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 and single colony isolation of TetR+B0015+PSB1K3
Electrophoresis of PCR product made at this morning and Plac(5/30 Ar) OK
Digestion:[Ter(B0015)]XP
Transformation of Ter(B0015) on LB-A plate & mGFP on LB-K plate & 37oC RBS+mGFP+Ter(B0015) on LB-C plate
Ligation: insert 37oC RBS[ES] & Ter(B0015)[XP]/ vector pSB1C3[EP]
Single colony isolation from Ter(B0015) LB-A plate, and cultivation in liquid LB-A

2

Ligation: insert [TetR]ES+[B0015]XP/vector [PSB1K3]EP and transformation of this part.
Ligation: B0032+ho1&B0034+ho1
Transformation of B0032+ho1&B0034+ho1
Cultivation of pSB1C3(C20&C10)with liquid LB.
PCR of insert fragment[37oC RBS+mGFP+Ter(B0015)]
Transformation of mRFP on LB-A plate& LB-C plate& LB-K plate-Mini-prep of cultivated Ter(B0015) E.coli.

3

PCR of Plac &TetR+B0015
Mini-prep of pSB1C3
Cultivation of B0032+ho1&B0034+ho1 with LB-A plate PCR of B0032+ho1&B0034+ho1
Electrophoresis of B0032+ho1&B0034+ho1──NOT OK.
Single colony isolation from 37oC RBS+mGFP+Ter(B0015) LB-K plate, and cultivation in liquid LB-K & mRFP LB-A, and cultivation in liquid LB-K
Mini-prep of cultivated 37oC RBS+mGFP+Ter(B0015) E.coli
Digestion:[37oC RBS+mGFP+Ter(B0015)]XP

4

Electrophoresis of Plac .TetR+B0015 OK
Cultivation of Plac.TetR+B0015 in liquid LB-A tubes. PCR of B0032+ho1&B0034+ho1
Electrophoresis of B0032+ho1(OK)&B0034+ho1(After PCR)
Mini-prep of B0032+ho1. Digestion: [B0032+ho1]XP.
Cultivation of ho1 with liquid LB-K
Single colony isolation from mRFP LB-A plate, and cultivation in liquid LB-A
Digestion:[37oC RBS+mGFP+Ter(B0015)]XP
PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)].

5

Mini-prep of cultivated Plac and TetR+B0015 E.coli and then conduct digestion :[Plac]P&[TetR+B0015]P
Electrophoresis of [Plac ]P and [TetR+B0015]P,[Plac]XP and [TetR+B0015]XP
Ligation: insert[PompC+B0034]ES+[LacI+J61048]XP/vector [PSB1A3]EP
Electrophoresis of B0032+ho1 mini&B0032+ho1(digested)&pSB1C3 mini&pSB1C3(digested)&ho1 mini&ho1(digested)
Mini-prep of ho1
Digestion: [ho1]XP
Digestion:[37oC RBS+mGFP+Ter(B0015)]XP
Transformation of mRFP on LB-A plate

6

Transformation of PompC+B0034+LacI+J61048+PSB1A3
Ligation: insert [PompC+B0034]ES+[LacI+J61048]XP/vector [PSB1A3]EP
Electrophoresis of B0032+ho1 mini&B0032+ho1(digested)&ho1 mini&ho1 (digested).
Single colony isolation from mRFP LB-A plate, and cultivation in liquid LB-A & LB-K & LB
Digestion:[Ter(B0015)]EX
Ligation: insert 37oC RBS+mGFP[ES]&Ter(B0015)[EX]
Transformation of 37oCRBS+mGFP+Ter(B0015) on LB-K plate & LB-A plate, mRFP on LB-A plate

7

Transformation of PompC+B0034+LacI+J61048 again
Single colony isolation of TetR+B0015 Kr E.coli
Digestion:[LacI+J61048]ES. PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)]
Single colony isolation from 37oCRBS+mGFP+Ter(B0015) LB-A plate, and cultivation in liquid LB-A.

8

PCR + single colony isolation of PompC+B0034+LacI+J61048 E.coli
Electrophoresis of PompC+B0034+LacI+J61048 OK
Ligation: insert [LacI+J61048]ES+[Plac]XP/vector[PSB1C3]EP
Transformation of LacI+J61048+Plac on LB-C plate
Cultivation of PompC+B0034+LacI+J61048 in LB-A tubes & TetR+B0015 in LB-K tube & PSB1A3 in LB-A tube &PSB1C3 in LB-C tube.
Mini-prep of cultivated 37oCRBS+mGFP+Ter(B0015) E.coli
Digestion:[ 37oCRBS+mGFP+Ter(B0015)]XP
PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)].

9

Mini-prep from cultivated PompC+B0034+LacI+J61048 Eco.li&PSB1C3 E.coli
digestion : [PompC+B0034+LacI+J61048]ES&[PSB1C3]EP
Electrophoresis of [PompC+B0034+LacI+J61048] ES& [LacI+J61048]ES&[PSB1C3]EP.
Single colony isolation from 37oCRBS + mGFP+Ter(B0015)

10

Mini-prep of cultivated 37oCRBS+mGFP+Ter(B0015) E.coli
Digestion:[ 37oCRBS+mGFP+Ter(B0015)]XP
Single colony isolation from 37oCRBS+mGFP+Ter(B0015) LB-A plate, and cultivation in liquid LB-A.

11

PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)]
Transformation of mRFP on LB-K plate.

12

Single colony isolation from mRFP LB-K plate, and cultivation in liquid LB-K

13

Ligation: insert[PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1C3]EP and insert[TetR]ES+[B0015]XP/vector[PSB1C3]EP.
Mini-prep of cultivated mRFP E.coli
Digestion:[mRFP]ES & [Ter(J61048)]XP & [37OCRBS+mGFP]XP
Ligation: insert 37oCRBS+LuxR[ES]&37oCRBS+mGFP[XP] /vector pSB1A3[EP]
Transformation of 37oCRBS +LuxR+37oCRBS+mGFP on LB-A plate & mRFP +Ter(J61048) on LB-C plate& Ter(J61048) on LB-A plate.

14

Transformation of PompC+B0034+LacI+J61048+PSB1C3 and TetR+B0015+PSB1C3
Cultivation of B0032+ho1 with liquid LB-A. PCR of insert fragment[Ter(B0015)]
Single colony isolation from Ter(J61048) LB-A plate, and cultivation in liquid LB-A & 37OCRBS+mGFP on LB-K plate, and cultivation in liquid LB-K
Transformation of 37oCRBS +LuxR+37oCRBS+mGFP on LB-A plate & mRFP +Ter(J61048) on LB-C plate
Mini-prep of cultivated Ter (J61048)& 37oCRBS+mGFP E.coli

15

Digestion:[B0032]XP
Transformation of PompC+B0034+LacI+J61048 +PSB1C3and TetR+B0015 +PSB1C3 again
Ligation: insert[PompC]ES+[B0032]XP/vector[PSB1K3]EP, then transformation of this part
Mini-prep of B0032+ho1. PCR of insert fragment[37oCRBS + LuxR + 37oCRBS+mGFP]&[mRFP+Ter(J61048)].

9

Mini-prep from cultivated PompC+B0034+LacI+J61048 Eco.li&PSB1C3 E.coli
digestion : [PompC+B0034+LacI+J61048]ES&[PSB1C3]EP
Electrophoresis of [PompC+B0034+LacI+J61048] ES& [LacI+J61048]ES&[PSB1C3]EP.
Single colony isolation from 37oCRBS + mGFP+Ter(B0015)

10

Mini-prep of cultivated 37oCRBS+mGFP+Ter(B0015) E.coli
Digestion:[ 37oCRBS+mGFP+Ter(B0015)]XP
Single colony isolation from 37oCRBS+mGFP+Ter(B0015) LB-A plate, and cultivation in liquid LB-A.

11

PCR of insert fragment[37oCRBS+mGFP+Ter(B0015)]
Transformation of mRFP on LB-K plate.

12

Single colony isolation from mRFP LB-K plate, and cultivation in liquid LB-K

13

Ligation: insert[PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1C3]EP and insert[TetR]ES+[B0015]XP/vector[PSB1C3]EP.
Mini-prep of cultivated mRFP E.coli
Digestion:[mRFP]ES & [Ter(J61048)]XP & [37OCRBS+mGFP]XP
Ligation: insert 37oCRBS+LuxR[ES]&37oCRBS+mGFP[XP] /vector pSB1A3[EP]
Transformation of 37oCRBS +LuxR+37oCRBS+mGFP on LB-A plate & mRFP +Ter(J61048) on LB-C plate& Ter(J61048) on LB-A plate.

14

Transformation of PompC+B0034+LacI+J61048+PSB1C3 and TetR+B0015+PSB1C3
Cultivation of B0032+ho1 with liquid LB-A. PCR of insert fragment[Ter(B0015)]
Single colony isolation from Ter(J61048) LB-A plate, and cultivation in liquid LB-A & 37OCRBS+mGFP on LB-K plate, and cultivation in liquid LB-K
Transformation of 37oCRBS +LuxR+37oCRBS+mGFP on LB-A plate & mRFP +Ter(J61048) on LB-C plate
Mini-prep of cultivated Ter (J61048)& 37oCRBS+mGFP E.coli

15

Digestion:[B0032]XP
Transformation of PompC+B0034+LacI+J61048 +PSB1C3and TetR+B0015 +PSB1C3 again
Ligation: insert[PompC]ES+[B0032]XP/vector[PSB1K3]EP, then transformation of this part
Mini-prep of B0032+ho1. PCR of insert fragment[37oCRBS + LuxR + 37oCRBS+mGFP]&[mRFP+Ter(J61048)].

16

PCR + single colony isolation of PompC+B003+LacI+J61048 Cr E.coli&TetR+B0015 Cr E.coli&PompC+B0032 Kr E.coli, then Electrophoresis of these parts

17

Mini-prep of cultivated PompC+B0034 E.coli and TetR+B0015 Cr E.coli

18

19

20

Ligation: Pcons+RBS+pcyA&B0032+ho1&pSB1A3.

21

Ligation: insert[PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1A3]EP
Transformation of Pcons+RBS+pcyA+B0032+ho1 on LB-A plate. PCR of insert fragment[37oCRBS + LuxR + 37oCRBS+mGFP]
Digestion:[pSB1C3]EP&[mRFP]ES&[Ter(J61048)]XP
Ligation: insert mRFP[ES]&Ter(J61048)[XP] /vector pSB1C3[EP]
Transformation of mRFP + Ter (J61048) on LB-C plate
Single colony isolation from 37oCRBS + LuxR +37oCRBS+mGFP LB-A plate, and cultivation in liquid LB-A.

22

Transformation of PompC+B0034+LacI+J61048+PSB1A3 but there is no colony appearing. PCR of insert fragment[mRFP+Ter(J61048)]
Mini-prep of cultivated 37o CRBS + LuxR + 37oCRBS+mGFP E.coli
Digestion:[ 37o CRBS + LuxR + 37oCRBS+mGFP]ES

23

Digestion:[PompC+B0032]ES and [TetR+B0015]XP,[LacI+J61048]XP
Ligation: insert [PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1C3]EP
Transformation of PompC+B0034+LacI+J61048 +PSB1C3
Electrophoresis of [PompC+B0032]ES & [TetR+B0015]XP
Digestion again:[PompC+B0032]ES&[LacI+J61048]XP&[LacI+B0015]XP
Transformation of mRFP + Ter (J61048) on LB-C plate

24

PCR and single colony isolation of PompC+B0034+LacI+J61048+PSB1C3 E.coli
Electrophoresis of [B0032]dig ES&[TetR+B0015]dig XP&[LacI+J61048]dig XP&PCR product [PompC+B0034+LacI+J61048] ,[TetR+B0015] OK
Decide to determine the sequence of [TetR+B0015] OK.
Single colony isolation from 6/21 plate.
Cultivation of Pcons+ RBS+pcyA+B0032+ho1 with liquid LB-A.
Electrophoresis of Pcons+RBS+PcyA+B0032+ho1.
PCR of insert fragment[mRFP+Ter(J61048)].

25

Ligation:insert[PompC+B0034]ES+[LacI+J61048]XP&[PompC]ES+[B0032]XP/vector[PSB1C3]EP and [PompC+B0034]ES+[LacI+J61048]XP/vector[PSB1A3]EP
Transformation of the ligation product made today
PCR of Pcons+RBS+pcyA+B0032+ho1.
Cultivation of Pcons+RBS+pcyA+B0032+ho1 with liquid LB-A
Electrophoresis of Pcons+RBS+pcyA+B0032+ho1.
Mini-prep of Pcons+RBS+pcyA+B0032+ho1.
Ligation: insert mRFP[ES]&Ter(J61048)[XP]/vector pSB1C3[EP]

26

PCR and single colony isolation of PompC+B0034+LacI+J61048+PSB1A3& PompC+B0034+LacI+J61048+PSB1C3&PompC+B0032+PSB1C3 E.coli
Electrophoresis of the PCR product made today
Electrophoresis of Pcons+RBS+pcyA+B0032+ho1──NOT OK
Ligation: Pcons+RBS+pcyA&B0032+ho1
Transformation of Pcons+RBS+pcyA+B0032+ho1. Transformation of mRFP + Ter (J61048) on LB-C plate

27

Cultivation of PompC+B0034+LacI+J61048+PSB1A3 E.coli in liquid LB-A tubes
PCR of Pcons+RBS+pcyA+B0032+ho1
Cultivation of Pcons+RBS+pcyA+B0032+ho1 with liquid LB-A
Electrophoresis of Pcons+RBS+pcyA+B0032+ho1──NOT OK
Transformation of mRFP + Ter (J61048) on LB-C plate

28

PCR of Pcons+RBS+pcyA+B0032+ho1
Electrophoresis of Pcons+RBS+pcAa+B0032+ho1──NOT OK
Ligation: insert mRFP[ES]&Ter(J61048)[XP] /vector pSB1C3[EP]

29

Cultivation of PompC+B0034+LacI+J61048+PSB1A3 E.coli in liquid LB-A tubes again.
Ligation: Pcons+RBS+pcyA&B0032+ho1&pSB1A3
Transformation of ligated Pcons+RBS+pcyA+B0032+ho1
Single colony isolation from 6/21 Pcons+RBS+pcyA+B0032+ho1 LB plate
PCR of 6/21 Pcons+RBS+pcyA+B0032+ho1
Electrophoresis of 621 Pcons+RBS+pcyA+B0032+ho1─NOT OK
Transformation of mRFP+Ter(J61048) on LB-C plate
Single colony isolation from 37oCRBS +LuxR +37oCRBS+mGFP LB-A plate, and cultivation in liquid LB-A

30

Mini-prep of cultivated PompC+B0034+LacI+J61048+PSB1A3 E.coli and then digest with EcoR1 and SpeI:[ PompC+B0034+LacI+J61048]dig ES
Electrophoresis of [PompC+B0034+LacI+J61048+PSB1A3] mini and [ PompC+B0034+LacI+J61048]dig ES OK
Decide to determine sequence of[ PompC+B0034+LacI+J61048]
Cultivation of 6/29 Pcons+RBS+PcyA+B0032+ho1 LB plate with liquid LB-A
PCR of 6/29 Pcons+RBS+PcyA+B0032+ho1
Cultivation of 6/29 re-ligated Pcons+RBS+PcyA+B0032+ho1
PCR of 6/29 re-ligated Pcons+RBS+pcyA+B0032+ho1
Electrophoresis of all above─NOT OK

July 2013

Sunday

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

1

PCR + single colony isolation of PompC+B003+LacI+J61048 Cr E.coli&TetR+B0015 Cr E.coli&PompC+B0032 Kr E.coli, then Electrophoresis of these parts

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