Well 8 Marker

Well 9 3Nh5

Well 10 3Nh6

Well 11 6X1

Well 12 6X2

Well 13 6X3

Well 14 6X4

Well 15 6X5

Well 16 6X6

Well 17 1K1

Gel 3

Well 1 Marker

Well 2 1K2

Well 3 1K3

Well 4 1K4

Well 5 1K5

Well 6 1K6

Well 7 1X1

Well 8 Marker

Well 9 1X2

Well 10 1X3

Well 11 1X4

Well 12 1X5

Well 13 1X6

Well 14 3K1

Well 15 3K2

Well 16 3K3

Well 17 3K4

Gel 4

Well 1 Marker

Well 2 3K5

Well 3 3K6

Well 4 3Sc1

Well 5 3Sc2

Well 6 3Sc3

Well 7 3Sc4

Well 8 Marker

Well 9 3Sc5

Well 10 3Sc6

Well 11 1Sc1

Well 12 1Sc2

Well 13 1Sc3

Well 14 1Sc4

Well 15 1Sc5

Well 16 1Sc6

Well 17 1Nc1

Gel 5

Well 1 Marker

Well 2 1Nc2

Well 3 1Nc3

Well 4 1Nc4

Well 5 1Nc5

Well 6 1Nc6

Well 7 1A1

Well 8 Marker

Well 9 1A2

Well 10 1A3

Well 11 1A4

Well 12 1A5

Well 13 1A6

Well 14 1Nd1

Well 15 1Nd2

Well 16 1Nd3

Well 17 1Nd4

Gel 6

Well 1 Marker

Well 2 1Nd5

Well 3 1Nd6

Well 4 3X1

Well 5 3X2

Well 6 3X3

Well 7 3X4

Well 8 Marker

Well 9 3X5

Well 10 3X6

Well 11 3A1

Well 12 3A2

Well 13 3A3

Well 14 3A4

Well 15 3A5

Well 16 3A6

Cultivation of

KpnI-1

NdeI-1

XhoI-1

SacI-1

AseI-1

Pcons+RBS+HindIII-1

Pcons+RBS+KpnI-4

Pcons+RBS+NheI-4

Pcons+RBS+SalI-5

Pcons+RBS+AseI-5

Pcons+RBS+XhoI-1

Pcons+RBS+SacI-1

Pcons+RBS+KpnI-1+RBS+BFP+Ter

Pcons+RBS+XhoI-6+RBS+BFP+Ter

Digestion of

SalI Kr dig(EP)

Ligation of

NdeI dig(EP)+C backbone

9/22

Transformation of

NheI Cr

Pcons+RBS+NheI+RBS+BFP+Ter Cr

Pcons+RBS+NcoI+RBS+BFP+Ter Cr

Pcons+RBS+Ndel+RBS+BFP+Ter Cr

Pcons+RBS+AseI+RBS+BFP+Ter Cr

Digestion of

HindIII(EP)

Cultivation of

SacI-3 Kr

NcoI-7 Cr

NcoI-8 Cr

Pcons+RBS+SacI-2

Pcons+RBS+XhoI-5+RBS+BFP+Ter

pbs3C5

Ligation of

Pcons+RBS+NcoI+RBS+BFP+Ter Cr

Pcons+RBS+HindIII+RBS+BFP+Ter Cr

Pcons+RBS+SacI+RBS+BFP+Ter Cr

Pcons+RBS+SalI+RBS+BFP+Ter Cr

9/23

Electrophoresis of

Well 1 Marker

Well 2 KpnI-1 Cr dig(EP)

Well 3 NdeI-1 Cr dig(EP)

Well 4 XhoI-1 Cr dig(EP)

Well 5 SacI-1 Cr dig(EP)

Well 6 AseI-1 Cr dig(EP)

Well 7 Pcons+RBS+HindIII-1 dig(EP)

Well 8 Pcons+RBS +KpnI-4 dig(EP)

Well 9 Pcons+RBS+NheI-4 dig(EP)

Well 10 Pcons+RBS+AseI-5 dig(EP)

Well 11 Pcons+RBS+XhoI-1 dig(EP)

Well 12 Pcons+RBS+SacI-1 dig(EP)

Well 13 Pcons+RBS+KpnI-7+RBS+BFP+Ter dig(EP)

Well 14 SalI Kr dig(EP) 9/21

Well 15 SalI Kr dig(EP) 9/19

DNA sequencing of

KpnI-1 Cr dig(EP)

NdeI-1 Cr dig(EP)

XhoI-1 Cr dig(EP)

SacI-1 Cr dig(EP)

AseI-1 Cr dig(EP)

Pcons+RBS+HindIII-1 dig(EP)

Pcons+RBS+KpnI-4 dig(EP)

Pcons+RBS+NheI-4 dig(EP)(fail)

Pcons+RBS+AseI-5 dig(EP)

Pcons+RBS+XhoI-1 dig(EP)

Pcons+RBS+SacI-1 dig(EP)

Pcons+RBS+KpnI-7+RBS+BFP+Ter dig(EP)

Transformation of

Pcons+RBS+HindIII+RBS+BFP+Ter

Pcons+RBS+NcoI+RBS+BFP+Ter

Pcons+RBS+SalI+RBS+BFP+Ter

Pcons+RBS+SacI+RBS+BFP+Ter

9/24

Cultivation of

Pcons+RBS+NheI+RBS+BFP+Ter Cr

Pcons+RBS+AseI+RBS+BFP+Ter Cr

Pcons+RBS+NcoI+RBS+BFP+Ter Cr

Pcons+RBS+NdeI+RBS+BFP+Ter Cr

NheI Cr

psb3C5 Cr

Cultivation of

Pcons+RBS+NheI+RBS+BFP+Ter Cr

Pcons+RBS+AseI+RBS+BFP+Ter Cr

Pcons+RBS+NcoI+RBS+BFP+Ter Cr

Pcons+RBS+NdeI+RBS+BFP+Ter Cr

NheI Cr

psb3C5 Cr

Electrophoresis of

Well 1 Marker

Well 2 SalI Kr dig(EP)

Well 3 Pcons+RBS+XhoI+RBS+BFP+Ter Cr dig(EP)

Well 4 Pcons+RBS+SacI Cr dig(EP)

Well 5 NcoI Cr dig(EP)

Digestion of

Pcons+RBS+XhoI+RBS+BFP+Ter Cr dig(EP)

Pcons+RBS+SacI-2 Cr dig(EP)

NcoI-8 Cr dig(EP)

SalI-3 Kr dig(EP)

Ligation of

HindIII+psb1C3

PCR of

SacI x8

SalI x5

HindIII x2

NheI x8

Mini-prep and digestion of

Pcons+RBS+AseI+RBS+BFP+Ter

Pcons+RBS+NheI+RBS+BFP+Ter

Pcons+RBS+NdeI+RBS+BFP+Ter

Pcons+RBS+NcoI+RBS+BFP+Ter

psb3C5

NheI

9/25

Electrophoresis and DNA sequencing of

Well 1 Marker

Well 2 NheI (fail)

Well 3 Pcons+RBS+AseI+RBS+BFP+Ter

Well 4 Pcons+RBS+NcoI+RBS+BFP+Ter

Well 5 Pcons+RBS+NdeI+RBS+BFP+Ter (fail)

Transformation of

HindIII

SalI

Pcons+RBS+SacI+RBS+BFP+Ter

Pcons+RBS+SalI+RBS+BFP+Ter

Pcons+RBS+HindIII+RBS+BFP+Ter

PCR of

NcoI x5(9.10.11.12.13)

Pcons+RBS+SacI x5(7.8.9.10.11)

Cultivation of

NheI

Pcons+RBS+SacI-2(9/22)

Pcons+RBS+NcoI-8(9/22)

Pcons+RBS+NheI-4

Pcons+RBS+NdeI+RBS+BFP+Ter x2

Pcons+RBS+NheI+RBS+BFP+Ter x2

9/26

Mini-prep and digestion(EP) of

NcoI

NheI

Pcons+RBS+SacI-2

Pcons+RBS+NheI-4

Pcons+RBS+NdeI-1+RBS+BFP+Ter

Pcons+RBS+NdeI-2+RBS+BFP+Ter

Electrophoresis of

Well 1 Marker

Well 2 Pcons+RBS+NdeI-1+RBS+BFP+Ter

Well 3 Pcons+RBS+NdeI-2+RBS+BFP+Ter

Well 4 NcoI

Well 5 NheI

Well 6 Pcons+RBS+NheI-4

Well 7 Pcons+RBS+SacI-2

Cultivation of

psb3C5 x2

HindIII-3

HindIII-4

SalI-3

SalI-4

Pcons+RBS+HindIII-3+RBS+BFP+Ter

Pcons+RBS+HindIII-4+RBS+BFP+Ter

Pcons+RBS+SalI-3+RBS+BFP+Ter

Pcons+RBS+SalI-4+RBS+BFP+Ter

Pcons+RBS+SacI-3+RBS+BFP+Ter

Pcons+RBS+SacI-4+RBS+BFP+Ter

Pcons+RBS+NcoI-3+RBS+BFP+Ter

Pcons+RBS+NcoI-4+RBS+BFP+Ter


9/28

Electrophoresis of

Well 1 HindIII-3

Well 2 HindIII-4

Well 3 SalI-3

Well 4 SalI-4

Well 5 Pcons+RBS+SacI-3+RBS+BFP+Ter

Well 6 Marker

Well 7 Pcons+RBS+NcoI-3+RBS+BFP+Ter

Well 8 Pcons+RBS+NcoI-4+RBS+BFP+Ter

Well 9 Pcons+RBS+SalI-3+RBS+BFP+Ter

Well 10 Pcons+RBS+SalI-4+RBS+BFP+Ter

Well 11 psb3C5

9/29

Electrophoresis of

Well 1 Marker

Well 2 Pcons+RBS+HindIII+RBS+BFP+Ter

Well 3 Pcons+RBS+HindIII+RBS+BFP+Ter

Well 4 Pcons+RBS+HindIII+RBS+BFP+Ter

Well 5 Pcons+RBS+HindIII+RBS+BFP+Ter

Well 6 Pcons+RBS+HindIII+RBS+BFP+Ter

Well 7 Pcons+RBS+HindIII+RBS+BFP+Ter

Well 8 Pcons+RBS+NcoI+RBS+BFP+Ter

Well 9 Pcons+RBS+NcoI+RBS+BFP+Ter

Well 10 Pcons+RBS+NcoI+RBS+BFP+Ter

Well 11 Pcons+RBS+NcoI+RBS+BFP+Ter

Well 12 Pcons+RBS+NcoI+RBS+BFP+Ter

Well 13 Pcons+RBS+NcoI+RBS+BFP+Ter

Well 14 Pcons+RBS+SalI+RBS+BFP+Ter

Well 15 Pcons+RBS+SalI+RBS+BFP+Ter

Well 16 Pcons+RBS+SalI+RBS+BFP+Ter

Well 17 Pcons+RBS+SalI+RBS+BFP+Ter

Well 18 Pcons+RBS+SalI+RBS+BFP+Ter

Well 19 Pcons+RBS+SalI+RBS+BFP+Ter

Well 20 psb3C5 dig

Well 21 Marker

Ligation of

Pcons+RBS+KpnI + pbs3C5

Pcons+RBS+SacI + pbs3C5

Pcons+RBS+AseI + pbs3C5

Pcons+RBS+HindIII + pbs3C5

Pcons+RBS+NheI + pbs3C5

Pcons+RBS+SalI + pbs3C5

Pcons+RBS+NcoI + pbs3C5

Pcons+RBS+NdeI + pbs3C5

Pcons+RBS+XhoI+RBS+BFP+Ter + pbs1C3

Pcons+RBS+NdeI+RBS+BFP+Ter + pbs1C3

Pcons+RBS+SacI + pbs1C3

Pcons+RBS+NcoI + pbs1C3

DNA sequencing of

SalI-4

HindIII-4

Pcons+RBS+SalI-3+RBS+BFP+Ter

Pcons+RBS+SalI-2

Pcons+RBS+NheI-4

NheI(9/26)

Pcons+RBS+HindIII+RBS+BFP+Ter(9/30)

Pcons+RBS+SalI+RBS+BFP+Ter(9/30)

9/30

PCR and Electrophoresis of

Pcons+RBS+NdeI+RBS+BFP+Ter

NcoI

Pcons+RBS+NheI+RBS+BFP+Ter

Pcons+RBS+XhoI+RBS+BFP+Ter

Pcons+RBS+SacI+RBS+BFP+Ter

Cultivation of

Pcons+RBS+NheI-11+RBS+BFP+Ter

Pcons+RBS+NheI-13+RBS+BFP+Ter

Pcons+RBS+NdeI-3+RBS+BFP+Ter

Pcons+RBS+NdeI-10+RBS+BFP+Ter

Pcons+RBS+XhoI-3+RBS+BFP+Ter

Pcons+RBS+XhoI-5+RBS+BFP+Ter

Pcons+RBS+SacI-2+RBS+BFP+Ter

Pcons+RBS+SacI-11+RBS+BFP+Ter

NcoI-3

NcoI-12


October 2014

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10/3

Cultivation of

[Pcons+RBS+NdeI]Cr

[Pcons+RBS+NcoI]Cr

[KpnI-1]Cr

[NdeI-1]Cr

[XhoI-1]Cr

[SacI-1]Cr

[AseI-1]Cr

[Pcons+RBS+HindIII-1]Cr

[Pcons+RBS+KpnI-4]Cr

[Pcons+RBS+AseI-5]Cr

[Pcons+RBS+XhoI-1]Cr

[Pcons+RBS+SacI-1]Cr

[Pcons+RBS+KpnI-1+J61048]Cr

[SalI-4]Cr

[Pcons+RBS+SalI-3+J61048]Cr

[HindIII-4]Cr

[Pcons+RBS+AseI+J61048]Cr

[NheI]

[NcoI]

Electrophoresis of

[NdeI](EP)

[NcoI](EP)

[SacI](EP)

Transformation of

[Pcons+RBS+NdeI]Cr

[Pcons+RBS+SacI]Cr

DNA Sequencing of

[Pcons+RBS+NdeI+J61048-1]

[Pcons+RBS+HindIII+J61048-1]

[Pcons+RBS+SalI+J61048-4]

[Pcons+RBS+SacI+J61048-1]

[Pcons+RBS+SacI+J61048-1](fail)

10/4

Digestion of

[Pcons+RBS+XhoI+J61048-2, -4, -5]

[Pcons+RBS+SacI+J61048-10, -11]

[Pcons+RBS+NheI+J61048-20]

[Pcons+RBS+NheI]Cr

[Pcons+RBS+NcoI]Cr

Cultivation of

[Pcons+RBS+NheI-1, -2, -4, -6, -10, -12]

[Pcons+RBS+SalI-1, -3, -6, -7, -11, -12]

Cultivation of

[Pcons+RBS+NcoI+J61048-1~8]

PCR of

[Pcons+RBS+NheI-1, -2, -4, -6, -10, -12]

[Pcons+RBS+SacI-1, -3, -6, -7, -11, -12]

[Pcons+RBS+NcoI+J61048-1~8]

Ligation of

[Pcons+RBS+NheI]Kr

[Pcons+RBS+NcoI]Kr

Electrophoresis of

[XhoI-2, -4, -5](dig)

[SacI-10, -11](dig)

[NheI-20](dig)

[Pcons+RBS+NdeI](dig)

[Pcons+RBS+NcoI](dig)

[PCR NcoI-1~8]

Mini-prep of

[Pcons+RBS+NheI-6]

[Pcons+RBS+SalI-1, -7, -11]

Digestion of

[Pcons+RBS+NheI-6](EP)

[Pcons+RBS+SalI-1, -7, -11](EP)

[Pcons+RBS+NcoI]

[Pcons+RBS+Ndel]

PCR of and cultivation of

[Pcons+RBS+NcoI+J61048]

Mini-prep of

[Pcons+RBS+NcoI+J61048-7]Cr

Electrophoresis of

[Pcons+RBS+KphI+J61048-1]Cr

[Pcons+RBS+SalI+J61048-3]Cr

[Pcons+RBS+AseI+J61048 ]Cr

[Pcons+RBS+NdeI+J61048-1]

[Pcons+RBS+HindIII+J61048-1]

[Pcons+RBS+SacI+J61048 -1]

[Pcons+RBS+NcoI+J61048](dig)

[SacI-11]

DNA Sequencing of

[Pcons+RBS+NdeI]-2 Cr

[Pcons+RBS+NcoI]-2 Cr

[Pcons+RBS+NheI]-6 Cr

[Pcons+RBS+SalI-1]Cr

[Pcons+RBS+SalI-11]Cr

[Pcons+RBS+HindIII-1]Cr

[NcoI-12]

[Pcons+RBS+KpnI-1+J61048]Cr

[Pcons+RBS+AseI+J61048]Cr

[Pcons+RBS+SalI-3+J61048]Cr

[Pcons+RBS+HindIII-1+J61048]Cr

[Pcons+RBS+XhoI-2+J61048]Cr

[Pcons+RBS+XhoI-5+J61048]Cr

[Pcons+RBS+NcoI-5+J61048]Cr

[Pcons+RBS+SacI-11-1+J61048]Cr

[Pcons+RBS+NdeI-1+J61048]Cr

[Pcons+RBS+NheI-20+J61048]Cr

[Pcons+RBS+AseI-7+J61048]Cr

[Pcons+RBS+HindIII-7+J61048]Ar

[Pcons+RBS+SacI-16+J61048]Ar

[Pcons+RBS+SalI-17+J61048]Ar

[Pcons+RBS+NdeI-2+J61048]Cr

[Pcons+RBS+NcoI-2+J61048]Cr

[Pcons+RBS+XhoI+J61048]Ar

[Pcons+RBS+KpnI-7+J61048]Ar

[Pcons+RBS+NheI-11+J61048]Ar


10/7

PCR of

[Pcons+RBS+NheI+J61048]

[Pcons+RBS+SalI in PSB3C5]

[Pcons+RBS+SacI in PSB3C5]

Cultivation of

[Pcons+RBS+NheI+J61048-1, -3, -4, -6, -8, -10, -15, -16]

Transformation of

[Pcons+RBS+XhoI-2 +J61048]

[Pcons+RBS+XhoI-5 +J61048-1]

[Pcons+RBS+SacI-11+J61048]

PCR of

[Pcons+RBS+ NdeI in PSB3C5]

[Pcons+RBS+KpnI in PSB3C5]

[Pcons+RBS+AseI in PSB3C5]

[Pcons+RBS+XhoI in PSB3C5]

[Pcons+RBS+NcoI in PSB3C5]

[Pcons+RBS+NheI in PSB3C5]

nine kinds of PBAN

DNA Sequencing of

[Pcons+RBS+NcoI-5+J61048]

10/8

DNA Sequencing of

[PBAN NcoI]

[Pcons+RBS+SalI -1]

[Pcons+RBS+SalI-11]

[Pcons+RBS+NheI-6]

[Pcons+RBS+xhol-2+RBS+BFP+TER]

[Pcons+RBS+SacI-11+RBS+BFP+TER]

[Pcons+RBS+ Nhel-20+RBS+BFP+TER]

Ligation of

Pcons+RBS+NcoI on psb3c5

Pcons+RBS+NcoI on psb3c5

10/9

ALL Nhel-9(10/7)x2

ALL SacI-11(10/7)x1

ALL xhol-2(10/7)x1

Electrophoresis of

Gel 1

Well 1 Marker

Well 2 Pcons+RBS+NheI-20+RBS+BFP+TER

Well 3 Pcons+RBS+Hind-1+RBS+BFP+TER

Well 4 Pcons+RBS+KpnI-1+RBS+BFP+TER

Well 5 Pcons+RBS+Ndel-1+RBS+BFP+TER

2 Well 6 Pcons+RBS+NcoI-5+RBS+BFP+TER

Well 7 Pcons+RBS+AseI ALL

Well 8 PBAN NcoI-12

Well 9 Pcons+RBS+NheI-6

Well 10 Pcons+RBS+Hind-1

Well 11 Pcons+RBS+NcoI-20

Well 12 Pcons+RBS+NcoI-2-2

Well 13 Pcons+RBS+Ndel-2-1

Well 14 Pcons+RBS+Nedl-2-2

Well 15 Pcons+RBS+SacI-1

Well 16 Pcons+RBS+SacI-11

Well 17 Marker

Gel 2

Well 1 marker

Well 2 Pcons+RBS+NheI-11

Well 3 Pcons+RBS+Hind3

Well 4 Pcons+RBS+KpnI-7

Well 5 Pcons+RBS+SacI-16

Well 6 Pcons+RBS+SalI-12

Well 7 Pcons+RBS+Xhol

Transformation of

Pcons+RBS+SacI-3 on psb3c5

Pcons+RBS+KpnI-1 on psb3c5

Pcons+RBS+NcoI-1 on psb3c5

Mini-prep of and sequencing of

ALL NheI-2 x1

ALL SacI-11 x1

ALL Xhol-2 X1

10/10

Transformation of

Pcons+RBS+Ndel on psb3c5

Pcons+RBS+NcoI on psb3c5

Electrophoresis of

Well 1 Marker

Well 2 Pcons+RBS+SacI-3 on psb3c5

Well 3 Pcons+RBS+KpnI-1 on psb3c5

Well 4 Pcons+RBS+NcoI-1 on psb3c5


10/12

PCR of and Electrophoresis of

Well 1 Pcons+RBS+NcoI+psb3c5

Well 2 Pcons+RBS+Ndel+psb3c5


None


None


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