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.

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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]

9.1

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

[TOP]

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

Upload and edit parts of our group. [TOP]

==go to her page==

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 [[https://2010.igem.org/Team:Peking/Notebook/YWChen TOP]]
 ===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==

Mon	Tue	Wed	Thu	Fri	Sat	Sun
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30	31	-	-	-	-

Mon	Tue	Wed	Thu	Fri	Sat	Sun
-	-	1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	-	-	-

Mon	Tue	Wed	Thu	Fri	Sat	Sun
-	-	-	-	1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	-	-	-	-	-	-

Mon	Tue	Wed	Thu	Fri	Sat	Sun
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30	31	-	-	-	-

Mon	Tue	Wed	Thu	Fri	Sat	Sun
-	-	1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	-	-	-

Mon	Tue	Wed	Thu	Fri	Sat	Sun
-	-	-	-	1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	-	-	-	-	-	-

Team:Peking/Notebook/YWChen

From 2010.igem.org

Revision as of 18:09, 25 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

9.1

9.2

9.3

9.6

9.7

9.8

9.9

9.12

9.14

9.15

9.16

9.17

9.21

9.22

9.23

9.24

9.25

9.26

9.27

9.28

9.29

9.30

October

10.1

10.3

10.4

10.5

10.7

10.8

10.9

10.10

10.11

10.12

10.13

10.15

10.16-10.21

10.21-10.25

Mon	Tue	Wed	Thu	Fri	Sat	Sun
1	2	3	4	5	6	7
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30	31	-	-	-	-

Mon	Tue	Wed	Thu	Fri	Sat	Sun
-	-	1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	-	-	-

Mon	Tue	Wed	Thu	Fri	Sat	Sun
-	-	-	-	1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	-	-	-	-	-	-