Team:NCTU Formosa/Notebook

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Notebook

Contents

About The Notes

Our notes are the abbreviations of the experimental process, including titles, genes we transformed, plasmids we used, and resistances on the plasmids. We categorized these notes by date. Moreover, team members followed the iGEM protocol in each experimental step in order to get better experimental efficiency and the results we had expected.

Material and Methods

Wet Lab Protocol

Insect Experiment Protocol

In order to test our PBAN device, we had to conduct many insect experiments, we chose Spodoptera Litura as experimental lead in modeling experiments and PBAN tests because the vitality of Spodoptera Litura in Taiwan just peaked at that time. Some things needed to be prepared well before every test and experiment, we needed many devices to keep and catch Spodoptera Litura and fed them and also made some simple instruments to conduct insect experiment, the devices we used in insect experiment was listed below:

  1. Several insect-catching nets as well as insect-catching boxes
    2. Insect-catching boxes
    Insect-catching nets

  2. A giant experimental box made by acrylic boards as the space for PBAN experiments and storing Spodoptera Litura , we called it "moth box".
    Moth box
  3. The "food" provided for Spodoptera Litura includs some flowers which contained nectar and 10%~15% sucrose solution, sucrose solution was put in the beaker, and beaker was covered by handi-wrap, tissue papers were used to help moths suck the solution
    Sucrose solution
  4. A barrel equipped with several lights, we used this barrel in experiment of Spodoptera Litura hobby corresponding to changes of temperature and light, we called it " CHOU CHUN-WEI No.1".
    CHUNG-WEI CHIOU No.1
  5. Spodoptera Litura, we caught many Spodoptera Litura from organic farm by light traps and insect-catching nets. In addition, we also bred them by ourselves, in a word, we had enough amount of Spodoptera Litura to conduct experiment
    6. Larvae of Spodoptera Litura
    Chrysalis of Spodoptera Litura

  6. PBAN product made by our E.coli device. After E.coli expresses PBAN, we used instrument to sonicate E.coli, and then sterilized with autoclave
  7. PBAN container, beakers which passed through changing became PBAN container, handi-wrap distributed a beaker into two layers, the PBAN product or sucrose solution was placed in the lower layer, one or more female moths were placed in the higher layer, and each beaker was covered by handi-wrap with many holes
  8. A professional digital camera, we used it to take many photos and videos, sometimes we also used smartphone to do that.
  9. Pheromone traps and pheromone, we bought these things from internet and used them to catch male Spodoptera Litura in order that we had enough moths to conduct insect experiment
    10. Pheromone trap 1.jpg
    Pheromone trap2.jpg
    Pheromone trap 3.jpg
    Pheromone trap 4.jpg
    Pheromone trap.jpg

  10. Last but not least, our pyramidal device .
    11. NCTU Formosa 2014 Project Device1.jpg

    As to the process of insect experiment, you can read the details and methods at the "Insect Aspect" in the Result region of our web page.


    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


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