Team:NTU-LIHPAO-Taiwan/Notebook

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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

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