Team:NCTU Formosa/notes

From 2013.igem.org

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.

Material and Methods

Minipreps of Plasmid DNA

We use Plasmid miniPREP kit (Genedirex Inc.) to obtain plasmid DNA for analysis, sequencing and cloning. Single colony is picked from LB agar plate and inoculated into LB medium with suitable antibiotics. Bacteria cells are harvest by centrifuge after incubation for 12 – 16 hours. Afterward, alkaline lysis is performed to release inside components, and DNA is purified by silicone resin-based column. The quality and amount of plasmid DNA is estimated by agarose gel electrophoresis stained by SyBr Safe (Invitrogen).

BioBrick assembly

To digest the desired DNA, appropriate restriction enzymes and reaction buffer (all perched from New England Biolabs) is used in 20 μL reaction volume. Upstream BioBrick is digested with EcoRI-HF and SpeI in CutSmart buffer. Downstream BioBrick and backbone part are digested with XbaI and PstI, EcoRI-HF and PstI respectively in NEBuffer 2. Reactions take place in 37 ℃ water bath for 2 hours or overnight.

Ligation is performed using BioBrick Assembly Kit Ligation Protocol. All the reagent and enzymes are perched from New England Biolabs). Reactions take place in 37 ℃ water bath for 2 hours or overnight in 16 ℃ incubator.

Transformation

DH5α E. coli competent cells (ECOSTM 101, Yeastern Biotech Co., Ltd.) were used for the propagation of plasmid DNA. Standard transformation protocol is used: First place 2 μL plasmid or 10μL product into 1.5 mL mini centrifuge tube and chilled on ice. Competent cells are melt on ice before transformation. After adding 33 – 50 μL competent cells, heat shock at 42 ℃ for 1 minutes. Place cells into LB medium to recover then plating on LB agar plate and incubate for 16 – 18 hours.

PCR reaction

Taq DNA Polymerase 2x Master Mix Red (Ampliqon) and VF2 and VR primers (BBa_G00100 and BBa_G00101 respectively) are used for colony PCR. Single colony is picked and place into 10 μL PCR reagent, and followed by PCR with condition described below:

  • Predenature: 95 ℃，10 min
  • Denature, annealing and extension: [98 ℃, 10 s; 55 ℃, 30 s; 72 ℃, 1 m/kb + 30 s] × 25 cycles
  • Hold: 4 ℃

Reagent and Medium

Lysogeny broth (LB): 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl with with suitable antibiotics.

LB agar plate: LB medium with 1.5% (w/v) agar.

50X TAE buffer: tris base 242 g/L, glacial acetic acid 57.1 mL/L, 0.05 M EDTA, pH 8.0 Antibiotics working concentration:

  • Kanamycin  : 25 μg/mL
  • Ampicillin  : 100 μg/mL
  • Chloramphenicol: 20 μg/mL


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

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

    • transformation of this part and cultivation of this part on LB-C plate

    • Digestion : mRFP [EP]

    • Ligation : insert mRFP [EP]/vector pSB1C3 [EP]

    • Transformation of mRFP ,and cultivation on LB-C plate

    2

    • PCR +single colony isolation B0030+TetR+B0015 Cr E.coli

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

    3

    • Electrophoresis of B0030+TetR+B0015 not OK

    • electrophoresis of B0030+TetR+B0015 again OK

    • Ligation: Pcons+B0030+pcyA+B0032+ho1

    • Transformation of Pcons+B0030+pcyA+B0032+ho1

    • Mini-prep of cultivated 37℃RBS+luxR+37℃RBS+mGFP and mRFP E.coli

    • Digestion : mRFP [ES] & pSB1A3 [EP] & pSB1C3 [EP] & pSB1K3 [EP]

    • Electrophoresis of insert fragment [mRFP & pSB1A3 & pSB1C3 & pSB1K3]---- OK

    4

    • Cultivation of B0030+TetR+B0015+PSB1C3 in liquid LB-C tubes. PCR of Pcons+B0030+pcyA+B0032+ho1

    • Electrophoresis of Pcons+B0030+pcyA+B0032+ho1

    • Cultivation of Pcons+B0030+pcyA+B0032+ho1 with liquid LB-A

    • Ligation : insert mRFP [ES] & J61048 [XP]/ vector pSB1K3 [EP]

    • Transformation of mRFP+J61048 ,and cultivation on LB-K plate

    5

    • Mini-prep of cultivated B0030+TetR+B0015+PSB1C3 Cr E.coli and then digest with XbaI and Pst1

    • Electrophoresis of[ B0030+TetR+B0015]mini&dig XP

    • Decide to determine the sequence of B0030+TetR+B0015

    • Digestion: [Pcons+B0030+pcyA+B0032+ho1]ES

    • Electrophoresis of Pcons+B0030+pcyA+B0032+ho1──NOT OK

    • PCR of insert fragment [mRFP+J61048]

    6

    • Ligation :insert [PompC]ES+[B0032]XP/vector [PSB1C3] EP and insert [LacI+J61048]ES&[Plac]XP/vector [PSB1C3]EP

    • Transformation of PompC+B0030+PSB1C3 and LacI+J61048+Plac+PSB1C3

    • Ligation : insert 37℃RBS+luxR+37℃RBS+mGFP [ES] & B0015 [XP]/ vector pSB1C3 [EP]

    • Transformation of 37℃RBS+luxR+37℃RBS+mGFP+B0015 ,and cultivation on LB-C plate

    • Electrophoresis of insert fragment [mRFP+J61048]----undetermined

    7

    8

    • PCR + single colony isolation of LacI+J61048+Plac +PSB1C3 E.coli

    • Electrophoresis of LacI+J61048+Plac OK

    9

    • Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K

    • Mini-prep of cultivated mRFP+J61048 E.coli

    • Digestion : mRFP+J61048 [XP]

    10

    • Cultivation of LacI+J61048+Plac in liquid LB-C tubes.

    • Electrophoresis of insert fragment [mRFP+J61048]----NOT OK

    11

    • Mini-prep of cultivated LacI+J61048+Plac E.coli

    12

    • Ligation again:Insert [PompC]ES&[B0032]XP/vector [PSB1K3]EP

    • Digestion: [LacI+J61048+Plac]XP

    • Electrophoresis of [LacI+J61048+Plac]mini&dig XP OK

    • Decide to determine the sequence of LacI+J61048+Plac

    • Transformation of PompC(Ar)+B0032(Ar)+PSB1K3 again but there is still no colony

    • Transformation of 37℃RBS+luxR+37℃RBS+mGFP+B0015, and cultivation on LB-C plate

    • Transformation of mRFP+J61048 ,and cultivation on LB-K plate.

    13

    • Ligation: Insert [PompC]ES&[B0032]XP/vector [PSB1K3]EP. PCR of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048].

    14

    • Transformation of the ligation product made yesterday on LB-K plate but there is no colony appearing

    • Electrophoresis of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015]----OK

    • Electrophoresis of insert fragment [mRFP+J61048]----NOT OK RBS+mGFP+B0015 LB-C plate, and cultivation of in liquid LB-C. PCR of insert fragment [mRFP+J61048]----NOT OK

    15

    • Ligation and trans formation of insert [ B0030]ES+ [TetR+B0015]XP/vector [PSB1C3]EP and cultivation on LB-C plate

    • Transformation of PSB1K3 for testing

    • Transformation of Pcons

    • Digestion: [B0030+pcyA]ES

    • Electrophoresis of Pcons──OK

    • Electrophoresis of insert fragment [mRFP+J61048]----NOT OK

    • Mini-prep of cultivated 37℃RBS+luxR+37℃RBS+mGFP+B0015 E.coli----NOT OK

    • PCR of insert fragment [mRFP+J61048]----OK

    16

    • Digestion:[PompC]ES,[PSB1K3]&[PSB1C3]EPand Electrophoresis for checking these parts OK

    • PCR + singles colony isolation of B0030+TetR+B0015 +PSB1C3 E.coli and electrophoresis of this insert fragment not OK Because the PCR electrophoresis result is strange，we conduct this experience again OK

    • Ligation: Pcons&Pcons+B0030+pcyA+B0032+ho1&pSB1K3.

    • Transformation of Pcons+B0032+pcyA+B0030+ho1.

    • Cultivation of Pcons with liquid LB-C

    • Electrophoresis of Pcons──NOT OK

    • Single colony isolation from 37℃RBS+luxR+37℃RBS+mGFP+B0015 LB-C plate, and cultivation of in liquid LB-C

    • Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K

    17

    • Transformation of PompC+B0032+PSB1C3 and Cultivation of B0030+TetR+B0015 Cr E.coli in liquid LB-C tube.

    • Transformation of Pcons

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

    • Digestion : pSB1C3 [ES] & pSB1C3 [XP]

    • Ligation : insert sRNA+target 1 [ES]/vector pSB1C3 [ES]

    • Transformation of 37℃RBS+luxR+37℃RBS+mGFP+B0015 & sRNA 1 & sRNA 2 & target 1 & target 2 ,and cultivation on LB-C plate

    • Transformation of mRFP+J61048 & sRNA+target 1 & sRNA+target 2 ,and cultivation on LB-K plate

    • Electrophoresis of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048 & pSB1A3 & pSB1C3 & pSB1K3]-- mRFP+J61048 NOT OK Digestion : mRFP+J61048 [XP]

    18

    • Mini-prep of cultivated B0030+TetR+B0015 Cr E.coli and then digest these mini with XbaI and PstI

    • PCR + single colony isolation of PompC+B0032 Cr E.coli and electrophoresis of this insert fragment OK Cultivation of [PompC+B0032] Cr E.coli in liquid LB-C Tubes.

    • Cultivation of Pcons with liquid LB-C

    • Digestion : pSB1C3 [ES] & pSB1C3 [XP]

    • Mini-prep of Pcons. Electrophoresis of Pcons──NOT OK

    • Four colonies isolation from Pcons LB plate and use streak plate method to isolate the pure cultures. Digestion : mRFP+J61048 [XP]

    • Single colony isolation from sRNA+target 1 & sRNA+target 2 LB-K plate, and cultivation of in liquid LB-K

    • Single colony isolation from Plux LB-A plate, and cultivation of in liquid LB-A----NOT OK

    19

    • Mini-prep of cultivated PompC+B0032 Cr E.coli, And then digest with EcoR1 and SpeI

    • Electrophoresis of PSB1K3 made at 7/16 and B0030+TetR+B0015, PompC+B0032 OK.

    • Cultivation of Pcons with liquid LB-C

    • Single colony isolation from37℃RBS+luxR+37℃RBS+mGFP+B0015 LB-C plate, and cultivation of in liquid LB-C

    • Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K(?)

    • PCR of insert fragment [sRNA 1 & sRNA 2 & target 1 & target 2]

    • Electrophoresis of insert fragment [sRNA 1 & sRNA 2 & target 1 & target 2]---- target 1?

    • Mini-prep of cultivated sRNA+target 1 & sRNA+target 2 E.coli.

    20

    • Mini-prep of Pcons. -Digestion: [Pcons]ES.

    • Electrophoresis of Pcons──NOT OK.

    • Single colony isolation from sRNA 1 & sRNA 2 & target 2 LB-C plate, and cultivation of in liquid LB-C

    • Single colony isolation from mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K

    • Transformation of Plux ,and cultivation on LB-A plate

    • Single colony isolation from Plux & PLac & Pcons LB-A plate, and cultivation of in liquid LB-A

    • Transformation of RBS+lacI+B0010+B0012+pLac ,and cultivation on LB-K plate

    • Mini-prep of cultivated 37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048 & sRNA 1 & sRNA 2 & target 2 E.coli

    21

    • Single colony isolation from 7/15 Pcons LB plate(Red one).

    • Cultivation of Pcons with LB-C. PCR of insert fragment [target 1]

    • Transformation of RBS+lacI+B0010+B0012+pLac ,and cultivation on LB-K plate

    • Electrophoresis of insert fragment [37℃RBS+luxR+37℃RBS+mGFP+B0015 & mRFP+J61048 & sRNA 1 & sRNA 2 & target 1 & target 2]----?

    • Mini-prep of cultivated Plux & PLac E.coli Digestion : Plux [ES] & Plac [ES] & mRFP+J61048 [XP].

    22

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

    • Transformation of the ligation product made at this morning and cultivation on LB-C plate.

    • Mini-prep of Pcons+mRFP

    • Digestion: [Pcons]ES.

    • Electrophoresis of Pcons+mRFP──NOT OK.

    • Cultivation of Pcons+mRFP with liquid LB-C. PCR of Pcons+mRFP

    • Electrophoresis of Pcons+mRFP──OK.

    • Electrophoresis of insert fragment [Plux & Plac & mRFP+J61048?]----?

    23

    • PCR +Single colony isolation of LacI+J61048+Plac and electrophoresis Of this insert fragment OK

    • Mini-prep of Pcons-mRFP.

    • Digestion: [B0030]XP&[B0032]XP&[B0023]XP&[Pcons]ES

    • Electrophoresis of B0030,B0032,B0034,Pcons──OK

    • Transformation of B0030+pcyA+B0032+ho1+B0030/B0032/B0034

    • Electrophoresis of insert fragment [mRFP+J61048]----?

    • Transformation of RBS+lacI+B0010+B0012+Plac ,and cultivation on LB-K plate----NOT OK

    • Single colony isolation from RBS+lacI+B0010+B0012+pLac & mRFP+J61048 LB-K plate, and cultivation of in liquid LB-K

    24

    • Cultivation of LacI+J61048+Plac E.coli in liquid LB-C tubes

    • Mini-prep of cultivated LacI+J61048+Plac E.coli.

    • Cultivation of B0030+pcyA+B0032+ho1+B0032 with liquid LB

    • Transformation of B0030+pcyA+B0032+ho1+B0030/B0032/B0034

    • Mini-prep of cultivated mRFP+J61048 E.coli

    • Digestion : mRFP+J61048 [XP]

    • Transformation of target 1 ,and cultivation on LB-C plate

    • Single colony isolation from RBS+lacI+B0010+B0012+pLac plate, and cultivation of in liquid LB-without antibiotics----NO E.coli?

    25

    • Digestion of LacI+J61048+Plac Cr [XP]

    • Electrophoresis of[ LacI+J61048 +Plac]dig XP OK

    • Decide to determine the sequence of LacI+J61048 +Plac

    • Ligation :insert [B0030]ES& [TetR+B0015]XP/vector [PSB1K3]EP.

    • Cultivation of B0030+pcyA+B0032+ho1+B0030/B0032/B0034 with liquid LB.

    • PCR of insert fragment [target 1]

    • Single colony isolation from RBS+lacI+B0010+B0012+pLac plate , and cultivation of in liquid LB-without antibiotics----NO E.coli?

    • Electrophoresis of insert fragment [mRFP+J61048 & target 1]----?

    26

    • Transformation of B0030+TetR+B0015+PSB1K3 and cultivation on LB-K plate. -Mini-prep of B0030+pcyAB0032+ho1+B0030/B0032/B0034.

    • Digestion: [B0030+pcyA+B0032+ho1+B0030/B0032/B0034]XP

    • Electrophoresis of digested B0030+pcyA+B0032+ho1+B0030/B0032/B0034──OK

    • Electrophoresis of B0030,B0032,B0034,Pcons──OK

    • Mini-prep of cultivated target 1 & mRFP+J61048 E.coli

    • Digestion: target 1 [ES] & mRFP [XP]?

    • Electrophoresis of insert fragment [RBS+lacI+B0010+B0012+pLac & mRFP & target 1]----?

    27

    • PCR and single colony isolation of B0030+TetR+B0015 Kr E.coli

    • Cultivation of B0030+TetR+B0015 in liquid LB-tube.

    • Ligation: pSB1C3&Pcons&B0030+pcyA+B0032+ho1+B0030

    28

    • Cultivation of B0030+TetR+B0015 in liquid LB-K tubes again

    • Ligation:insert[PompC+B0032]ES+[LacI+J61048+Plac]XP/vector[PSB1K3]EP

    • Mini-prep of cultivated B0030+TetR+B0015 Kr E.coli

    • Transformation of Pcons+B0030+pcyA+B0032+ho1+B0030

    29

    • Transformation of PompC+B0032+LacI+J61048+Plac+PSB1K3. -Transformation of Cph8.

    • Digestion: [Pcons]XP.

    30

    • Ligation:insert[PompC+B0032]ES+[LacI+J61048+Plac]XP/vector[PSB1K3]EP&[PSB1A3]EP

    • Transformation of PompC+B0032+LacI+J61048+Plac+PSB1K3&PSB1A3.

    • Electrophoresis of Pcons ─OK. Digestion: [Pcons]SP.

    31

    • PCR + single colony isolation of PompC+B0032+LacI+J61048+Plac+PSB1K3&PSB1A3 E.coli

    • Electrophoresis of PompC+B0032+LacI+J61048+Plac OK.

    • Ligation: Pcons&B0030+pcyA+B0032+ho1+B0030.

    • Transformation of Pcons+B0030+pcyA+B0032+ho1+B0030

    August 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • Cultivation of PompC+B0032+LacI+J61048+Plac Kr&Ar E.coli in liquid LB-K&LB-A tubes.

    • Cultivation of Pcons+B0030+pcyA+ B0032+ho1+B0030 with liquid LB-K

    2

    • Mini-prep of cultivated PompC+B0032+LacI+J61048+Plac Kr&Ar E.coli

    • Digestion: [PompC+B0032+LacI+J61048]ES. Mini-prep of Pcons+B0030+pcyA+ B0032+ho1+B0030.

    • Digestion: [Pcons+B0030+pcyA+ B0032+ho1+B0030]ES

    • Electrophoresis of Pcons+B0030+pcyA+ B0032+ho1+B0030──OK

    3

    4

    5

    6

    7

    8

    9

    • Ligation: insert[PompC+B0032]ES+[LacI+J61048+Plac]XP/vector[PSB1K3]EP, then transformation of this ligation product

    10

    11

    • PCR + single colony isolation of PompC+B0032+LacI+J61048+Plac Kr E.coli

    • Electrophoresis of PompC+B0032+LacI+J61048+Plac not OK

    12

    • Colony PCR of 37。C RBS+LuxR+37。C RBS+mGFP Ar E.coli&Plux+sRNA2 Kr E.coli&mRFP+J61048 Kr E.coli

    • Electrophoresis of [target site 1]PCR&[37。C RBS+LuxR+37。C RBS+mGFP]PCR&[LacI+J61048+Plac+sRNA2]dig XP&[target site2+mRFP]dig ES&[Plux+sRNA2]PCR&[mRFP+J61048]PCR

    • Cutivation of Plux+sRNA2 Kr E.coli in liquid LB-K tube

    13

    • Mini-prep of cultivated Plux+sRNA2 Kr E.coli(fault)

    • Transformation of 37。C RBS+LuxR+37。C RBS+mGFP in PSB1A3

    • Ligation:insert[sRNA+target site1]XP/vector[PSB1C3]XP Colony PCR of target site2+mRFP Ar E.coli

    • Cultivation of Plux+sRNA2 Kr E.coli in liquid LB-K tube

    14

    • Mini-prep of cultivated Plux+sRNA2 Kr E.coli

    • Digestion:[Plux+sRNA2]XP

    • Digestion:[Plux+sRNA2]XP

    • Colony PCR of 37。C RBS+LuxR+37。C RBS+mGFP Ar E.coli

    • Electrophoresis of [target site2+mRFP]PCR

    • Transformation of target site1 in PSB1C3

    • Electrophoresis of [target site2+mRFP]PCR&[LacI+J61048+Plac+sRNA2]mini

    15

    • Electrophoresis of [LacI+J61048+ Plac+sRNA2]dig XP&[Plux+sRNA2]dig XP&[37。C RBS+LuxR+37。C RBS+mGFP]PCR

    • Colony PCR of target 1 Cr E.coli&mRFP+J61048 Kr E.coli and then electrophoresis of these PCR products

    16

    • Colony PCR of target 1 Cr E.coli and then electrophoresis of PCR products

    • Transformation of mRFP+J610148 in PSB1K3(failed)

    17

    • Colony PCR of mRFP+J61048 Kr E.coli and then electrophoresis of the PCR product

    • Colony PCR of target 1 Cr E.coli and then electrophoresis of the PCR product

    • Transformation of mRFP+J610148 in PSB1K3(failed)

    • Cultivation of mRFP+J61048 Kr E.coli in liquid LB-K tube(failed)

    18

    • Colony PCR of mRFP+J61048 Kr E.coli

    • Digestion:[J61048]XP&[target site1]XP

    • Ligation:insert[target site2]ES+[J61048]XP/vector[PSB1C3]EP

    • Electrophoresis of [mRFP+J61048]PCR&[target site1]PCR

    19

    • Cultivation of mRFP+J61048 Kr E.coli in liquid LB-K tube

    • Transformation of target site2+mRFP+J61048 in PSB1C3

    • Ligation:insert[target site 1]/vector[PSB1C3]

    • Mini-prep of cultivated mRFP+J61048 Kr E.coli

    • Digestion:[mRFP+J61048]XP

    20

    • Colony PCR of target site2+mRFP+J61048 Cr E.coli

    • Transformation of target site1 in PSBC3 Cr E.coli

    • Electrophoresis of[mRFP+J61048]dig XP&[Plux+sRNA]digXP

    • Mini-prep of cultivated Plux+sRNA2 Kr E.coli Digestion:[Plux+sRNA2]XP

    21

    • Electeophoresis of [mRFP+J61048]dig XP&[ target site2+mRFP+J61048]PCR

    22

    • Cultivation of target site2+mRFP+J61048 Cr E.coli

    • Transformation of target site1 in PSB1C3

    • Ligation:insert[mRFP]ES+[J61048]XP/vector[PSB1K3]EP

    • Dig:LuxI(BBa_C0061)Transformation of mRFP+J61048 in PSb1K3&LuxI inPSB1A3

    • Mini-prep of cultivated target site2+mRFP+J61048 Cr E.coli and then digestion:[target site2+mRFP+J61048]XP

    23

    • Electrophoresis of [target site 2+mRFP+J61048]dig XP&[37。C RBS+LuxR+37。C RBS+GFP]PCR&[Pompc+B0032+LacI+J61048+Plac]PCR&[mRFP+J61048]PCR&[target site1]PCR

    • Cultivation of LuxI Ar E.coli in liquid LB-A tube

    • Colony PCR of target site1 Cr E.coli&mRFP+J61048Kr E.coli

    • Dig:B0034+LuxI(BBa_C0261)Transformation of B0034+LuxI in PSB1A3

    24

    • Cultivation of 37。C RBS+LuxR+37。C RBS+mGFP Ar E.coli&B0034+LuxI Ar E.coli in liquid LB-A tubes and Pompc+B0032+LacI+J61048+Plac Kr E.coli in liquid LB-K tube

    • Digestion:[37。C RBS+mGFP]ES&[37。C RBS+LuxR]XP&[sRNA1]ES&[Pcons]XP

    • ligation:insert[target site 1]XP/vector[PSB1C3]XP&insert[mRFP]ES+[B0015]XP/vector[PSB1C3]EP&insert[Pcons]ES+[target site 2+mRFP+J61048]XP/vector[PSB1C3]EP&insert[37。C RBS+mGFP]ES+[37。C RBS+LuxR]XP/vector[PSB1C3]EP&insert[sRNA1]ES+[Pcons]XP/vector[PSB1K3]EP&insert[Pcons]ES+[32。C RBS+mGFP]XP/vector[PSB1C3]EP and then transformation

    • mini-prep of cultivated LuxI Ar E.coli&B0034+LuxI Ar E.coli&Pompc+B0032+LacI+J61048+Plac Kr E.coli&37。C RBS+LuxR+37。C RBS+mGFP Ar E.coli

    25

    • Digestion:[LuxI]XP&[B0034+LuxI]ES&[ Pompc+B0032+LacI+J61048+Plac]ES&[7。C RBS+LuxR+37。C RBS+mGFP]&[PSB1A3.PSB1C3.PSB1K3]EP&[PSB1C3]ES and XP&[37。C RBS+mGFP]EP and then electrophoresis

    • Ligation:insert[B0034+LuxI]ES+[B0015]XP/vector[PSB1C3]EP&[Pcons]ES+[target site 2+mRFP+J61048]XP+[37。C RBS+mGFP]EP

    • Transformation of B0034+LuxI+B0015 inPSB1C3&Pcons+target site2+mRFP+J61048&Pcons+32。C RBS+mGFP in PSB1C3

    • Colony PCR of sRNA1+Pcons Kr E.coli&target site1 Cr E.coli&mRFP+B0015 Cr E.coli&37。C RBS+mGFP+37。C RBS+LuxR Cr E.coli

    • Electrophoresis of [target site1]PCR&[mRFP+B0015]PCR&[37。C RBS+mGFP+37。C RBS+LuxR]PCR+[sRNA1+Pcons]PCR

    26

    • Mini-prep of cultivate target site1Cr E.coli&mRFP+B0015 Cr E.coli&37。C RBS+mGFP+37。C RBS+LuxR Cr E.coli&sRNA+Pcons Kr E.coli

    • Digestion:[target site 1]ES&[ mRFP+B0015]XP&[37。C RBS+mGFP+37。C RBS+LuxR]ES&[ sRNA+Pcons]ES&[37。C　RBS+LuxR+37。C RBS+mGFP]ES

    • Colony PCR of B0034+LuxI+B0015 Cr E.coli&Pcons+target site 2+mRFP+J61048 Kr E.coli

    • Electrophoresis of those digestion and PCR products made today

    27

    • Mini-prep of cultivated Pcons+target site2+mRFP+J61048 K r E.coli

    • Digestion:[ Pcons+target site2+mRFP+J61048]ES&[Pompc+B0032+LacI+J61048+Plac]XP

    • Ligation insert[target site 1]ES+[mRFP+B0015]XP/vector[PSB1A3]EP&insert[Pcons+target site2+mRFP+J61048]ES+[Pcons+B0032+LacI+J61048+Plac+sRNA2]XP/vector[PSB1A3]EP&insert[Pcons]ES+[37。C RBS+mGFP+37。CRBS+LuxR]XP/vector[PSB1K3]EP&insert[Pcons]ES+[37。C RBS+LuxR+37。C RBS+mGFP]XP/vector[PSB1K3]EP&insert[Pcons+target site 2+mRFP+J61048]ES+[Plux+sRNA2]XP/vector[PSB1A3]EP&insert[Pompc+B0032+LacI+J61048+Plac]ES+[37。C RBS+mGFP+37。C RBS+LuxR]XP/vector[PSB1A3]EP&insert[Pompc+B0032+LacI+J61048+Plac]ES+[37。CＲＢＳ＋LuxR+37。C RBS+mGFP]XP/vector[PSB1C3]EP&insert[sRNA1(With Plux)]ES+[Pompc+B0032+LacI+J61048+Plac]XP/vector[PSB1A3]EP&insert[Pompc+B0032+LacI+J61048+Plac]ES+[target site2+mRFP]XP/vector[PSB1A3]&insert[Pcons+target site 2+mRFP+J61048]ES+[Pcons+B0032+LacI+J61048+Plac+sRNA2]XP/vector[mGFP]EP&insert[Pcons+target site2+mRFP+J61048]ES+[Plux+sRNA]XP/vector[mGFP]EP

    • Transformation of these ligation product and B0034+LuxI+B0015 in PSB1C3

    • Electrophoresis of [Pcons+tar get site 2+mRFP+J61048]PCR&[PSB1K3]dig EP&[Pcons+target site2+mRFP+J61048]dig ES&[Pompc+B0032+LacI+J61048+Plac]XP&[37CRBS+mGFP+37C

    28

    • Cultivation of B0034+LuxI+B0015 E.coli on LB-C plate&Pcons+37。C RBS+mGFP+37。C RBS+LuxR E.coli on LB-K plate&Pcons+37。C RBS+LuxR+37。C RBS+mGFP E.coli on LB-K plate&sRNA1(with Plux)+Pompc+B0032+LacI+J61048+Plac on LB-A plate

    • Colony PCR of target site 1+mRFP+B0015 Ar& Pcons+target site2+mRFP +J61048+Pcons+B0032+LacI+J61048+Plac+sRNA2 Ar & Pcons+37。C RBS+mGFP+37。C RBS+LuxR Kr&Pcons+target site 2+mRFP+J61048+Plac+sRNA2 Ar&Pompc+B0032+LacI+J61048+Plac+37。C RBS+mGFP+37。C RBS+LuxR Ar&PomPC +B0032+LacI+J61048+Plac+37。C RBS+LuxR+37。C RBS+mGFP Cr &sRNA2(with Plac)+Pompc+B0032+LacI+J61048+Plac Ar& Pompc+B0032+LacI+J61048+Plac+target site 2+mRFP+J61048 Ar E.coli and then electrophoresis of these PCR products

    29

    • Mini-prep of cultivated target site 1+mRFP+B0015 Ar &Pcons+target site 2+mRFP+J61048+Pcons+B0032+LacI+J61048+Plac+sRNA 2 Ar&Pcons+37。C RBS＋mGFP+37。C RBS+LuxR Kr & Pompc+B0032+LacI+J61048+Plac+37。C RBS+LuxR+37。C RBS+mGFP Cr & Pcons+target site 2+mRFP+J61048+Plux+sRNA2 Kr &Pcons+37。C RBS+LuxR+37。C RBS+mGFP Kr E.coli

    • Digestion:[ target site 1+mRFP+B0015]XP&[Pcons+target site 2+mRFP+J61048+Pcons+B0032+LacI+J61048+Plac+sRNA 2]XP&[ Pcons+37。C＋mGFP+37。C RBS+LuxR ]ES&[ Pompc+B0032+LacI+J61048+Plac+37。C RBS+LuxR+37。C RBS+mGFP]ES and then electrophoresis of these digestion products

    • Colony PCR of Pcons+target site mRFP+J61048+Pcons+B0032+LacI+J61048+Plac+sRNA2 Ar &Pcons+target site2+mRFP+J61048+Plac+sRNA2 Ar &Pompc+B0032+LacI+J61048+Plac+37。 C RBS+mGFP+37。C RBS+LuxR Ar &Pompc+B0032+LacI+J61048+Plac+target site2+mRFP+J61048 Ar &sRNA1(with Plac)+Pompc+B0032+LacI+J61048+Plac Ar &Pcons+37。C RBS+LuxR+37。C RBS+mGFP Kr &B0034+LacI+B0015 Cr E.coli and then electrophoresis of these PCR products

    • Ligation:insert[Pcons]ES+[37。 C RBS+mGFP]XP/vector[PSB1C3]EP&insert[37。C RBS]ES+[LacI+J61048+Plac+sRNA2]XP/vector[PSB1K3]EP&insert[B0034+LuxI]ES+[J61048]

    30

    • Cultivation of target site1+mRFP+B0015 Ar and Pcons+target site2+mRFP+J61048+Pcons+B0032+LacI+J61048+Plac+sRNA2 Ar E.coli in liquid LB-A tubes &Pcons+37。C RBS+mGFP+37。C RBS+LuxR Kr E.coli in liquid LB-K tube &Pompc+B0032+LacI+J+61048+Plac+37。C RBS+LuxR+37。C RBS+mGFP Cr E.coli in liquid LB-C tube &Plux+sRNA 2 in liquid LB tube

    • Mini-prep of those cultivated E.coli which was cultivated this morning

    • Electrophoresis of the mini-prep products made today and [Pcons+target site 2+mRFP+J61048+Pcons+B0032+LacI+J61048+Plac+sRNA 2]dig ES &[ Pcons+target site 2+mRFP+J61048+Plux+sRNA2]dig ES&[cons+37。C RBS+LuxR+37。C RBS+mGFP]XP

    • digestion:[B0034]XP&[target site 2]XP colony PCR of Pcons+37。C RBS Cr E.coli&37。C RBS+LacI+J61048+Plac+sRNA2 Kr E.coli&B0034+LacI+J61048 Kr E.coli&B0032+LacI Kr E.coli and then electrophoresis of these PCR products

    31

    • Ligation:insert[Pcons]ES+[target site 2]XP/vector[PSB1K3]EP&insert[Pcons]ES+[B0034]XP/vector[PSB1K3]EP

    • Transformation of Pcons+target site 2 in PSB1K3&Pcons+B0034 in PSB1K3 & BBa_K516030(mRFP protein generator) in PSB1C3&BBa_K516132(Constitutive promoter [J23101] with mRFP, RBS B0032) in PSB1C3&Pcons+LacI+Plac+sRNA2 in PSB1K3

    • Mini-prep of cultivated Pcons+37。C RBS+mGFP Cr E.coli&37。C RBS+LacI+J61048+Plac+sRNA2 Cr E.coli&B0032+LuxI Kr E.coli

    • digestion:[ Pcons+37。C RBS+mGFP]ES&[37。C RBS+LacI+J61048+Plac+sRNA2]XP&[ B0032+LuxI]ES &[Pcons+37。C RBS+mGFP+37。C RBS+LuxR]ES&[Pompc+B0032+LacI+J61048+Plac+37。C RBS+LuxR+37。C RBS+mGFP]ES

    • cultivation of Pompc+B0032+LacI+J61048+Plac+37。C RBS+mGFP+37。C RBS+LuxR Ar &Pompc+B0032+LacI+J61048+Plac+target site 2+mRFP=J61048 Ar E.coli on LB-A plates

    September 2013

    Sunday

    Monday

    Tuesday

    Wednesday

    Thursday

    Friday

    Saturday

    1

    • Ligation:insert[Pcons+37。C RBS+mGFP]ES+[37。C RBS+LacI+J61048+Plac+sRNA2]XP/vector[PSB1A3]EP&inert[Pcons+37。C RBS+mGFP]ES+[37。C RBS+LuxR]XP/vector[PSB1A3]EP

    2

    • Mini-prep of cultivated Pcons+37。C RBS+mGFP+37。C RBS+LacI+J61048+Plac+sRNA2 Ar E.coli Colony PCR of Pompc+B0032+LacI+J61048+Plac+37。C RBS+mGFP+37。C RBS+LuxR Ar &sRNA(with Plux)+Pompc+B0032+LacI+J61048+Plac Ar &Pompc+B0032+LacI+J61048+Plac+target site2+mRFP+J61048 Ar &Pcons+37。C RBS+mGFP+37。C RBS+LuxR Ar E.coli Electrophoresis of PCR products today

    3

    • Mini-prep of cultivated Pcons+37。C RBS+mGFP+37。C RBS+LacI+J61048+Plac+sRNA2 Ar E.coli Colony PCR of Pompc+B0032+LacI+J61048+Plac+37。C RBS+mGFP+37。C RBS+LuxR Ar &sRNA(with Plux)+Pompc+B0032+LacI+J61048+Plac Ar &Pompc+B0032+LacI+J61048+Plac+target site2+mRFP+J61048 Ar &Pcons+37。C RBS+mGFP+37。C RBS+LuxR Ar E.coli Electrophoresis of PCR products today

    • Digestion:[ Pompc+B0032+LacI+J61048+Plac+37。C RBS+mGFP+37。C RBS+LuxR]ES&[ sRNA1(with Plux)+Pompc+B0032+LacI+J61048+Plac]ES&[ Pompc+B0032+LacI+J61048+Plac+target site2+mRFP+J61048]ES&[Pcons+37。C RBS+mGFP+37。C RBS+Lux]ES&[BBa_K516030(Constitutive promoter [J23101] with mRFP, RBS B0030) ]XP and then electrophoresis

    • Ligation:insert[Pcons]ES+[target site 2]XP/vector[PSB1K3]&insert[Pcons]ES+[B0034]XP/vector[PSB1K3]EP&insert[B0034+LuxI]ES+[B0015]XP/vector[PSB1C3]EP

    4

    • Colony PCR of Pcons+B0034 Kr&Pcons+target site Kr&Pcons+K516030 Kr&B0034+LacI+B0015 Cr E.coli and then electrophoresis of these PCR products

    • Ligation: insert[Pcons+B0032]ES+[LacI+J61048+Plac+sRNA2]XP/vector[PSB1K3]&insert[37。C RBS]ES+[LacI+J61048+Plac+sRNA2]XP/vector[PSB1K3]EP&insert[Pompc+B0032+LacI+J61048+Plac]ES+[37。C RBS+mGFP]XP/vector[PSB1C3]EPBackbone changing:[37。C RBS+mGFP]XP&[37。C RBS+LuxR]XP&[37。C RBS+LuxR+37。C RBS+mGFP]XP&[Pcons+B0030+PcyA+B0032+LacI+B0030]ES&[Pcons+target site2+mRFP+J61048]ES /vector[PSB1C3]ES and [sRNA+target site2]EP/vector[PSB1C3]EP

    • Digestion:[sRNA+target site2]EP Transformation of Pcons+B0032+LacI+J61048+Plac+sRNA2 in PSB1K3&37。C RBS+LacI+J61048+Plac+sRNA2 in PSB1K3&Pompc+B0032+LacI+J61048+Plac+37。C RBS+mGFP in PSB1C3&37。C RBS+mGFP in PSB1C3&37。C RBS+LuxR in PSB1C3&37。C RBS+LuxR+37。C RBS+mGFP in PSB1C3&Pcons+B0034+PcyA+B0032+LacI+B0030 in PSB1C3&Pcons+target site2+mRFP+J61048 in PSB1C3&sRNA+target site 2 in PSB1C3

    • Mini-prep of cultivated Pcons+B0034 Kr E.coli&Pcons+target site2 Kr E.coli&Pcons+K516030 Kr E.coli and then digestion:[ Pcons+B0034]ES and E&[ Pcons+target site2]ES and E&[Pcons+K516030]ES

    5

    6

    7

    8

    9

    10

    11

    12

    13

    14

    15

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