Team:Peking/Notebook/YWChen

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Yiwei Chen's Notes

I expanded the MerR protein engineering strategy to lead-responsive regulator, PbrR. Outer membrane protein A (OmpA) was fused with Pb binding peptide at its C-terminal via the same method in mercury binding peptide construction. I also participated the construction of mercury absorption facilitation module and inductive aggregation module.

download her notes

July

7.26

PbrR MBP construction plan

August

[TOP]

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

[TOP]

9.5

Assembly:

T7p+RBS(B0034)+DsbA+MBP+TER(B0015)+T7p+RBS(B0034)+MBP+TER(B0015)+

T7p+RBS(B0034)+Lpp_OmpA+MBP+TER(B0015)

Ⅰ.Digestion:RBS with SpeI and PstI

1-23L terminator with EcoRI and XbaI

Ⅱ.Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel

9.9

Ligation: RBS+MBP

DsbA+MBP

FORGET DIGESTION!!!!

9.10

Digestion:MBD with XbaI and PstI

DsbA-MBP with EcoRI and SpeI

Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel

9.11

Transformation to BL21 for western blotting

Omni for preserve

9.13

Digestion :with XbaI and PstI (for ligation)

Miniprep T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)&RBS+MBP（1~3）

9.15

Ⅰ.Sent the plasmids for sequencing【T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)&RBS+MBP（1~3）】

T7p+RBS(B0034)+DsbA+MBP+Ter 1 got right sequence

Ⅱ.Digestion:

T7p+RBS(B0034)+DsbA+MBP+Ter with EcoRI and SpeI

Identify them using agarose gel electrophoresis.Then retrieve the gel(got the right size)

Ⅲ.Digestion:

RBS+MBP with XbaI and PstI

After Identify them by using agarose gel electrophoresis,the results turns wrong.

Digestion AGAIN!:

RBS+MBP with XbaI and PstI

Meanwhile: Pick 10 single clone for plate PCR by using tagmix

Digest the PCR products with XbaI and PstI ,10 clones all got the right size,so minipret them.

9.16

Ligation :RBS-MBP(XP)+Ter(SP)

Transformation it.

Culture the plates.

9.17

Pick the single clone to PCR, but failed

Meanwhile: Digest the RBS-MBP1 for backup

9.18

Digest the RBS-MBP(2&3)

Ligation: RBS-MBP(2&3) +T7p

Transformation the Ligation products

9.19

Use RBS-MBP(2&3) as templete,PCR(Fast pfu),got a mass of RBS-MBP

Identify them using agarose gel electrophoresis.Then go to retrieve the gel

9.20

Transformation:RBS-MBP-T7p

Culture the plates

9.21

Pick 3 single clone T7p-RBS-MBP(1~3)

Some for PCR some for Miniprep

9.22

Miniprep T7p-RBS-MBP(1~3)

Digestion:T7p-RBS-MBP(1~3) with EcoRI and SpeI

9.23

Wiki writing

Retrieve the digestion products T7p-RBS-MBP(1~3)ES

Ligation:

T7p-RBS-MBP(1~3)+Ter

Transformation:T7p-RBS-MBP-Ter(1~3)

Culture the plates

9.24

Pick 3 single clone from each plate(except1)

Miniprep T7p-RBS-MBP-Ter(2-1~3&3-1~3)

10µI for sequencing

10µI for digestion

Left 20µI

9.25

Digestion:T7p-RBS-MBP-Ter with SpeI and PstI

Gel for identification ,the size of T7p-RBS-MBP-Ter(2-1~3) are right

9.26

Digestion:PSB1C3 with EcoRI and PstI

Ligation:PSB1C3 + T7p-RBS-MBP-Ter+T7p+RBS+Lpp-OmpA+MBP+Ter

Gel retrieving

9.27

Transformation:Positive cloning

T7p+RBS+Lpp-OmpA+MBP+Ter&T7p+RBS(B0034)+DsbA+MBP+Ter

Culture the plates

9.28

The sequence of T7p-RBS-MBP-Ter(2-1~3&3-1~3) came out ,but without T7p inside.

October

[TOP]

10.3

There is something wrong with PSBIC3(RFP inside )

Ligation Again!:

PSB1C3 + T7p-RBS-MBP-Ter+T7p+RBS+Lpp-OmpA+MBP+Ter

10.4

Pick 3 single clone from the plate

Miniprep them.

10.5

Transformation:Positive cloning:T7p+RBS(B0034)+DsbA+MBP+Ter

Digestion:RBS-MBP-Ter+T7p with XbaI and PstI

Identify them using agarose gel electrophoresis.Then go to retrieve the gel

10.6

Pick 3 single clone from the plate（T7p+RBS(B0034)+DsbA+MBP+Ter）

Digestion:PSB1C3 with XbaI and PstI （Change backbone）

Identify them using agarose gel electrophoresis.Then go to retrieve the gel

10.7

Miniprep T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)

Digestion：RBS-MBP-Ter with XbaI and PstI

Ligation:RBS-MBP-Ter+T7p(sp) then transformation

10.8

Ligation again

T7p+RBS-MBP-Ter-+T7p+RBS+Lpp-OmpA+MBP+Ter

Transformation it

10.9

Transformation T7p+RBS+Lpp-OmpA+MBP+Ter(xp)-PSB1C3(xp)

PCR(tag mix) for identification T7p+RBS(B0034)+DsbA+MBP+Ter

Culture the plates, but it did not grow up

10.10

Ligation:T7p+RBS+Lpp-OmpA+MBP+Ter(EP)+PSB1C3

T7p+RBS+DsbA+MBP+Ter(EP)+PSB1C3

Transformation

Easy Mix PCR.

Culture the plates

10.11

ⅠDigestion:T7p+RBS+Lpp-OmpA+MBP+Ter(EP)

T7p+RBS+DsbA+MBP+Ter(EP)

Identify them using agarose gel electrophoresis.Then go to retrieve the gel

10.16

Ⅰ.Ligation: T7p-RBS-MBP-Ter

Ⅱ.Transformation :T7p-RBS-MBP-Ter

Ⅲ.Culture the plates

Pick 3 single clones

10.17

T7p-RBS-MBP-Ter for sequencing

10.20

Ⅰ.Liagation:T7p+RBS+DsbA+MBP+TER+T7p+RBS+MBP+TER+T7p+RBS+Lpp-OmpA+MBP+TER+PSBIC3

Ⅱ.Transformation

Ⅲ.Culture the plates

Pick 6 Single clones

Ⅳ.T7p+RBS+DsbA+MBP+TER+T7p+RBS+MBP+TER+T7p+RBS+Lpp-OmpA+MBP+TER+PSBIC3 for sequencing

[TOP]

==go to her page==

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 ==July==
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+===7.26===
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+PbrR MBP construction plan
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-===7.3===
-Antigen 43 PCR, 20ul system. T3 polymerase PCR.
-Identify them by 1% agarose gel electrophoresis.
-===7.4===
-Antigen 43 PCR using different annealing temperature, with gradient 1 ℃.
-Attend the group seminar.
-Retrieve the PCR product of T3 polymerase and digest it with EcoRI and PstI. Digest for the whole night.
-===7.5===
-Identify the product by agarose gel electrophoresis. Retrieve the PCR product of Antigen 43.
-Retrieve the digested T3 polymerase and ligated it to the plasmid PSB1A2.
-Transform the plasmid to Trans 5α strain.
-PCR the antigen 43 with much larger annealing temperature gradient. Identify it with electrophoresis.
-===7.6===
-Design the primer of antigen 43 again by Primer Premier 5.0.
-===7.8===
-PCR antigen 43 using nest-PCR procedure. Using Taq DNA polymerase to do the first step nest PCR.
-Identify it by electrophoresis. Retrieve the 3k band and do the second step of nest PCR by using the product of the
-first step as template.
-===7.9===
-Identify the second PCR product by electrophoresis. Retrieve the 3k band and digest it with EcoRI and SpeI to put it
-into plasmid. Digest it with PstI to test whether ther is antigen 43.( antigen 43 have 6 PstI digestion sites.)
-Design the six point mutation primer.
-===7.10===
-Do the transformation and ligation.
-===7.12===
-Prepare plasmid DNA.
-Digest the plasmid DNA with PstI to identify it.
-PCR the product using Easytaq DNA polymerase to identify the molecule weight of the product.
-===7.13===
-Learn to do the western blotting. Write the protocols.
-Digest merT, merP and merC with Xbal and PstI. Meanwhile digest B0034(RBS) with SpeI and PstI.
-Connect the two digested product together.
-Transform the ligation product into Trans5αstrain.
-Pick the single clone from the plate.
-Send the PCR product of Antigen 43 for sequencing.
-===7.14===
-Prepare the plasmid DNA of rbs+merT, rbs+merP, rbs+merC.
-Do the western blotting.
-===7.15===
-Digest the plasmid DNA by EcoRI and PstI to identify it.
-Digest rbs+merT with Spel and Pstl and digest rbs+merP with Xbal and Pstl.
-Identify them using agarose gel electrophoresis.
-===7.16===
-Connect rbs with merT, merP and merC again.
-Do the first point mutation. First PCR the antigen 43 gene with designed point mutation primers, then retrieve it by
-electrophoresis. Then do the blunting kination, finally do the ligation and put it into strains by transformation.
-===7.18===
-Prepare the plasmid DNA for rbs+merT, rbs+merP and rbs+merC, Antigen 43 mutant 1.
-Identify them using electrophoresis.
-Connect rbs+merT with rbs+merP.
-Do the second step of point mutation for antigen 43. Do the PCR step. Identify it by electrophoresis.
-Blunting kination, ligation and transformation.
-Send the first point mutation product for sequencing.
-The rbs+merT,rbs+merP and rbs+merC sequenced correct.
-===7.19===
-Retrieve the product of digested rbs+merT and rbs+merP by electrophoresis. Connect the rbs+merT with rbs+merP,. Do the transformation.
-Pick the single clone of antigen 43 second step point mutation strain.
-===7.20===
-Prepare the plasmid DNA of antigen 43 second point mutation strain. Send it for sequencing.
-Do the third step point mutation.
-Identify the plasmid DNA by electrophoresis. Retrieve the third point mutation PCR product by electrophoresis.
-Digest rbs+merT+rbs+merP with Spel and Pstl, digest rbs+merC with Xbal and Pstl.
-===7.21===
-Identify the product of the digestion using electrophoresis.
-Pick the single clone of the 3rd point mutation of antigen 43 strain on the plate.
-===7.22===
-Connect rbs+merT+rbs+merP with rbs+merC.
-Using easyPFu to PCR antigen 43.The forward primer contains a rbs. Digest it with EcoRl and Spel to put it into PSB1A2 plasmid.
-Prepare the plasmid DNA of the product of the third point mutation of antigen 43.digest it with EcoRl and SpeI to identify it.
-Do the 4th point mutation PCR step.
-===7.23===
-Identify the plasmid using agarose gel electrophoresis.
-Send the plasmid DNA for sequencing.
-Put the rbs+agn43 into plasmid by ligation.
-===7.29===
-Attend the group seminar.
-Prepare the plasmid DNA for rbs+merT+rbs+merP+rbs+merC. Digest it with EcoRl and Pstl for identification. Digest it with Xbal and Pstl and digest the plasmid contains T7 promoter with Spel and Pstl.
-Do the 5th point mutation of antigen 43. Do the PCR step.
-Identify the product of the 4th point mutation and the rbs+merT+rbs+merP+rbs+merC by electrophoresis. Collect those correct ones and send them for sequencing.
-See the point mutation result in the sequence.
-Identify the T7+rbs+merT+rbs+merP+rbs+merC by electrophoresis.
-===7.30===
-Digest rbs+agn43 with EcoRl and Xbal. Digest phiR73+Po promoter with EcoRl and Spel.
-Retrieve the digested T7+rbs+merT+rbs+merP+rbs+merC gel.
-Do the transformation.
-===7.31===
-Digest 2-2E constitutive promoter with EcoRl and Spel. Digest rbs+agn43 with EcoRl and Xbal.
-Connect 2-2E promoter with rbs+agn43.
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 ===8.1===
-Prepare the plasmid DNA of T7+rbs+merT+rbs+merP+rbs+merC and PhiR73+Po promoter+rbs+antigen 43.
-Prepare the plasmid DNA of 2-2E promoter+rbs+antigen 43.
+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
-Identify PhiR73+Po promoter+rbs+antigen 43 and 2-2E promoter+rbs+antigen 43 using electrophoresis.
 ===8.2===
-Send the correct ones for sequencing.
-Digest rbs+antigen 43 again.
+Lpp-OmpA-MBP construction plan
-===8.4===
-Pick the clone of PhiR73+Po promoter+rbs+antigen 43 on the plate for autoaggregation assay.
-Plan the assay.
+Ⅰ.Use Pfu Mix PCR Nde1+Lpp-OmpA(437bp)+Sal1 & E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1
-Design new primers for antigen 43 which contains different promoters in the forward primer.
+Ⅱ.Digest the PCR products by Ndel,Sall&EcoR,Sall ,respectively
-===8.5===
-Preliminary autoaggregation assay.
-Send PhiR73+Po promoter+rbs+antigen 43 for sequencing.
+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.
-Transform PhiR73+Po promoter+rbs+antigen 43 into BL21 strains.
+===8.3===
-===8.6===
-Pick the single clone.
-Induce the antigen 43 gene’s expression by adding 10^-5 M IPTG for 4 hours.
+MBP construction plan
-Do the auto-aggregation assay( for detail, see the inductive aggregation page or antigen 43 part.)
+Ⅰ.Pfu PCR Sall+N-MBP+Bspel
-PCR the rbs+agn43 using easyPFU.
+Bspel+C-MBP+SNP
-Identify it using electrophoresis.
+Bspel+C-MBP+Xhol
-Digest the PCR product using EcoRl and Spel to put it into PSB1A2.
+Ⅱ.Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel, if not ,back to Ⅰ
-Do the ligation and transformation.
+===8.4===
-Send some of the PCR product for sequencing.
+Lpp-OmpA,N-MBP,+C-MBP with backbone PET21a and PSBIK3
-===8.7===
-Digest merT+merC with Spel and Pstl, digest merP with Xbal and Pstl.
-Retrieve the gel. Do the ligation.
+Ⅰ.Ligation,Nde1+Lpp-OmpA(437bp)+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+Xhol & PET21a
-Pick three clones of rbs+merP.
+Ⅱ.Ligation, E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+SNP & PSBIK3
-===8.8===
-Prepare the plasmid DNA for rbs+merP and merT+merC.
-Pick single clones of merT+merP+merC. Do the colony PCR.
+Ⅲ.Learn to do the western blotting. Write the protocols.
-Retrieve the rbs+agn43.
+===8.5===
-Do the ligation and transformation.
+Transformation the products of 4 fragments above.
-===8.9===
-PCR antigen 43.
-Nest PCR using nest primers. Do the first step.identify it using electrophoresis.
+===8.6===
-===8.10===
-PCR antigen 43 using nest primers. Using different template.
-Using better template to run PCR.
+There is something wrong with the primer ,so it doesn't work, back to the work from 8.2
-Do the transformation of antigen 43.
+===8.7===
-===8.11===
-Retrieve the product of rbs+agn43. Digest it and do the ligation and transformation.
-===8.12===
-Pick the single clone of rbs+agn43.
-Digest 1-23L with EcoRl and Xbal. Digest T7+merT+merP+merC with EcoRl and Spel.
+Ⅰ.Use Pfu Mix PCR Nde1+Lpp-OmpA(437bp)+Sal1 & E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1
-Digest pmerT+GFP with Spel and Pstl, digest TPC with Xbal and Pstl.
+Ⅱ.Digest the PCR products by Ndel,Sall&EcoR,Sall ,respectively
-Identify them using agarose gel electrophoresis.
+===8.8===
-Do the ligation and transformation.
+Ⅰ.Pfu PCR Sall+N-MBP+Bspel
-===8.13===
-Identify rbs+agn using electrophoresis.
-Digest 1-23L with EcoRl and Xbal. Digest T7+merT+merP+merC with EcoRl and Spel.
+Bspel+C-MBP+SNP
-Do the ligation and transformation.
+Bspel+C-MBP+Xhol
-===8.14===
-Prepare the plasmid DNA.
-Send the correct ones for sequencing.
+Ⅱ.Gel to identification
-===8.15===
-See the sequencing result.
-===8.16===
-Digest PhiR73+Po promoter with EcoRl and Spel. Digest RBS with EcoRl and Xbal.
-Retract the whole genome of K12 strain.
+===8.9===
-PCR antigen 43 using new template.
+Ⅰ.Ligation,Nde1+Lpp-OmpA(437bp)+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+Xhol & PET21a
-Pick single clones of antigen 43.
+Ⅱ.Ligation, E(EcoR1)NX(Xba1)+Lpp-OmpA+Sal1 with Sall+N-MBP+Bspel & Bspel+C-MBP+SNP & PSBIK3
-===8.17===
-T7+TCP+Terminator done.
-Transform it to BL21 strain.
+===8.10===
-Send antigen 43 for sequencing.
+Transform the ligation product into Trans5α strain.
-PCR antigen 43 using new template and better protocol.
+===8.11===
-Do the colony PCR of PhiR73+Po promoter+rbs.
+Help a teammate transform P(RBS+T3pol 1)&PmerR-PSB1C3 Plasmid to OmniMAX2-T1 Competent cell
-Prepare the plasmid DNA and digest it for identification.
+Digestion:
-Do the ligation and transformation.
+Ⅰ.RBS+T3pol 1 with XbaI and pstI(insert)
-Digest pmerT+GFP with Spel and Pstl, digest TPC with Xbal and Pstl.
+Ⅱ.PmerR-PSB1C3 with SpeI and PstI (vector)
-Digest pPbrA with Spel and Pstl. Digest rbs+T3 pol with Xbal and Pstl.
+Retrieve the gel
-===8.18===
-Retrieve the digested product .
-Identify them using electrophoresis.
+Ligation the parts above.
-Pick the single clone of PCR of PhiR73+Po promoter+rbs, do the colony PCR.
+===8.12===
-Prepare the plasmid DNA.
+Digestion MBP construct parts
-Send the correct ones for sequencing.
+Ligation 4 fragments
-Connect pPbra with rbs+T3 pol. Connect PhiR73+Po promoter+rbs with antigen 43.
+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
-Connect merp+GFP with TCP.
+===8.13===
-Do the ligation and transformation.
+Digestion: PET21a 20 µl with NdeI and XhoI
-===8.19===
-Identify them using electrophoresis.
-Retrieve the gel.
+Ligation again
-Do the ligation and transformation.
+Ⅰ.RBS+T3pol 1 with XbaI and pstI(insert)
-PCR antigen 43. Digest it using EcoRl and Spel.
+Ⅱ.PmerR-PSB1C3 with SpeI and PstI (vector)
-===8.20===
-Pick the colony of PhiR73+Po promoter+rbs+antigen 43.
-Put rbs+antigen 43 into PSB1A2 plasmid. Do the ligation and transformation.
+Insert:vector=1:7&2:6
-Digest merp+GFP and T3 pol.
+Transform the ligation product
-===8.21===
-Identify the colony PCR product using electrophoresis.
-Do the transformation.
+Finally got clone ,2:6 better than 1:7
-Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter using EcoRl and Spel.
+Ⅲ.transformation 4 pieces fragments
-===8.22===
-Do the autoaggregation assay of antigen 43.
-Induce its expression by adding IPTG.
+===8.14===
-===8.23===
-Do the autoaggregation assay.
-===8.25===
-Prepare the plasmid DNA for TPC+Terminator.
-Send the correct ones for sequencing.
+Ⅰ.Plate PCR for identification 4 pieces fragments ligation products
-Construct pPbra using primers annealing.
+Ⅱ.Miniprep
-===8.26===
-Transform antigen 43.
-===8.27===
-Pick single clone of antigen 43.
-Do the transformation of TPC+terminator.
+a-(1~3) 1K3(1~3) 26-(1~2) 17-(3~4)
+Ⅲ.Sent the plasmids for sequencing
+===8.15===
-Do the transformation of pPbrA.
+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
-Connect pBAD with rbs. Do the ligation and transformation.
+Ⅱ.transformation
-===8.30===
-Do the functional test of antigen 43.
-Pick the clone of antigen 43.
+K3(1~3) 26-(1~2) 17-(3~4)
-Pick the clone of merP+GFP+TCP.
+Ⅲ.Pick 20 single clones on 21a plate
-Do the ligation of pPbra+T3 pol again.
+Ⅳ.PCR for identification
-Pick the single clone of Pbad+rbs.
+Got the right size from agarose gel electrophoresis ( Lpp-OmpA+MBP=735bp)
-Do the transformation.
+Ⅴ.Digestion with NdeI and XhoI,then use PCR purification kit to retrieve products.
-===8.31===
-Send the correct merp+GFP and Pbad+rbs for sequencing.
-Do the transformation.
+Ⅵ.Ligation 1(PET-21a):7
-Induce the expression of antigen 43.
+Ⅶ.Transformation
 ==September==
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-[[https://2010.igem.org/Team:Peking/Notebook/YWChen TOP]]
+[<html><a href="#top">TOP</a></html>]
-===9.1===
+===9.5===
-Connect the antigen 43 into plasmid.
+Assembly:
-Send merP+GFP+TCP for sequencing.
+T7p+RBS(B0034)+DsbA+MBP+TER(B0015)+T7p+RBS(B0034)+MBP+TER(B0015)+
-Connect pPbra+T3 pol using primers annealing.
+T7p+RBS(B0034)+Lpp_OmpA+MBP+TER(B0015)
-Do the ligation and transformation.
+Ⅰ.Digestion:RBS with SpeI and PstI
-===9.2===
-Prepare the plasmid DNA for rbs+agn43.
-See the sequencing result of merp+GFP+TCP.
+-23L terminator with EcoRI and XbaI
-Pick the clone of pPbra+T3 pol.
+Ⅱ.Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel
-===9.3===
-Send the rbs+agn43 for sequencing.
-Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter with EcoRl and Spel.
+===9.9===
-Pick the clone of pPbra+T3 pol. Prepare the plasmid DNA.
+Ligation: RBS+MBP
-===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.
+DsbA+MBP
-Connect pPbra+T3 pol with 1-23L. pick the single clone. Prepare the plasmid DNA.
+FORGET DIGESTION!!!!
-Do the ligation and transformation.
+===9.10===
-Pick the clone of Pbad+T3 pol.
+Digestion:MBD with XbaI and PstI
-Make competent cell containing pc+merR+merp+rbs+T3pol. Transform T3 promoter into it.
+DsbA-MBP with EcoRI and SpeI
-Induce the expression using different concentration of IPTG.
+Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel
-Re-suspend the cell. Measure the GFP intensity by a microplate reader.
+===9.11===
-===9.7===
-Digest merP+GFP+TCP with EcoRl and Pstl to put it into PSB3K3.
-Send pbra+T3 pol+terminator for sequencing.
+Transformation to BL21 for western blotting
-Identify the pBAD+T3 pol using electrophoresis.
+Omni for preserve
-Make competent cells of pc+merR+merp+rbs+T3pol.
+===9.13===
-Transform T3 promoter into it.
+Digestion :with XbaI and PstI (for ligation)
-===9.8===
-Re-suspend the cell.
-Measure the GFP intensity using microplate reader.
+Miniprep T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)&RBS+MBP（1~3）
-PCR antigen 43 using hifi Taq DNA polymerase.
+===9.15===
-===9.9===
-PCR Rbs+agn 43 using agn for/rev primer to identify it.
-Digest the correct ones using EcoRl and Spel.
+Ⅰ.Sent the plasmids for sequencing【T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)&RBS+MBP（1~3）】
-Send the correct ones for sequencing.
+T7p+RBS(B0034)+DsbA+MBP+Ter 1 got right sequence
-Transform T3 promoter into cells containing pc+merR+merp+rbs+T3pol.
+Ⅱ.Digestion:
-Send pBAD+T3 pol for sequencing.
+T7p+RBS(B0034)+DsbA+MBP+Ter with EcoRI and SpeI
-pPbra+T3pol+terminator done.
+Identify them using agarose gel electrophoresis.Then retrieve the gel(got the right size)
-Measure the GFP intensity using a microplate reader.
+Ⅲ.Digestion:
-===9.12===
-Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter with EcoRl and Spel.
-Identify them using electrophoresis.
+RBS+MBP with XbaI and PstI
-Retrieve the gel.
+After Identify them by using agarose gel electrophoresis,the results turns wrong.
-Do the ligation and transformation.
+Digestion AGAIN!:
-Pick the clone.
+RBS+MBP with XbaI and PstI
-===9.13===.
-Digest Antigen 43 with EcoRl and Xbal. Digest PhiR73+Po promoter+rbs with EcoRl and Spel.
-Identify them using electrophoresis.
+Meanwhile: Pick 10 single clone for plate PCR by using tagmix
-===9.14===
-PCR antigen 43.
-Get the candidate of rbs+agn43.
+Digest the PCR products with XbaI and PstI ,10 clones all got the right size,so minipret them.
-Send the correct ones for sequencing.
-===9.15===
-Put antigen 43 into PSB1A2.
-Do the ligation and transformation.
 ===9.16===
-Digest PSB1A2 and PSB1A3.
+Ligation :RBS-MBP(XP)+Ter(SP)
+Transformation it.
+Culture the plates.
 ===9.17===
-Digest PSB1C3 with EcoRI and Spel.
-Digest antigen 43 with EcoRl and Spel.
+Pick the single clone to PCR, but failed
+Meanwhile: Digest the RBS-MBP1 for backup
+===9.18===
+Digest the RBS-MBP(2&3)
+Ligation: RBS-MBP(2&3) +T7p
+Transformation the Ligation products
+===9.19===
+Use RBS-MBP(2&3) as templete,PCR(Fast pfu),got a mass of RBS-MBP
+Identify them using agarose gel electrophoresis.Then go to retrieve the gel
+===9.20===
+Transformation:RBS-MBP-T7p
+Culture the plates
-Do the ligation and transformation.
 ===9.21===
-Retract the whole genome of K12 strain.
+Pick 3 single clone T7p-RBS-MBP(1~3)
+Some for PCR some for Miniprep
 ===9.22===
-Nest PCR of antigen 43 using new template and new primers.
-Put antigen 43 into PSB1C3.
+Miniprep T7p-RBS-MBP(1~3)
+Digestion:T7p-RBS-MBP(1~3) with EcoRI and SpeI
-Pick the single clone of antigen 43.
 ===9.23===
-Prepare the plasmid DNA for antigen43.
-Digest the plasmid DNA using EcoRl and Spel.
+Wiki writing
-Identify them using electrophoresis.
+Retrieve the digestion products T7p-RBS-MBP(1~3)ES
-Make the competent cell contains PBAD+ T3 pol. Transform T3 promoter+GFP into it.
+Ligation:
-Pick the clone of PMERT+T3 pol.
+T7p-RBS-MBP(1~3)+Ter
+Transformation:T7p-RBS-MBP-Ter(1~3)
+Culture the plates
-Digest PSB3C5 using EcoRl and Pstl.
 ===9.24===
-PSB3K5: EcoRl and Pstl.
-T7+TPC+Ter: Xbal and Pstl.
+Pick 3 single clone from each plate(except1)
-MerP+GFP:EcoRl and Spel.
+Miniprep T7p-RBS-MBP-Ter(2-1~3&3-1~3)
+µI for sequencing
+µI for digestion
+Left 20µI
-Do the ligation and transformation.
 ===9.25===
-Retrieve the digested merp+GFP.
-Induce the expression of PBAD+T3 pol using 10^-5 M arabinose.
+Digestion:T7p-RBS-MBP-Ter with SpeI and PstI
-Do the antigen 43 PCR using touchdown PCR.
+Gel for identification ,the size of T7p-RBS-MBP-Ter(2-1~3) are right
-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.
+Digestion:PSB1C3 with EcoRI and PstI
-===9.27===
-Attend the seminar.
-===9.28===
-Connect T7+PhiR73+Po promoter+rbs+antigen 43.
-Do the ligation and transformation.
+Ligation:PSB1C3 + T7p-RBS-MBP-Ter+T7p+RBS+Lpp-OmpA+MBP+Ter
-PCR the T3 promoter+PhiR73+Po promoter.
+Gel retrieving
-===9.29===
-Retrieve the PCR product.
-Digest the product using EcoRl and Spel.
+===9.27===
-Digest TCP with Xbal and Pstl. Identify it using electrophoresis.
+Transformation:Positive cloning
-Digest merp+GFP using Spel and Pstl.
+T7p+RBS+Lpp-OmpA+MBP+Ter&T7p+RBS(B0034)+DsbA+MBP+Ter
-Do the transformation.
+Culture the plates
-===9.30===
-Do the ligation and transformation.
+===9.28===
-Prepare the plasmid DNA and identify it using PCR.
+The sequence of T7p-RBS-MBP-Ter(2-1~3&3-1~3) came out ,but without T7p inside.
 ==October==
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-[[https://2010.igem.org/Team:Peking/Notebook/YWChen TOP]]
+[<html><a href="#top">TOP</a></html>]
-===10.1===
+===10.3===
-Digest pTET+T7 pol using EcoRl and Spel.
-Retrieve the gel.
+There is something wrong with PSBIC3(RFP inside )
-Connect T3 promoter+PhiR73+Po promoter+ antigen 43.
+Ligation Again!:
-Do the ligation and transformation.
+PSB1C3 + T7p-RBS-MBP-Ter+T7p+RBS+Lpp-OmpA+MBP+Ter
-===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.
+Pick 3 single clone from the plate
+Miniprep them.
 ===10.5===
-Do the auto-aggregation assay.
+Transformation:Positive cloning:T7p+RBS(B0034)+DsbA+MBP+Ter
+Digestion:RBS-MBP-Ter+T7p with XbaI and PstI
+Identify them using agarose gel electrophoresis.Then go to retrieve the gel
+===10.6===
+Pick 3 single clone from the plate（T7p+RBS(B0034)+DsbA+MBP+Ter）
+Digestion:PSB1C3 with XbaI and PstI （Change backbone）
+Identify them using agarose gel electrophoresis.Then go to retrieve the gel
 ===10.7===
-Digest merp+GFP using Spel and Pstl.
-Digest TCP using Xbal and Pstl.
+Miniprep T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)
+Digestion：RBS-MBP-Ter with XbaI and PstI
+Ligation:RBS-MBP-Ter+T7p(sp) then transformation
-Do the ligation and transformation.
 ===10.8===
-Identify the digested product using electrophoresis.
-Do the ligation and transformation.
+Ligation again
+T7p+RBS-MBP-Ter-+T7p+RBS+Lpp-OmpA+MBP+Ter
+Transformation it
 ===10.9===
-Prepare the plasmid DNA.
-Identify them using PCR.
+Transformation T7p+RBS+Lpp-OmpA+MBP+Ter(xp)-PSB1C3(xp)
+PCR(tag mix) for identification T7p+RBS(B0034)+DsbA+MBP+Ter
+Culture the plates, but it did not grow up
-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.
+Ligation:T7p+RBS+Lpp-OmpA+MBP+Ter(EP)+PSB1C3
+T7p+RBS+DsbA+MBP+Ter(EP)+PSB1C3
+Transformation
+Easy Mix PCR.
+Culture the plates
-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.
+ⅠDigestion:T7p+RBS+Lpp-OmpA+MBP+Ter(EP)
-===10.16-10.21===
-Connect merp+GFP with TCP.
+T7p+RBS+DsbA+MBP+Ter(EP)
+Identify them using agarose gel electrophoresis.Then go to retrieve the gel
+===10.16===
+Ⅰ.Ligation: T7p-RBS-MBP-Ter
+Ⅱ.Transformation :T7p-RBS-MBP-Ter
+Ⅲ.Culture the plates
+Pick 3 single clones
+===10.17===
+T7p-RBS-MBP-Ter for sequencing
+===10.20===
+Ⅰ.Liagation:T7p+RBS+DsbA+MBP+TER+T7p+RBS+MBP+TER+T7p+RBS+Lpp-OmpA+MBP+TER+PSBIC3
+Ⅱ.Transformation
+Ⅲ.Culture the plates
-Connect pc+merR with terminator.
+Pick 6 Single clones
-Transform these two plasmid into one single strain.
+Ⅳ.T7p+RBS+DsbA+MBP+TER+T7p+RBS+MBP+TER+T7p+RBS+Lpp-OmpA+MBP+TER+PSBIC3 for sequencing
-Antigen 43 auto-aggregation assay.  Analyse the result.
-===10.21-10.25===
-Upload and edit parts of our group.
-[[https://2010.igem.org/Team:Peking/Notebook/YWChen TOP]]
+[<html><a href="#top">TOP</a></html>]
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Team:Peking/Notebook/YWChen

From 2010.igem.org

Latest revision as of 10:29, 27 October 2010

Contents

July

7.26

August

8.1

8.2

8.3

8.4

8.5

8.6

8.7

8.8

8.9

8.10

8.11

8.12

8.13

8.14

8.15

September

9.5

9.9

9.10

9.11

9.13

9.15

9.16

9.17

9.18

9.19

9.20

9.21

9.22

9.23

9.24

9.25

9.26

9.27

9.28

October

10.3

10.4

10.5

10.6

10.7

10.8

10.9

10.10

10.11

10.16

10.17

10.20

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