Team:NTU-LIHPAO-Taiwan/Notebook

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  • July
  • August

7/4

Manufacture CP+ plate

7/6

Learn how to transform

Suicide Part:

Transformation of:

[RBS] (BBa_B0034), [C2] (BBa_C0053)

7/7

Learn how to manufacture competent cell

Manufacture competent cell

PYY part:

Transformation of:

[promoter] (BBa_J23100), [signal peptide] (BBa_K554002), [terminator] (BBa_B0015)

Suicide Part:

Transformation of:

[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806), [C1] (BBa_C0051)

7/8

Learn how to extract the plasmid

Learn how to conduct the gel extraction and electrophoresis

Suicide Part:

Extract the plasmid:

[RBS] (BBa_B0034), [C2] (BBa_C0053)

Restriction enzyme:

[RBS] cleave by EcoRI, [C2] cleave by EcoRI and PstI

Gel electrophoresis:

[RBS] ----- Result: Failed (Smeared band was too blurred)

[C2] ----- Result: Succeeded

Transformation of:

[pC1] (BBa_R0051), [pC2] (BBa_R0053), [endolysin] (BBa_K112806) again

7/9

Suicide Part:

Extract the plasmid:

[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)

Gel electrophoresis:

[RBS] ----- Result: Failed (Smeared band was too blurred)

[C1] ----- Result: Succeeded

[endolysin] ----- Result: Failed (Smeared band was too blurred)

PYY part:

Extract the plasmid:

[signal peptide] (BBa_K554002), [terminator] (BBa_B0015)

Gel electrophoresis:

[signal peptide] ----- Result: Succeeded

[terminator] ----- Result: Succeeded

7/10

Manufacture CP+ plate

Manufacture Amp+ plate

Suicide Part:

Gel electrophoresis:

[RBS] (BBa_B0034) ----- Result: Failed (Smeared band was too blurred)

[C2] (BBa_C0053) ----- Result: Succeeded

Gel extraction:

[C2]

7/11

Suicide Part:

Extract the plasmid:

[RBS] (BBa_B0034), [C1] (BBa_C0051), [endolysin] (BBa_K112806)

PYY part:

Transformation of:

[promoter] (BBa_J23101)

7/12

Suicide Part:

Extract the plasmid:

[pC1] (BBa_R0051)

7/13

PYY part:

Extract the plasmid:

[promoter] (BBa_J23101)

Gel electrophoresis (two white colonies & two red colonies):

[promoter] ------ Result: Failed (white colonies didn’t have band, red colonies had band but not the correct size, we speculated that was pollution)

7/20

Discuss about total synthesis

7/13

PYY part:

Transformation of:

[terminator] (BBa_B0015)

7/24

Discuss our project with professor

PYY part:

Extract the plasmid:

[terminator] (BBa_B0015)

Gel electrophoresis:

[terminator] ----- Result: Failed

7/25

Discuss our problems with Instructor

7/26

PYY part:

Cultured [terminator] again

7/27

PYY part:

Extract the plasmid:

[terminator] (BBa_B0015)

Gel electrophoresis:

[terminator] ----- Result: Succeeded

8/2

Suicide Part:

Extract the plasmid:

[RFP](BBa_E1010)

Restriction enzyme:

[RFP] cleave by EcoRI and SpeI

[Cl] cleave by XbaI and PstI

[pSB1A3] cleave by EcoRI and PstI

8/5

Passaged L.c.393

Manufacture MRS plate

PYY part

Redissoluted DNA of total synthesis

Restriction enzyme:

[signalpeptide -PYY] cleave by EcoRI and PstI

Ligation:

[signalpeptide-PYY]+ [pSB1C3]

Transformation

8/6

Passaged L.c.393

PYY part

Restriction enzyme:

[PYY] cleave by EcoRI and PstI

[TAT-PYY] cleave by EcoRI and PstI

[Y2R] cleave by KpnI and XhoI

Ligation:

[PYY]+ [pSB1C3]

[TAT-PYY]+ [pSB1C3]

[Y2R]+[pcDNA]

Transformation

8/7

PYY part

Gel electrophoresis:

Result:

signalpeptide-PYY] ----- Failed (Smeared band was too blurred)

[PYY] ----- Failed (Smeared band was too blurred)

Transform again

8/10

Manufacture CP⁺ plate

Suicide part

Gel electrophoresis:

Result:

[RFP+Terminator] ----- Failed (Confirmed it was pollution)

[Cl+Terminator] ----- Failed

Clean up:

[RFP+Terminator]

PYY part

Clean up:

[signalpeptide-PYY]+ [pSB1A3]

[PYY]+ [pSB1A3]

[TAT-PYY]+ [pSB1A3]

[Y2R]+[pcDNA]

Transformation

8/11

Suicide part

Clean up:

[Cl+Terminator]

Gel electrophoresis

Result:

[RFP+Terminator]----- Failed (Smeared band was too blurred)

[Cl+Terminator] ----- Failed (Smeared band was too blurred)

PYY part

Extract the plasmid:

[TAT-PYY], [Y2R]

8/12

Suicide part

Redissoluted DNA of total synthesis

Restriction enzyme:

[Lac-RBS] cleave by EcoRI and PstI

[NucA-Terminator] cleave by EcoRI and PstI

[NIsI] cleave by EcoRI and PstI

[LcSPddhol] cleave by EcoRI and PstI

Ligation:

[Lac-RBS]+ [pSB1C3]

[NucA-Terminator]+ [pSB1C3]

[NIsI]+ [pSB1C3]

[LcSPddhol]+ [pSB1C3]

Transformation

PYY part

Gel electrophoresis:

Result:

[TAT-PYY]----- Failed(Confirmed it was pollution)

[Cll] ----- Succeeded

8/13

Suicide part

Extract the plasmid:

[Lac-RBS]+ [pSB1C3]

[NucA-Terminator]+ [pSB1C3]

[NIsI]+ [pSB1C3]

Clean up:

[Cl], [terminator], [RFP]

Ligation:

[Cl]+ [terminator]

[RFP]+ [terminator]

Gel electrophoresis:

Result:

[Cl]+ [terminator] ----- Succeeded

[RFP]+ [terminator] ----- Succeeded

PYY part

Extract the plasmid:

[LcSPddhol]+ [pSB1C3]

Clean up:

[TAT-PYY]

8/14

DNA sequencing:

[SP-PYY], [PYY], [Y2R], [Lac-RBS], [NucA-Terminator], [NIsI], [LcSPddhol]

Suicide part

Ligation:

[Cl]+ [terminator]

Gel electrophoresis:

Result:

[Lac-RBS] ----- Succeeded

[NucA-Terminator] ----- Succeeded

[NIsI] ----- Succeeded

[LcSPddhol] ----- Succeeded

Extract the plasmid:

[RFP-terminator]

8/15

Passaged L.c.393

Suicide part

Extract the plasmid:

[Cl-terminator]

Gel electrophoresis:

Result:

[RFP-terminator] ----- Succeeded

[Cl-terminator] ----- Succeeded

PYY part

Extract the plasmid:

[TAT-PYY]

Transformation of [TAT-PYY]

Restriction enzyme:

[TAT-PYY] cleave by EcoRI and PstI

Gel electrophoresis:

Result:

[TAT-PYY] ----- Succeeded

8/16

Passaged L.c.393

Manufacture LB broth

Manufacture MRS broth

Suicide part

Restriction enzyme:

[Cl-Terminator] cleave by EcoRI and PstI

[RFP-Terminator] cleave by EcoRI and PstI

PYY part

Transformation of [SP-PYY], [PYY], [TAT-PYY]

8/17

Suicide part

Ligation:

[plac-RFP-T], [plac-Cl-T]

Transformation of [plac-RFP-T], [plac-Cl-T]

PYY part

Culture [SP-PYY], [PYY], [TAT-PYY] in BL21 Competent

E. coli and Rosetta Competent E. coli

8/18

Suicide part

Culture [plac-RFP], [plac-Cl], [NucA] in LB broth

Extract the plasmid:

[plac-RFP], [plac-Cl]

Gel electrophoresis:

Result:

[plac-RFP] ----- Succeeded

[plac-Cl] ----- Succeeded

PYY part

Western blot ([SP-PYY], [PYY], [TAT-PYY]):

Sonication of Competent E. coli

8/19

Suicide part

Extract the plasmid:

[NucA]

Gel electrophoresis:

Result:

[NucA] ----- Succeeded

PYY part

Western blot ([SP-PYY], [PYY], [TAT-PYY]):

SDS-page

Transfer

Blocking

Primary antibody

8/20

Passaged Caco2 cell

Suicide part

Gel Purification:

[plac-Cl-T], [pCl-NucA-T]

Ligation:

[plac-Cl-T], [pCl-NucA-T]

Transformation of [plac-Cl-T], [pCl-NucA-T]

PYY part

Western blot ([SP-PYY], [PYY], [TAT-PYY]):

Secondary antibody

Chemiluminescent detection

Result:

[SP-PYY] ----- Failed (no band displayed)

[PYY] ----- Failed (no band displayed)

[TAT-PYY] ----- Failed (no band displayed)

Western blot ([SP-PYY], [PYY], [TAT-PYY]):

SDS-page

Transfer

Blocking

Primary antibody

8/21

Suicide part

Culture [[plac-Cl-T], [pCl-NucA-T] in LB broth

Extract the plasmid:

[plac-Cl-T], [pCl-NucA-T]

Gel electrophoresis:

Result:

[plac-Cl-T] ----- Succeeded

[pCl-NucA-T] ----- Failed (band was too blurred)

PYY part

Western blot ([SP-PYY], [PYY], [TAT-PYY]):

Secondary antibody

Chemiluminescent detection

Result:

[SP-PYY] ----- Failed (band was too blurred)

[PYY] ----- Failed (no band displayed)

[TAT-PYY] ----- Failed (no band displayed)

Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again. (use [RFP] as negative control)

Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21

Competent E. coli and Rosetta Competent E. coli

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