iGEM 2015 — Team NTU-LIHPAO-Taiwan

# Team:NTU-LIHPAO-Taiwan/Notebook

NTU-LIHPAO-Taiwan

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* [Home](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan)
* Team

  + [Team](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Team)
  + [Attributions](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Attributions)
* Project

  + [Description](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Description)
  + [Design](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Design)
  + [Results](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Results)
  + [Modeling](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Modeling)
  + [Protocols](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Experiments)
* Parts

  + [Team Parts](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Parts)
  + [Basic Parts](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Basic_Part)
  + [Composite Parts](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Composite_Part)
* [Notebook](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Notebook)
* [Safety](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Safety)
* Society

  + [Human Practices](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Practices)
  + [Collaborations](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Collaborations)
  + [Entrepreneurship](https://2015.igem.org/Team%3ANTU-LIHPAO-Taiwan/Entrepreneurship)

![](https://static.igem.org/mediawiki/2015/6/69/Healthin_White.png)

* Months
  + [July](#First1)
  + [August](#First2)
  + [September](#First3)

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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