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Revision as of 05:29, 16 September 2015

NTU-LIHPAO-Taiwan

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
8/22
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY] , [RFP]):
Sonication of Competent E. coli
8/23
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY] , [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
8/24
Suicide part
Restriction enzyme:
[NucA] cleave by SpeI and PstI
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (band was not specificity)
[PYY] ----- Failed (band was not specificity)
[TAT-PYY] ----- Failed (band was not specificity)
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP]in mini
8/25
Suicide part
Restriction enzyme:
[plac-C1-T-backbone] cleave by EcoRI
Gel electrophoresis:
Result:
[plac-C1-T] ----- Failed
Redissoluted [pJ23102]
Transformation of [pCl], [pCll], [RBS], [pJ23102]
Culture [pCl], [pCll], [RBS] in LB broth
PYY part
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in midi
Centrifugal and took the supernatant
Bradford protein assay
8/26
Passaged Caco2 cell
Suicide part
Ligation:
[plac-Cl-T]
Transformation of [plac-Cl-T]
Extract the plasmid:
[plac], [pCl], [pCll], [RBS]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
8/27
Suicide part
Transformation of [RBS-Cl-T], [RBS-RFP-T]
Culture [plac-Cl-T],[RBS-Cl-T],[RBS-RFP-T],[pJ23102] in LB broth
Extract the plasmid:
[plac-Cl-T], [pJ23102]
Gel electrophoresis:
Result:
[plac-Cl-T] ----- Succeeded
[pJ23102] ----- Succeeded
Restriction enzyme:
[pCl-T] cleave by SpeI and PstI
Gel Purification:
[pCll]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (no band displayed)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
Transformation of [SP-PYY], [PYY], [TAT-PYY], [RFP] again
Culture [SP-PYY], [PYY], [TAT-PYY] , [RFP] in BL21
Competent E. coli
8/28
Suicide part
Ligation:
[pCIt]+[NucA]
Extract the plasmid:
[Cl-T], [RFP-T]
Gel electrophoresis:
Result:
[Cl-T] ----- Failed (band size was wrong)
[RFP-T] ----- Failed (band size was wrong)
Restriction enzyme:
[plac-T] cleave by SpeI and PstI
[pJ23102] cleave by SpeI and PstI
Gel Purification:
[pCI-T]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
8/29
Passaged Caco2 cell
Suicide part
Extract the plasmid:
[RBS-Cl-T], [RBS-RFP-T]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (no band displayed)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
Histag column([SP-PYY], [PYY], [TAT-PYY] , [RFP])
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
8/30
Suicide part
Extract the plasmid:
[RBS], [pCl]
Gel electrophoresis:
Result:
[RBS] ----- Succeeded
[pCl] ----- Failed
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (no band displayed)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
8/31
Passaged hek293 cell
Suicide part
Gel Purification:
[RBS], [CI-T]
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
SDS-page
Transfer
Blocking
Primary antibody
9/1
Suicide part
Ligation:
[RBS] + [CI-T]
Culture [RBS- CI-T] in LB broth
PYY part
Western blot ([SP-PYY], [PYY], [TAT-PYY], [RFP]):
Secondary antibody
Chemiluminescent detection
Result:
[SP-PYY] ----- Failed (no band displayed)
[PYY] ----- Failed (no band displayed)
[TAT-PYY] ----- Failed (no band displayed)
Transformation of [SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Selection part
Culture [nisI] in MRS broth
9/2
Suicide part
Extract the plasmid:
[RBS- CI-T]
Gel electrophoresis:
Result:
[RBS- CI-T] ----- Succeeded
PYY part
Extract the plasmid:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Restriction enzyme:
[SP-PYY] cleave by EcoRI
[PYY] cleave by EcoRI
[TAT-PYY] cleave by EcoRI
[pETDuet-1] cleave by EcoRI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Selection part
Nisin Immunity Test
Culture [nisI] in LB broth
9/3
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Failed (small band didn’t display)
[PYY] ----- Failed (small band didn’t display)
[TAT-PYY] ----- Failed(small band didn’t display)
[pETDuet-1] ----- Succeeded
Again -->
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
16℃ Ligation 4hr:
[SP-PYY]+ [pETDuet-1] ----- Failed
[PYY]+ [pETDuet-1] ----- Failed
[TAT-PYY]+ [pETDuet-1] ----- Failed
Selection part
Extract the plasmid:
[nisI-pSB1C3]
Restriction enzyme:
[nisI-pSB1C3] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[nisI-pSB1C3] ----- Succeeded
Nisin Immunity Test
9/4
Suicide part
Ligation:
[pJ23102] + [RBS-CI-T]
[plac] + [RBS-CI-T]
Transformation of [pJ23102-RBS-CI-T], [plac-RBS-CI-T]
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
16℃ Ligation 4hr:
[SP-PYY]+ [pETDuet-1] ----- Failed
[PYY]+ [pETDuet-1] ----- Failed
[TAT-PYY]+ [pETDuet-1] ----- Failed
9/5
Suicide part
Culture[pJ23102-RBS-CI-T], [plac-RBS-CI-T] in LB broth
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
16℃ Ligation 4hr:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Transformation of [SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Measure the resistance of Caco2 cell
9/6
Suicide part
Extract the plasmid:
[placE-RBS-CI-T], [pJ23102-RBS-CI-T], [placE]
Gel electrophoresis:
Result:
[placE-RBS-CI-T] ----- Failed
[pJ23102-RBS-CI-T] ----- Succeeded
[placE] ----- Succeeded
PYY part
Extract the plasmid:
[SP-PYY-pETDuet-1],[PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet-1] cleave by XbaI and PstI
[PYY-pETDuet-1] cleave by XbaI and PstI
[TAT-PYY-pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Extract the plasmid:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1]-----Failed(Ligation failed)
[PYY-pETDuet-1] ----- Failed(Ligation failed)
[TAT-PYY-pETDuet-1]-----Failed(Ligation failed)
Measure the resistance of Caco2 cell
9/7
Suicide part
Restriction enzyme:
[placE] cleave by SpeI and PstI
[RBS-CI-T] cleave by XbaI and PstI
Transformation of [pCLP7]
Restriction enzyme:
[pCLP7] cleave by XbaI and PstI
[nisi] cleave by XbaI and PstI
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Restriction enzyme:
[SP-PYY-pETDuet-1] cleave by XbaI and PstI
[PYY-pETDuet-1] cleave by XbaI and PstI
[TAT-PYY-pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Failed(small band didn’t display)
[PYY] ----- Failed(small band didn’t display)
[TAT-PYY] ----- Failed(small band didn’t display)
[pETDuet-1]-----Failed(small band didn’t display)
[SP-PYY-pETDuet-1]-----Failed(Ligation failed)
[PYY-pETDuet-1] ----- Failed(Ligation failed)
[TAT-PYY-pETDuet-1]-----Failed(Ligation failed)
Measure the resistance of Caco2 cell
Selection part
Construction of the plasmid:
[nisI- pCLP7]
Restriction enzyme:
[nisI- pCLP7] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[nisI- pCLP7] ----- Succeeded
Clean up:
[pCLP7]
Ligation:
[nisI] + [pCLP7]
Transformation of [nisI- pCLP7]
9/8
Suicide part
Restriction enzyme:
[pCLP7] cleave by XbaI and PstI
[plac-RBS-RFP-T] cleave by XbaI and PstI
Ligation:
[pCLP7] + [plac-RBS-RFP-T]
Manufacture competent cell with [pJ23102-RBS-CI-T]
PYY part
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1]-----Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
4℃ Ligation overnight:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Measure the resistance of Caco2 cell
Transfect hek293 cell
Selection part
Culture [nisI- pCLP7] in LB broth
9/9
Passaged hek293 cell
Suicide part
Extract the plasmid:
[pCLP7], [place-RBS-CI-T]
Gel electrophoresis:
Result:
[pCLP7] ----- Succeeded
[place-RBS-CI-T]-----Failed(concentration to low)
Transformation of [plac-RBS-RFP-T- pCIp]
Transformation of [plac-RBS-CI-T-NucA] in competent cell with [pJ23102-RBS-CI-T]
PYY part
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1]-----Succeeded
Measure the resistance of Caco2 cell
Selection part
Extract the plasmid:
[nisI- pCLP7]
Restriction enzyme:
[nisI- pCLP7] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[nisI- pCLP7] ----- Failed
9/10
Suicide part
Extract the plasmid:
[plac-RBS-RFP-T- pCLP7]
Gel electrophoresis:
Result:
[plac-RBS-RFP-T- pCLP7] ----- Failed
Manufacture L.casei competent cell
Electroporation of [pCIp] in L.casei competent cell
PYY part
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
4℃ Ligation overnight:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Extract the plasmid:
[SP-PYY-pETDuet-1],[PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by EcoRI and SpeI
[PYY-pETDuet-1] cleave by EcoRI and SpeI
[TAT-PYY-pETDuet1] cleave by EcoRI and SpeI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Measure the resistance of Caco2 cell
Selection part
Extract the plasmid:
[nisI- pCLP7]
Nisin Immunity Test
9/11
Suicide part
Extract the plasmid:
[plac+RBS-RFP-T- pCLP7]
Gel electrophoresis:
Result:
[plac+RBS-RFP-T- pCLP7] ----- Failed
Test electroporation of L.casei
PYY part
4℃ Ligation overnight:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1] in XL1blue
Extract the plasmid:
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
[PYY-pETDuet-1] cleave by XbaI and KpnI
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Extract the plasmid:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
Restriction enzyme:
[SP-PYY] cleave by EcoRI
[PYY] cleave by EcoRI
[TAT-PYY] cleave by EcoRI
[pETDuet-1] cleave by EcoRI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Restriction enzyme:
[SP-PYY] cleave by XbaI and PstI
[PYY] cleave by XbaI and PstI
[TAT-PYY] cleave by XbaI and PstI
[pETDuet-1] cleave by XbaI and PstI
Gel electrophoresis:
Result:
[SP-PYY] ----- Succeeded
[PYY] ----- Succeeded
[TAT-PYY] ----- Succeeded
[pETDuet-1] ----- Succeeded
Gel Purification:
[SP-PYY], [PYY], [TAT-PYY], [pETDuet-1]
4℃ Ligation overnight:
[SP-PYY]+ [pETDuet-1]
[PYY]+ [pETDuet-1]
[TAT-PYY]+ [pETDuet-1]
Measure the resistance of Caco2 cell
Selection part
Nisin Immunity Test
9/12
Suicide part
Test absorbance(O.D. 600nm) of E.coli in Suicide circuit
PYY part
Extract the plasmid:
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
[PYY-pETDuet-1] cleave by XbaI and KpnI
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Transformation of [SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1] in XL1blue
Extract the plasmid:
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
[PYY-pETDuet-1] cleave by XbaI and KpnI
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
Gel electrophoresis:
Result:
[SP-PYY-pETDuet-1] ----- Failed
[PYY-pETDuet-1] ----- Failed
[TAT-PYY-pETDuet-1] ----- Failed
Selection part
Nisin Immunity Test
9/13
PYY part
Extract the plasmid:
[SP-PYY-pETDuet-1], [PYY-pETDuet-1], [TAT-PYY-pETDuet-1]
Restriction enzyme:
[SP-PYY-pETDuet1] cleave by XbaI and KpnI
[PYY-pETDuet-1] cleave by XbaI and KpnI
[TAT-PYY-pETDuet1] cleave by XbaI and KpnI
Selection part
Nisin Immunity Test
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