Team:Peking/Notebook/YWChen

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Contents

• July, 2010

• August, 2010

• September, 2010

• October, 2010

July

7.26

PbrR MBP construction plan

August

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8.1

Ⅰ.Use Tag PCR "PbrR MBP(≈500bp)" for commercial plasmid(PET-21a) and standard plasmid (PSB1K3)

Ⅱ.Digestion:The products of PCR(PbrR MBP)[backbone 4100bp;digest site PstⅠ&NdeⅠ]

Ⅲ.Gel for identification & Retrieve the gel

Ⅳ.Miniprep pbrR-mbp-commercial for backups

8.2

Lpp-OmpA-MBP construction plan

Ⅰ.Use Pfu Mix PCR Nde1+Lpp-OmpA(437bp)+Sal1 & E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1

Ⅱ.Digest the PCR products by Ndel,Sall&EcoR,Sall ,respectively

Ⅲ.if the products above got right ,Retrieve the gel ,if not ,back to Ⅰuse gel retrieve kit then digestion for 2h,after that, go to purification.

8.3

MBP construction plan

Ⅰ.Pfu PCR Sall+N-MBP+Bspel

Bspel+C-MBP+SNP

Bspel+C-MBP+Xhol

Ⅱ.Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel, if not ,back to Ⅰ

8.4

Lpp-OmpA,N-MBP,+C-MBP with backbone PET21a and PSBIK3

Ⅰ.Ligation,Nde1+Lpp-OmpA(437bp)+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+Xhol & PET21a

Ⅱ.Ligation, E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+SNP & PSBIK3

Ⅲ.Learn to do the western blotting. Write the protocols.

8.5

Transformation the products of 4 fragments above.

8.6

There is something wrong with the primer ,so it doesn't work, back to the work from 8.2

8.7

Ⅰ.Use Pfu Mix PCR Nde1+Lpp-OmpA(437bp)+Sal1 & E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1

Ⅱ.Digest the PCR products by Ndel,Sall&EcoR,Sall ,respectively

8.8

Ⅰ.Pfu PCR Sall+N-MBP+Bspel

Bspel+C-MBP+SNP

Bspel+C-MBP+Xhol

Ⅱ.Gel to identification

8.9

Ⅰ.Ligation,Nde1+Lpp-OmpA(437bp)+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+Xhol & PET21a

Ⅱ.Ligation, E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+SNP & PSBIK3

8.10

Transform the ligation product into Trans5α strain.

8.11

Help a teammate transform P(RBS+T3pol 1)&PmerR-PSB1C3 Plasmid to OmniMAX2-T1 Competent cell

Digestion:

Ⅰ.RBS+T3pol 1 with XbaI and pstI(insert)

Ⅱ.PmerR-PSB1C3 with SpeI and PstI (vector)

Retrieve the gel

Ligation the parts above.

8.12

Digestion MBP construct parts

Ligation 4 fragments

Nde1+Lpp-OmpA(437bp)+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+Xhol & PET21a//E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+SNP & PSBIK3 overnight

8.13

Digestion: PET21a 20 µl with NdeI and XhoI

Ligation again

Ⅰ.RBS+T3pol 1 with XbaI and pstI(insert)

Ⅱ.PmerR-PSB1C3 with SpeI and PstI (vector)

Insert:vector=1:7&2:6

Transform the ligation product

Finally got clone ,2:6 better than 1:7

Ⅲ.transformation 4 pieces fragments

8.14

Ⅰ.Plate PCR for identification 4 pieces fragments ligation products

Ⅱ.Miniprep

21a-(1~3) 1K3(1~3) 26-(1~2) 17-(3~4)

Ⅲ.Sent the plasmids for sequencing

8.15

Ⅰ.Got the right sequence 1K3(1~3) 26-(1~2) 17-(3~4) while 21a-(1~3) without MBP but have Lpp-OmpA inside

Ⅱ.transformation

1K3(1~3) 26-(1~2) 17-(3~4) 

Ⅲ.Pick 20 single clones on 21a plate

Ⅳ.PCR for identification

Got the right size from agarose gel electrophoresis ( Lpp-OmpA+MBP=735bp)

Ⅴ.Digestion with NdeI and XhoI,then use PCR purification kit to retrieve products.

Ⅵ.Ligation 1(PET-21a):7

Ⅶ.Transformation

September

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9.1

Connect the antigen 43 into plasmid.

Send merP+GFP+TCP for sequencing.

Connect pPbra+T3 pol using primers annealing.

Do the ligation and transformation.

9.2

Prepare the plasmid DNA for rbs+agn43.

See the sequencing result of merp+GFP+TCP.

Pick the clone of pPbra+T3 pol.

9.3

Send the rbs+agn43 for sequencing.

Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter with EcoRl and Spel.

Pick the clone of pPbra+T3 pol. Prepare the plasmid DNA.

9.6

Do the PCR of antigen 43 using Hifi Taq DNA polymerase.

Digest merP+GFP+TCP with EcoRl and Spel. Put it into PSB3K3 backbone.

Connect pPbra+T3 pol with 1-23L. pick the single clone. Prepare the plasmid DNA.

Do the ligation and transformation.

Pick the clone of Pbad+T3 pol.

Make competent cell containing pc+merR+merp+rbs+T3pol. Transform T3 promoter into it.

Induce the expression using different concentration of IPTG.

Re-suspend the cell. Measure the GFP intensity by a microplate reader.

9.7

Digest merP+GFP+TCP with EcoRl and Pstl to put it into PSB3K3.

Send pbra+T3 pol+terminator for sequencing.

Identify the pBAD+T3 pol using electrophoresis.

Make competent cells of pc+merR+merp+rbs+T3pol.

Transform T3 promoter into it.

9.8

Re-suspend the cell.

Measure the GFP intensity using microplate reader.

PCR antigen 43 using hifi Taq DNA polymerase.

9.9

PCR Rbs+agn 43 using agn for/rev primer to identify it.

Digest the correct ones using EcoRl and Spel.

Send the correct ones for sequencing.

Transform T3 promoter into cells containing pc+merR+merp+rbs+T3pol.

Send pBAD+T3 pol for sequencing.

pPbra+T3pol+terminator done.

Measure the GFP intensity using a microplate reader.

9.12

Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter with EcoRl and Spel.

Identify them using electrophoresis.

Retrieve the gel.

Do the ligation and transformation.

Pick the clone. ===9.13===. Digest Antigen 43 with EcoRl and Xbal. Digest PhiR73+Po promoter+rbs with EcoRl and Spel.

Identify them using electrophoresis.

9.14

PCR antigen 43.

Get the candidate of rbs+agn43.

Send the correct ones for sequencing.

9.15

Put antigen 43 into PSB1A2.

Do the ligation and transformation.

9.16

Digest PSB1A2 and PSB1A3.

9.17

Digest PSB1C3 with EcoRI and Spel.

Digest antigen 43 with EcoRl and Spel.

Do the ligation and transformation.

9.21

Retract the whole genome of K12 strain.

9.22

Nest PCR of antigen 43 using new template and new primers.

Put antigen 43 into PSB1C3.

Pick the single clone of antigen 43.

9.23

Prepare the plasmid DNA for antigen43.

Digest the plasmid DNA using EcoRl and Spel.

Identify them using electrophoresis.

Make the competent cell contains PBAD+ T3 pol. Transform T3 promoter+GFP into it.

Pick the clone of PMERT+T3 pol.

Digest PSB3C5 using EcoRl and Pstl.

9.24

PSB3K5: EcoRl and Pstl.

T7+TPC+Ter: Xbal and Pstl.

MerP+GFP:EcoRl and Spel.

Do the ligation and transformation.

9.25

Retrieve the digested merp+GFP.

Induce the expression of PBAD+T3 pol using 10^-5 M arabinose.

Do the antigen 43 PCR using touchdown PCR.

Do the ligation and transformation.

9.26

Make the competent cell contains T3 promoter+GFP. Transform pmert+T3pol and 1-18i+merR.

Do the colony PCR to identify it.

9.27

Attend the seminar.

9.28

Connect T7+PhiR73+Po promoter+rbs+antigen 43.

Do the ligation and transformation.

PCR the T3 promoter+PhiR73+Po promoter.

9.29

Retrieve the PCR product.

Digest the product using EcoRl and Spel.

Digest TCP with Xbal and Pstl. Identify it using electrophoresis.

Digest merp+GFP using Spel and Pstl.

Do the transformation.

9.30

Do the ligation and transformation.

Prepare the plasmid DNA and identify it using PCR.

October

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10.1

Digest pTET+T7 pol using EcoRl and Spel.

Retrieve the gel.

Connect T3 promoter+PhiR73+Po promoter+ antigen 43.

Do the ligation and transformation.

10.3

Connect pTET+T7 pol and T7 promoter+PhiR73+Po promoter+ antigen 43.

Do the ligation and transformation.

10.4

Do the auto-aggregation assay of antigen 43.

10.5

Do the auto-aggregation assay.

10.7

Digest merp+GFP using Spel and Pstl.

Digest TCP using Xbal and Pstl.

Do the ligation and transformation.

10.8

Identify the digested product using electrophoresis.

Do the ligation and transformation.

10.9

Prepare the plasmid DNA.

Identify them using PCR.

Send the correct ones for sequencing.

10.10

Antigen 43 clone step done.

Connect ptet+T7 pol with T7 promoter+PhiR73+Po promoter+ antigen 43.

Do the ligation and transformation.

10.11

Do the auto-aggregation assay.

10.12

Do the auto-aggregation assay.

10.13

Measure the result.

10.15

Attend group seminar.

Do the auto-aggregation assay.

10.16-10.21

Connect merp+GFP with TCP.

Connect pc+merR with terminator.

Transform these two plasmid into one single strain.

Antigen 43 auto-aggregation assay. Analyse the result.

10.21-10.25

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