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]

10.1

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]]
-===9.1===
+===9.5===
-Connect the antigen 43 into plasmid.
-Send merP+GFP+TCP for sequencing.
+Assembly:T7p+RBS(B0034)+DsbA+MBP+TER(B0015)+T7p+RBS(B0034)+MBP+TER(B0015)+T7p+RBS(B0034)+Lpp-OmpA+MBP+TER(B0015)
-Connect pPbra+T3 pol using primers annealing.
+Ⅰ.Digestion:RBS with SpeI and PstI
-Do the ligation and transformation.
+-23L terminator with EcoRI and XbaI
-===9.2===
-Prepare the plasmid DNA for rbs+agn43.
-See the sequencing result of merp+GFP+TCP.
+Ⅱ.Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel
-Pick the clone of pPbra+T3 pol.
+===9.9===
-===9.3===
-Send the rbs+agn43 for sequencing.
-Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter with EcoRl and Spel.
+Ligation: RBS+MBP
-Pick the clone of pPbra+T3 pol. Prepare the plasmid DNA.
+DsbA+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.
+FORGET DIGESTION!!!!
-Connect pPbra+T3 pol with 1-23L. pick the single clone. Prepare the plasmid DNA.
+===9.10===
-Do the ligation and transformation.
+Digestion:MBD with XbaI and PstI
-Pick the clone of Pbad+T3 pol.
+DsbA-MBP with EcoRI and SpeI
-Make competent cell containing pc+merR+merp+rbs+T3pol. Transform T3 promoter into it.
+Identify them using agarose gel electrophoresis.if right ,go to retrieve the gel
-Induce the expression using different concentration of IPTG.
+===9.11===
-Re-suspend the cell. Measure the GFP intensity by a microplate reader.
+Transformation to BL21 for western blotting
-===9.7===
-Digest merP+GFP+TCP with EcoRl and Pstl to put it into PSB3K3.
-Send pbra+T3 pol+terminator for sequencing.
+Omni for preserve
-Identify the pBAD+T3 pol using electrophoresis.
+===9.13===
-Make competent cells of pc+merR+merp+rbs+T3pol.
+Digestion :with XbaI and PstI (for ligation)
-Transform T3 promoter into it.
+Miniprep T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)&RBS+MBP（1~3）
-===9.8===
-Re-suspend the cell.
-Measure the GFP intensity using microplate reader.
+===9.15===
-PCR antigen 43 using hifi Taq DNA polymerase.
+Ⅰ.Sent the plasmids for sequencing【T7p+RBS(B0034)+DsbA+MBP+Ter(1~3)&RBS+MBP（1~3）】
-===9.9===
-PCR Rbs+agn 43 using agn for/rev primer to identify it.
-Digest the correct ones using EcoRl and Spel.
+T7p+RBS(B0034)+DsbA+MBP+Ter 1 got right sequence
-Send the correct ones for sequencing.
+Ⅱ.Digestion:
-Transform T3 promoter into cells containing pc+merR+merp+rbs+T3pol.
+T7p+RBS(B0034)+DsbA+MBP+Ter with EcoRI and SpeI
-Send pBAD+T3 pol for sequencing.
+Identify them using agarose gel electrophoresis.Then retrieve the gel(got the right size)
-pPbra+T3pol+terminator done.
+Ⅲ.Digestion:
-Measure the GFP intensity using a microplate reader.
+RBS+MBP with XbaI and PstI
-===9.12===
-Digest rbs+agn43 with EcoRl and Xbal. Digest PhiR73+Po promoter with EcoRl and Spel.
-Identify them using electrophoresis.
+After Identify them by using agarose gel electrophoresis,the results turns wrong.
-Retrieve the gel.
+Digestion AGAIN!:
-Do the ligation and transformation.
+RBS+MBP with XbaI and PstI
-Pick the clone.
+Meanwhile: Pick 10 single clone for plate PCR by using tagmix
-===9.13===.
-Digest Antigen 43 with EcoRl and Xbal. Digest PhiR73+Po promoter+rbs with EcoRl and Spel.
-Identify them using electrophoresis.
+Digest the PCR products with XbaI and PstI ,10 clones all got the right size,so minipret them.
-===9.14===
-PCR antigen 43.
-Get the candidate of rbs+agn43.
+===9.16===
-Send the correct ones for sequencing.
+Ligation :RBS-MBP(XP)+Ter(SP)
-===9.15===
-Put antigen 43 into PSB1A2.
+Transformation it.
+Culture the plates.
-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.
+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==

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 18:16, 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

9.5

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

10.1

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	-	-	-	-	-	-