Team:NCTU Formosa/Notebook

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====Insect Experiment 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:
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:
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1.Several insect-catching nets as well as insect-catching boxes
1.Several insect-catching nets as well as insect-catching boxes
[[File:Insect-catching box.jpg|thumb|left|300px| Insect-catching boxes]]
[[File:Insect-catching box.jpg|thumb|left|300px| Insect-catching boxes]]
[[File:Insect-catching nets.jpg|thumb|left|100px| Insect-catching nets]]
[[File:Insect-catching nets.jpg|thumb|left|100px| Insect-catching nets]]
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2.A giant experimental box made by acrylic boards as the space for PBAN experiments and storing ''Spodoptera Litura'' , we called it "moth box".[[File:Moth box.jpg|thumb|center|300px|Moth box]]  
2.A giant experimental box made by acrylic boards as the space for PBAN experiments and storing ''Spodoptera Litura'' , we called it "moth box".[[File:Moth box.jpg|thumb|center|300px|Moth box]]  
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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 [[File:Sucrose solution.jpg|thumb|center|100px| Sucrose solution]]
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 [[File:Sucrose solution.jpg|thumb|center|100px| Sucrose solution]]
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</div><div class="left">
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".
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".
[[File:CCW_Model.JPG|thumb|center|300px|CHOU CHUN-WEI No.1 (CCW No.1)]]
[[File:CCW_Model.JPG|thumb|center|300px|CHOU CHUN-WEI No.1 (CCW No.1)]]
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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 [[File:Larvae of Spodoptera Litura .jpg|thumb|left|300px|Larvae of ''Spodoptera Litura'' ]][[File:Chrysalis o fSpodoptera Litura .jpg|thumb|left|300px|Chrysalis of ''Spodoptera Litura'' ]][[File:Spodoptera Litura.jpg|thumb|left|200px| ''Spodoptera Litura'' ]]
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 [[File:Larvae of Spodoptera Litura .jpg|thumb|left|300px|Larvae of ''Spodoptera Litura'' ]][[File:Chrysalis o fSpodoptera Litura .jpg|thumb|left|300px|Chrysalis of ''Spodoptera Litura'' ]][[File:Spodoptera Litura.jpg|thumb|left|200px| ''Spodoptera Litura'' ]]
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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  
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  
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</div><div class="left">
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
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
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8.A professional digital camera, we used it to take many photos and videos, sometimes we also used smartphone to do that.
8.A professional digital camera, we used it to take many photos and videos, sometimes we also used smartphone to do that.
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9.Last but not least, our pyramidal device.
9.Last but not least, our pyramidal device.
[[File:NCTU_Formosa_2014_Project_Device1.jpg|thumb|center|300px|Our essential device]]
[[File:NCTU_Formosa_2014_Project_Device1.jpg|thumb|center|300px|Our essential device]]
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.
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.
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Revision as of 13:36, 15 October 2014

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

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".

CHOU CHUN-WEI No.1 (CCW 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
Larvae of Spodoptera Litura
Chrysalis of Spodoptera Litura
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.Last but not least, our pyramidal device.

Our essential device

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


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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