Team:NCTU Formosa/notes

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Notes

The experimental log that records down the purpose, method, result and date of each experiment that we have conducted.

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.

March 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

24

25

26

27

28

29

  • Transformation of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) and cultivation on three LB plates.

  • transformation of PompC and cultivation on LB-A plate

30

  • Single colony isolation from three plates and cultivation them in liquid LB

  • Single colony isolation from PompC LB-C plate and cultivation in liquid LB-A

  • mini-prep of cultivated PompC E.coli

31

  • Transformation of RBS B0030+PSB1A3&B0034+PSB1A3 and cultivation on LB-A plate.

  • Mini-prep for cultivated ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) PCR of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar).

  • Electrophoresis of ho1(Kr), Cph8(Cr), 32℃ RBS(Ar) ──NOT OK.

  • PCR of insert fragment [PompC+psB1A2]

April 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

  • Cultivation of B0030&B0034 Ar E.coli in liquid LB-A tubes.

2

  • Mini-prep of cultivated B0030&B0034 E.coli and then digestion: [B0030]XP&[B0034]XP.

3

4

5

6

  • Electrophoresis of [B0030]XP&[B0034]XP Not OK.

  • PCR of ho1 to check ho1 and transform to test the resistance.

7

  • Electrophoresis of [B0030]mini&dig XP and [B0034]mini&dig XP OK

  • Cultivation of B0030&B0034 Ar E.coli in liquid LB-A tubes.

  • Cultivation of ho1 in liquid LB-K and LB-K plate

  • Cultivation of Cph8+RBS in liquid LB-C.

    • 8

    • Mini-prep of cultivated B0030&B0034 Ar E.coli. Mini-prep of ho1 & Cph8+RBS.

    9

    • Electrophoresis of mini ho1&Cph8+RBS.

    10

    11

    • PCR of mini ho1&Cph8+RBS

    • Electrophoresis of ho1&Cph8+RBS(After PCR)

    • Digestion: [ho1]EP&[Cph8+RBS]EP.

    12

    • Transformation of Plac&Ptet& pcyA but there is no colony appearing in pcyA plate.

    • Electrophoresis of digested ho1&Cph8+RBS (NOT OK).

    13

    14

    • Cultivation of Ptet&Plac E.coli in LB tubes.

    • Transformation: LuxR,B0015,J61048 and cultivation on LB-A plate

    15

    • Mini-prep of cultivated Ptet&Plac E.coli

    • Digestion:[Ptet]ES&[Plac]ES PCR of insert fragment pcyA

    • Electrophoresis of [Ptet]dig ES&[pcyA]PCR&[Plac]dig ES.

    • Single colony isolation from J61048,B0015,LuxR plate and cultivation in liquid LB-A mini-prep of cultivated LuxR,J61048,B0015

    16

    • PCR of mini Cph8+RBS

    • Electrophoresis of Cph8+RBS(After PCR)──NOT OK.

    • digestion: [J61048]EP,[B0015]EP,[LuxR]EP

    • Electrophoresis of ter. mini [J61048] dig E/EP and t er. mini [B0015] dig E/EP and luxR mini dig E/EP

    17

    • PCR of mini Cph8+RBS Electrophoresis of Cph8+RBS(After PCR)──NOT OK.

    • Electrophoresis of the products of digestion of ter. [J61048], ter. [B0015] , luxR

    • transformation of tetR plasmid and cultivation on LB –A plate 3. Digestion: ter. [J61048], ter. [B0015] dig EP

    18

    19

    • Cultivation of pcyA Kr E.coli on LB-K plates

    • mini-prep of cultivated tetR E.coli

    • digestion: tetR+pSB1A2 dig EP

    • electrophoresis of tetR dig EP,ter.[J61048 ] dig EP and ter.[B0015] dig EP

    20

    • Cultivation of pcyA Kr E.coli in liquid LB-K tubes.

    • PCR of tetR,ter.[J61048] and ter.[B0015 ]mini

    • electrophoresis of PCR products of tetR mini,J61048 mini and B0015 mini

    21

    • Single colony isolation of pcyA Kr E.coli

    • Mini-prep of cultivated pcyA Kr E.coli

    • Digestion:[pcyA]ES

    • Electrophoresis of [pcyA]mini&dig ES OK

    • PCR of tetR,ter.[J61048] and ter.[B0015 ]mini

    • electrophoresis of PCR products of tetR mini,J61048 mini and B0015 mini

    • electrophoresis of the digestion products of tetR dig EP and B0015 dig EP

    • aliquot every 20ul of PompC,J61048 and LuxR

    22

    23

    24

    25

    26

    27

    28

    • Transformation of PompC mini and cultivation on LB-A plate

    29

    • transformation of PompC , Ar mini and cultivation on LB-A plate

    • single colony isolation from PompC , Ar

    30

    • Transformation of 37ﾟC RBS and ho1.

    • Mini-prep of cultivated PompC , Ar E.coli

    May 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • Cultivation of 37ﾟC RBS and ho1

    • Digestion: [pSB1C3] EP&[B0030]ES&{pcyA]XP

    • Transformation of pSB1A3 &pSB1K3

    • Transformation of mGFP[pSB1A2] and cultivation on LB-A plate.

    2

    • Cultivation of PompC&B0034&LacI&Plac&B0030&TetR&J61048&B0015 Ar E.coli in liquid LB-A tubes.

    • Cultivation of pSB1A3&pSB1K3 with liquid LB

    • Mini-prep of 37ﾟC RBS and ho1──Fail

    • Ligation: [pSB1C3] EP&[B0030]ES& {pcyA]XP

    • Transformation of B0030

    • Cultivation of 37ﾟC RBS and ho1 with liquid LB

    • Transformation of RBS+pcyA+psB1C3.

    • Transformation of mGFP[pSB1A2] and cultivation on LB-A plate

    3

    • Mini-prep of cultivated PompC &B0034&LacI&Plac&B0030&TetR&J61048&B0015,and then digestion:[PompC ]ES&[B0034]XP&[LacI]ES&[Plac]XP&[B0030]ES&[TetR]ES&[TetR]XP&[J61048]XP&[J61048]ES&[B0015]XP.

    • Mini-prep of ho1,pSB1A3, pSB1K3 and 37ﾟC RBS

    • Digestion: [B0030+pcyA]XP

    • Cultivation of B0030 with liquid LB-C

    • Check RBS+pcyA+psB1C3(PCR & Electrophoresis)──FAIL

    • Digestion: [Pcons]ES& [ho1]XP.

    • Single colony isolation from 37。C RBS Ar,pSB1A3,pSB1C3 and pSB1K3

    • electrophoresis of mGFP dig E

    4

    • Electrophoresis of [PompC]ES&[B0030]ES&[LacI]ES&[TetR]ES&[J61048]XP&[TetR]ES&[B0015]XP OK

    • Ligation: insert [PompC]ES+[B0030]XP&[LacI]ES+[J61048]XP&[J61048]ES+[Plac]XP&[B0030]ES+[TetR]XP&[TetR]ES+[B0015]XP/vector[PSB1C3]EP

    • Transformation of B0030+ho1.

    • Mini-prep of cultivated 37。C RBS Ar pSB1A3,37。C RBS+mGFP, Kr and pSB1K3 E.coli

    • digestion:37。C RBS dig ES,mGFP dig XP,LuxR dig XP and pSB1K3 dig EP

    • electrophoresis of digestion products of LuxR,37。C RBS, mGFP and pSB1K3

    • ligation:37。C RBS+luxR+pSB1K3

    • transformation of 37。C RBS+mGFP, Kr->pSB1K3 and cultivation on LB-K plate

    5

    • Electrophoresis of digested RBS+pcyA──OK

    • Ligation: RBS+ho1

    • Cultivation of J61048 with liquid LB-C

    • Digestion: [pSB1A3]EP &[pSB1K3]EP

    • Transformation of ligated RBS+ho1.

    • Ligation:37。C RBS+luxR+pSB1K3 and 37。C RBS+mGFP+pSB1K3

    • transformation of 37。C RBS+luxR, Kr and 37。C RBS+mGFP ,Kr and cultivation on LB-K plate

    6

    • Mini-prep of J61048

    • Ligation: RBS+pcyA+Pcons&pSB1K3

    • Electrophoresis of J61048.

    • Digestion: [J61048]XP

    • Transformation of RBS+pcyA+Pcons.

    • Check LB-A&C&K plates.

    7

    • Mini-prep of J61048 -Ligation: RBS+pcyA+Pcons&pSB1K3

    • Electrophoresis of J61048.

    • Digestion: [J61048]XP

    • Transformation of RBS+pcyA+Pcons.-Check LB-A&C&K plates.

    8

    • Ligation:insert[PompC]ES+[B0034]XP&[LacI]ES+[J61048]XP&[B0030]ES+[TetR]XP&[TetR]ES+[B0015]XP/vector[PSB1C3]E

    • PCR of 37。RBS+luxR, Kr and 37。C RBS+mGFP ,Kr

    • electrophoresis of 37。RBS+luxR ,Kr and 37。C RBS+mGFP ,Kr

    9

    • Transformation and cultivation of PompC+B0034&LacI+J61048&B0030+TetR&TetR+B0015(PSB1C3) on LB-C plates.

    • PCR of 37。RBS+luxR, Kr and 37。C RBS+mGFP ,Kr

    • electrophoresis of 37。RBS+luxR, Kr and 37。C RBS+mGFP ,Kr

    • single colony isolation from37。RBS+luxR, Kr and 37。C RBS+mGFP ,Kr

    10

    • Cultivation of Pcons+RBS+pcyA and B0030 with liquid LB.

    • Mini-prep of cultivated 37。C RBS+luxR, Kr E.coli and 37。C RBS+mGFP ,Kr E.coli

    • digestion: 37。RBS+luxR, dig ES and 37。C RBS+mGFP dig ES

    11

    • Mini-prep of Pcons+RBS+pcyA.

    • Electrophoresis of Pcons+RBS+pcyA──NOT OK.

    • Electrophoresis of 37。C RBS+mGFP, Kr mini, 37。C RBS+mGFP dig ES, 37。C RBS+luxR, Kr mini and 37。C RBS+luxR dig ES

    12

    • Electrophoresis of 37。C+luxR mini, dig ES and 37。C RBS+mGFP mini,dig ES

    13

    • Cultivation of TetR+B0015 Cr E.coli in liquid LB-C tube

    • Single colony isolation of backbone PSB1K3 E.coli

    • Ligation:insert[B0030]ES+[TetR]XP&[TetR]ES+[B0015]XP/vector[PSB1C3]EP.

    • Electrophoresis of Pcons+RBS+pcyA──NOT OK

    • Transformation of B0032&B0034.

    14

    • Ligation:insert[PompC]ES+[B0034]XP&[LacI]ES+[J61048]XP/vector[PSB1C3]EP.

    • Cultivation of Pcons+RBS+pcyA & B0032&B0034 with liquid LB.

    15

    • Mini-prep of cultivated PSB1K3 E.coli

    • Cultivation of B0015+TetR+PSB1C3 and TetR+B0015+PSB1C3 in liquid LB-C plates and Plac+PSB1A3 in liquid LB-A plate. Mini-prep of Pcons+RBS+pcyA&B0032&B0034.

    16

    • Ligation:insert[PompC]ES+[B0034]XP&[LacI]ES+[J61048]XP/vector[PSB1K3]EP

    • Transformation and cultivation of PompC+B0034&LacI+J61048 (PSB1K3) on LB-K plates.

    17

    • cultivation of PompC+B0034&LacI+J61048 Kr E.coli on liquid LB-K tubes.

    • single colony isolation of PompC+B0034&LacI+J61048 Kr E.coli.

    • Electrophoresis of Pcons+RBS+pcyA(NOT OK) &B0032&B0034

    • Cultivation of Pcons+RBS+pcyA.Digestion: [B0032]ES &[B0023]ES.

    18

    • Cultivation of PompC+B0034&LacI+J61048 Kr E.coli from single colony isolation plate made yesterday

    • Min-prep of cultivated [B0030+TetR]Cr&[ Plac]Ar&[PompC+B0034]Kr&[LacI+J61048]Kr E.coli and then digestion:[PompC+B0034]ES &[LacI+J61048]XP. Mini-prep Pcons+RBS+pcyA

    • Electrophoresis of Pcons+RBS+pcyA& digested B0032&B0034──NOT OK

    • Digestion: [B0032]ES &[B0034]ES &[Pcons+RBS+pcyA]E.

    19

    • Electrophoresis of [LacI+J61048]mini & dig XP and [PompC+B0034]mini & dig ES and [PSB1K3]mini & dig EP.

    • Electrophoresis of digested B0032&B0034&Pcons+RBS+pcyA──OK

    • Transformation of pSB1C3

    20

    • Digestion:[Plac]P.

    • Ligation: Pcons+RBS+pcyA&pSB1K3, B0032+ho1&pSB1C3, B0034+ho1&pSB1C1

    • Transformation of Pcons+RBS+pcyA&pSB1K3, B0032+ho1&pSB1C3, B0034+ho1&pSB1C1

    • Single colony isolation from pSB1C3

    21

    • Single colony isolation of TetR+B0015 Cr Ecoli

    • Electrophoresis of [PompC+B0034]mini&dig ES and[Plac]mini&dig P.

    • Cultivation of Pcons+RBS+pcyA &B0032+ho1 with liquid LB

    • Mini-prep of cultivated pSB1C3 E.coli

    22

    • Digestion:[Plac]XP

    • Electrophoresis of [PompC+B0034]mini&dig ES and[Plac]mini&dig XP

    • Ligation: insert[TetR]ES+[B]XP/vector[PSBC3]EP

    • Mini-prep of Pcons+RBS+pcyA

    • Mini-prep of cultivated 37。RBS+mGFP E.coli

    • digestion:37。C RBS+mGFP dig ES and mini

    • electrophoresis of 37。C RBS+mGFP mini and 37。C RBS+mGFP dig ES.

    23

    • Single colony isolation of TetR+B0015 transformation of TetR +B0015 +PSB1C3 on LB-C plates

    • Ligation +transformation on: insert[TetR]ES+[B0015]XP&[PompC]ES+[B0034]/vector[PSB1K3]EP on LB-K plates

    24

    • Single colony isolation of PompC+B0034+PSB1K3 E.coli

    • Electrophoresis of Pcons+RBS+pcyA.

    • -Ligation: B0032+ho1&B0034+ho1.

    • Digestion; [Pcons+RBS+pcyA]ES &[ho1]XP&[pSB1C3]EP.

    • Electrophoresis of 37。 RBS+mGFP dig ES

    25

    • Ligation and transformation: insert [PompC]ES+[B0034]XP/vector[PSB1K3]EP on LB-K plates

    • Single colony isolation of PompC+B0034+PSB1K3 E.coli again

    • Mini-prep of cultivated TetR+B0015 Cr E.coli and digestion:[TetR+B0015]P

    • Electrophoresis of :[TetR+B0015]P

    • Digestion: [TetR+B0015]XP

    • Cultivation of PompC+B0034 in liquid LB-K tubes.

    • Mini-prep of B0032+ho1

    • Cultivation of B0034+ho1 with liquid LB-A

    26

    • Cultivation of PompC+B0034Kr E.coli in liquid LB-K tubes again

    • Mini-prep of cultivate PompC+B0034 Kr E.coli

    • Electrophoresis of [TetR+B0015] dig XP not OK

    • Mini-prep of B0034+ho1 Electrophoresis of Pcons+RBS+pcyA & B0032+ho1 & B0034+ho1.

    27

    • Digestion:[TetR+B0015]XP

    • Electrophoresis of [TetR+B0015]mini&dig XP

    • Digestion:[PompC+B0034]E first

    • Electrophoresis of [PompC+B0034]E

    • Digestion: [PompC+B0034]E, S later

    • Ligation: insert[PompC]ES+[B0034]XP/vector[PSB1K3]EP

    28

    • Electrophoresis of [PompC+B0034]ES not OK

    • Transformation of PompC+B0034+PSB1K3.

    • Electrophoresis of B0034+ho1

    • Digestion: [B0034+ho1]XP &[Pcons+RBS+pcyA]E

    29

    • PCR and electrophoresis test. Electrophoresis of digested B0034+ho1 &Pcons+RBS+pcyA.

    30

    • Cultivation of PompC+B0034+PSB1K3 E.coli in liquid LB-K tubes

    • Ligation: insert[TetR]ES+[B0015]XP/vector[PSB1K3]EP

    • Transformation of Plac+PSB1A3 on LB-A plate

    • Digestion:B0015 Ar dig XP, pSB1A3 Ar dig EP and pSB1C3 Cr dig EP

    • ligation:37。C RBS+mGFP+ter. [B0015], Cr

    • transformation of 37。C RBS+mGFP+ter., Cr

    31

    • Mini-prep of cultivated PompC+B0034 Kr E.coli, then

    • Digestion and electrophoresis of [PompC+B0034]E

    • Decide to determine the sequence of PompC+B0015.

    • Cultivation of ho1 with liquid LB-K and pSB1C3 with liquid LB-C.

    • PCR of 37。C RBS+mGFP+ter. Cr

    • electrophoresis of 37。C RBS+mGFP+ter., ter.[B0015] digXP, pSB1C3 dig EP and pSB1A3 dig EP

    • transformation of BBa _E1010 Kr

    June 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • PCR of insert fragment [B0034+PBSII+ilvC] OK!

    • DNA Sequencing OK!

    2

    3

    • mini-prep of cultivated B0034+PBSII+ilvC E. coli & pLac+B0034+Zif268+AlsS E. coli

    • mini-prep of cultivated HivC+ilvD-12,20 E. coli

    4

    • digestion : HivC+ilvD-12,20 [XP]

    5

    • Design the primer for pLac+B0034+Zif268+AlsS point mutation (pm)

    6

    • digestion : B0034 [ES] & HivC [XP] & pSB1C3 [EP] & HivC [EP]

    • ligation : insert B0034 [ES] & HivC [XP]/vector pSB1C3 [EP]

    • ligation : insert HivC [EP]/vector pSB1C3 [EP]

    • transformation of B0034+ HivC & HivC ,and cultivation on LB-C plate

    7

    • PCR of insert fragment [B0034+ HivC] & [HivC] OK

    8

    • Design the primer for B0034+PBSII+ilvC point mutation (pm)

    • Single colony isolation from HivC, B0034+HivC & ilvD+37℃ RBS LB-C plate, and cultivation in liquid LB-C

    9

    • mini-prep of cultivated HivC & B0034+HivC & ilvD+37℃ RBS E. coli

    • Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR

    • ligation: insert B0034+HivC [ES] & ilvD [XP]/vector pSB1A3 [EP];insert B0034+HivC [ES] & ilvD+37℃ RBS [XP]/vector pSB1A3 [EP]

    10

    • transformation of B0034+ HivC+ ilvD & B0034+ HivC+ ilvD+37℃ RBS ,and cultivation on LB-A plate

    • Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 55℃ of PCR failed

    11

    • DNA Sequencing B0034+HivC & HivC OK

    • PCR of insert fragment [B0034+ HivC+ilvD] & [B0034+ HivC+ilvD+37℃ RBS] OK

    12

    • Single colony isolation from B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD LB-A plate, and cultivation in liquid LB-A

    • Single colony isolation from HivC(TA) LB-A plate, and cultivation in liquid LB-A

    13

    • mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS , B0034+ HivC+ ilvD & HivC(TA) E. coli

    • Testing the temperature of the point mutation between of pLac+B0034+Zif268 & AlsS by the m.p 50 & 52℃ of PCR----50℃ is better

    14

    • PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation -----NOT OK

    15

    • DNA Sequencing B0034+ HivC+ilvD+37℃ RBS & B0034+ HivC+ ilvD OK

    16

    17

    18

    19

    • PCR of insert fragment [pLac+B0034+Zif268+AlsS] with designed primer for point mutation OK

    • digestion: pLac+B0034+Zif268+AlsS [DPn1]

    20

    • transformation of pLac+B0034+Zif268+AlsS (point mutation), and cultivation on LB-A plate

    21

    • Single colony isolation from pLac+B0034+Zif268+AlsS (pm) LB-A plate, and cultivation in liquid LB-A

    22

    • mini-prep of cultivated pLac+B0034+Zif268+AlsS (pm) E. coli

    • digestion : pLac+B0034+Zif268+AlsS (pm) [EP]

    23

    • DNA sequencing OK

    24

    25

    • PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK

    • digestion : B0034+ PBSII+ilvC [DPn1]

    26

    • transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate------Fail

    27

    • PCR of insert fragment [B0034+ PBSII+ilvC] with designed primer for point mutation OK

    28

    • digestion : B0034+ PBSII+ilvC [DPn1]

    29

    • transformation of B0034+ PBSII+ilvC (pm), and cultivation on LB-C plate

    30

    • PCR of insert fragment [B0034+ PBSII+ilvC] (pm) OK

    • Single colony isolation from B0034+ PBSII+ilvC (pm) LB-C plate, and cultivation in liquid LB-C

    July 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • mini-prep of cultivated B0034+ PBSII+ilvC (pm) E. coli

    • digestion : B0034+ PBSII+ilvC (pm) [EP]

    2

    • DNA sequencing OK

    3

    4

    5

    6

    7

    • digestion : pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]

    8

    9

    • Testing the temperature of the point mutation between of HivC & ilvD by the m.p 50℃ of PCR

    • digestion : pSB1K3 [EP]

    • ligation : insert pLac+B0034+zif268+AlsS (pm) [ES] & B0034+ PBSII+ilvC (pm) [XP]/vector pSB1K3 [EP]

    • transformation of pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) and cultivation on LB-K plate

    10

    • Testing the temperature of the point mutation between of HivC & ilvD by the m.p 48℃ of PCR

    • Digestion : B0034+ HivC+ilvD+37℃ RBS [DPn1]

    • PCR of insert fragment [pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC] (pm) OK

    • SCI from pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) LB-K plate, and cultivation in liquid LB-K

    11

    • mini-prep of cultivated pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm) E. coli

    • DNA sequencing OK

    • culture condition test: activation DH5αovernight

    12

    • transfer to new medium(1/100), OD0.2 start counting culture time

    • transfer to 30゜C and 27゜C

    13

    • transfer to 30゜C and 27゜C

    14

    15

    • transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A plate

    16

    • PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm)-----NOT OK

    17

    • Point mutation by PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS]

    • Digestion: B0034+ HivC+ilvD+37℃ RBS [DPn1]

    18

    19

    • transformation of B0034+ HivC+ ilvD+37℃ RBS (pm),and cultivation on LB-A,K,C plate (A sucessful)

    20

    • PCR of insert fragment [B0034+ HivC+ilvD+37℃ RBS](pm) ---- OK

    • Single colony isolation from B0034+ HivC+ilvD+37℃ RBS (pm) LB-A plate, and cultivation in liquid LB-A

    21

    • mini-prep of cultivated B0034+ HivC+ilvD+37℃ RBS (pm) E. coli

    • Digestion: pLac+B0034+zif268+AlsS+ B0034+ PBSII+ilvC (pm)(G1) [ES] & B0034+ HivC+ilvD+37℃ RBS (pm)(G2) [XP]

    • ligation : insert G1[ES] & G2[XP] vector pSB1C3[EP]

    22

    • transformation of G1+G2,and cultivation on LB- C plate failed

    23

    • ligation : insert G1[ES] & G2[XP]/vector pSB1C3[EP]

    24

    • transformation of G1+G2,and cultivation on LB- C plate

    • Digestion: G2’(B0034+HIVC+ilvD) [XP]

    • ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]

    25

    • PCR of insert fragment [kivD+B0015]

    • transformation of G1+G2’, and cultivation on LB- C plate failed

    26

    • Single colony isolation from kivD+B0015 LB-C plate, and cultivation in liquid LB-C

    • sample and do GC

    27

    • mini-prep of cultivated kivD+B0015 E. coli

    28

    29

    • Point mutation by PCR of insert fragment [B0034+ HivC+ilvD(G2’)]

    • Digestion: G2’ [DPn1]

    30

    • transformation of G2’, and cultivation on LB- A plate

    31

    • Digestion: kivD+B0015 [EP] (checking bp----failed)

    • Single colony isolation from G2’ (pm) LB-A plate, and cultivation in liquid LB-A

    August 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • mini-prep of cultivated G2’ E. coli

    2

    3

    4

    5

    6

    7

    8

    9

    • Digestion: G1 [ES] & G2‘ [XP] & pSB1C3 [EP]

    • ligation : insert G1[ES] & G2‘[XP]/vector pSB1C3[EP]

    10

    • strain test: activation of different strains overnight

    11

    • transfer to new medium, OD0.2 IPTGinduction, culture in 37゜C

    12

    • Single colony isolation from G1+G2’ LB-C plate, and cultivation in liquid LB-C

    • transfer to 27゜C

    13

    • mini-prep of cultivated G1+G2’ E. coli

    14

    15

    • Digestion: Ptet+B0032[ES] & GliI+KivD[XP]

    • sample and do GC

    16

    • ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]

    • Single colony isolation from G1+G2 LB-C plate, and cultivation in liquid LB-C

    17

    • transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate

    • mini-prep of cultivated G1+G2 E. coli

    18

    • PCR of insert fragment [Ptet+B0032+ GliI+KivD]-----OK

    • DNA sequencing------NOT OK

    19

    20

    21

    22

    23

    24

    • carbon source test: activation DH5α overnight

    25

    • transfer to new medium(1/100), OD0.2 start counting culture time

    26

    • culturing for 72hours, inject feeding solution per 24hours

    27

    • culturing for 72hours, inject feeding solution per 24hours

    28

    • transformation of DNA program, and cultivation on LB- A plate

    • culturing for 72hours, inject feeding solution per 24hours

    29

    • ligation : insert Ptet+B0032[ES] & GliI+KivD[XP]/vector pSB1K3[EP]

    • Single colony isolation from DNA program LB-C plate, and cultivation in liquid LB-C

    • sample & do GC

    30

    • transformation of Ptet+B0032+ GliI+KivD, and cultivation on LB- K plate

    • mini-prep of cultivated DNA program E. coli

    • Do GC

    31

    • PCR of insert fragment [Ptet+B0032+ GliI+KivD] OK

    • DNA sequencing NOT OK

    September 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • ligation : insert Zif268 [ES] & AlsS [XP]/Vector pSB1K3 [EP]

    2

    • transformation of Zif268+AlsS+pSB1K3 and cultivation on LB-K plate

    3

    4

    • digestion : [pSB1K3] EP

    • digestion : [pSB1K3] EP

    5

    6

    7

    • PCR of insert fragment [Zif268+AlsS] OK

    • DNA Sequencing OK

    • ligation : point mutation HivC

    • TA cloning : point mutation HivC

    8

    9

    • Single colony isolation from Zif268+AlsS LB-K plate, and cultivation of in liquid LB-K

    10

    • mini-prep of cultivated Zif268+ AlsS E. coli

    • transformation of point mutation HivC and cultivation on LB-A plate

    • transformation of B0034 and cultivation on LB-A plate

    11

    12

    • Single colony isolation from HivC LB-A plate, and in liquid LB-A

    • Single colony isolation from B0034 LB-A plate, and in liquid LB-A

    13

    • mini-prep of cultivated point mutation HivC E. coli & B0034 E. coli

    14

    • digestion : Zif268+AlsS [XP]

    15

    16

    17

    • Single colony isolation from ilvD LB-A plate, and cultivation in liquid LB-A

    18

    • mini-prep of cultivated ilvD E. coli

    • digestion : ilvD [EP] & pSB1K3 [EP]

    • transformation of 37℃ RBS and cultivation on LB-C plate

    19

    • Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

    20

    • digestion : B0034 [SP]

    • gel extraction

    • ligation :insert Zif268+AlsS [XP]/Vector B0034 [SP]

    • mini-prep of cultivated Hivc E. coli

    21

    • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

    • transformation of HivC and cultivation on LB-A plate

    22

    • PCR of insert fragment [B0034+Zif268+AlsS] OK

    • DNA Sequencing NOT OK

    • digestion : B0034 [SP] & Zif268+AlsS [XP]

    • gel extraction

    • ligation :insert Zif268+AlsS [XP]/Vector B0034+pSB1A3 [SP]

    • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

    • Single colony isolation from B0034 LB-A plate, and cultivation in liquid LB-A

    • SCI from ilvD LB-K plate, and cultivation in liquid LB-K

    23

    • PCR of insert fragment [B0034+Zif268+AlsS] NOT OK

    • transformation of B0034+Zif268+AlsS and cultivation on LB-A plate

    • mini-prep of cultivated B0034 and ilvD E. coli

    • Single colony isolation from HivC LB-A plate, and cultivation in liquid LB-A

    24

    • mini-prep of cultivated HivC E. coli

    • digestion : B0034 [SP] & ilvD [XP]

    25

    • digestion : pSB1C3 [EP] & HivC [ES] & HivC [SP]

    • ligation :insert HivC [ES] & ilvD [XP]/Vector pSB1C3 [EP]

    • ligation :insert ilvD [XP]/Vector HivC+pSB1A3 [SP]

    26

    • transformation of HivC+ilvD and cultivation on LB-A & LB-C plate

    27

    • PCR of insert fragment [B0034+Zif268+AlsS] NOT OK

    28

    • transformation of pSB1A3 & pSB1C3 and cultivation on LB-A & LB-C plate

    • PCR of insert fragment [HivC+ilvD] OK

    • DNA sequencing NOT OK

    29

    • Single colony isolation from pSB1C3 LB-C plate, and cultivation in liquid LB-C

    30

    • mini-prep of cultivated & pSB1C3 E. coli

    Retrieved from "http://2013.igem.org/Team:NCTU_Formosa/notes"