Team:Peking/Notebook/ZRLiu

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Contents

• July, 2010

• August, 2010

• September, 2010

• October, 2010

July

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7.5

Purification of digested PCR product: merP, merT, and merC

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert (merP / merT / merC digested with EcoRI and PstI): 7μL

Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL

Transformation: Trans5α

7.6

No recognizable colonies grew on the agar plates (T_T)

Digestion (20μL):

pSB1A2: 5μL

EcoRI: 1μL

PstI: 1μL

Buffer (EcoRI Buffer): 2μL

ddH2O: 12μL

Electrophoresis

Gel Extraction

PCR to get merP, merT and merC fragments by Phusion (20μL)

5*phusionHF buffer: 4μL

2.5mM dNTPs: 1.6μL

Polymerase: 0.2μL

Primer_For:1μL

Primer_Rev: 1μL

Template: 0.5μL

ddH2O: 11.7μL

7.7

Electrophoresis

Gel Extraction

merT: 351bp

merP: 256bp

merC: 423bp

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert (merP / merT / merC digested with EcoRI and PstI): 7μL

Vector (pSB1A2 backbone digested with EcoRI and PstI): 1μL

Transformation: Trans5α

7.8

Pick up 3 colonies from each agar plate

Grow the culture overnight

[7.9-7.19 Field Practices @ Yantai & Beijing (^_<)] [7.20-7.21 Home @ Nanjing (#_#)] [7.22-7.24 World Exhibit @ Shanghai (*~*)Orz] [7.25 Home @ Nanjing (#_#)]

7.26

Design the PbrR Metal Binding Peptide (MBP) Construction

PCR from pbrR to get the N terminal and C terminal of MBP by PFUEasyMix (20μL)

EasyMix: 10μL

ddH2O: 9μL

Primer_For_N: 0.5μL

Primer_Rev_N:0.5μL

Template (pbrR): 0.5μL

=>Get the metal binding domain of pbrR as the N terminal for MBP_STD

EasyMix: 10μL

ddH2O: 9μL

Primer_For_C: 0.5μL

Primer_Rev_C:0.5μL

Template (pbrR): 0.5μL

=>Get the metal binding domain of pbrR as the C terminal for MBP_STD

Linker are designed into Primer_Rev_N and Primer_For_C

7.27

Electrophoresis

Gel Extraction

N C

Digestion:

N terminal: EcoRI+BspEI+NEBuffer3

C terminal: BspEI+PstI+NEBuffer3

Purification of digestion product

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert1 (N terminal digested with EcoRI and BspEI): 3μL

Insert2 (C terminal digested with BspEI and PstI): 3μL

Vector (pSB1K3 backbone digested with EcoRI and PstI): 2μL

7.28

Transformation: OmniMAX

Pick up 3 colonies from the agar plate

Grow the culture overnight

7.29

Mini-Prep

Verification

DNA Sequencing

To get the plasmid: pbrR_MBP_STD (pSB1K3)

PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU (50μL)

10*EasyPFU buffer: 5μL

2.5mM dNTPs: 5μL

Polymerase: 1μL

Primer_For_N’:1μL

Primer_Rev_N’: 1μL

Template (pbrR): 0.2μL

ddH2O: 36.8μL

=>Get the metal binding domain of pbrR as the N terminal for MBP_COM

10*EasyPFU buffer: 5μL

2.5mM dNTPs: 5μL

Polymerase: 1μL

Primer_For_C’:1μL

Primer_Rev_C’: 1μL

Template (pbrR): 0.2μL

ddH2O: 36.8μL

=>Get the metal binding domain of pbrR as the C terminal for MBP_COM

Linker are designed into Primer_Rev_N’ and Primer_For_C’

Electrophoresis

Gel Extraction

Digestion:

N terminal: NdeI+BspEI+NEBuffer3

C terminal: BspEI+XhoI+NEBuffer3

7.30

Purification of digested product

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert1 (N terminal digested with NdeI and BspEI): 3μL

Insert2 (C terminal digested with BspEI and XhoI): 3μL

Vector (pET21a backbone digested with NdeI and XhoI): 2μL

Transformation: OmniMAX

7.31

Pick up 3 colonies from the agar plate

Grow the culture for 12hrs

Mini-Prep

Verification

DNA Sequencing

To get the plasmid: pbrR_MBP_COM (pET21a)

August

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8.3

Design the DsbA-MBP Construction

PCR from pbrR to get the N terminal and C terminal of MBP by EasyPFU SuperMix (50μL)

2*EasyPFU SuperMix: 25μL

ddH2O: 20.8μL

Primer_For_N: 2μL

Primer_Rev_N:2μL

Template (pbrR): 0.2μL

=>Get the metal binding domain of pbrR as the N terminal for DsbA-MBP

2*EasyPFU SuperMix: 25μL

ddH2O: 20.8μL

Primer_For_C: 2μL

Primer_Rev_C:2μL

Template (pbrR): 0.2μL

=>Get the metal binding domain of pbrR as the C terminal for DsbA-MBP

Linker are designed into Primer_Rev_N and Primer_For_C

Electrophoresis to verify

Purification of PCR product

Digestion:

N terminal: XbaI+BspEI+NEBuffer3

C terminal: BspEI+XhoI+NEBuffer3

8.4

Purification of digested product

Digestion (20μL):

pET39b: 5μL

SpeI: 1μL

XhoI: 1μL

Buffer (NEBuffer4): 2μL

ddH2O: 12μL

Electrophoresis

Gel Extraction

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert1 (N terminal digested with XbaI and BspEI): 3μL

Insert2 (C terminal digested with BspEI and XhoI): 3μL

Vector (pET39b backbone digested with SpeI and XhoI): 2μL

Transformation: OmniMAX

8.5

Mini-Prep

Verification: Digestion (20μL):

Plasmid: 5μL

NdeI: 1μL

XhoI: 1μL

Buffer (NEBuffer4): 2μL

ddH2O: 12μL

Electrophoresis to verify

NdeI(1030) XhoI(174) MBP(abt 500bp)

Correct band=1030-174+500=1356

DNA Sequencing

To get the plasmid: DsbA-MBP (pET39b)

8.10

Design and the T7-RBS-DsbA-MBP Construction

1st Round of Nest PCR by EasyPFU SuperMix (50μL)

2*EasyPFU SuperMix: 25μL

ddH2O: 20.8μL

Primer_For (T7-RBS-DsbA-F1): 2μL

Primer_Rev (PbrR-MBP-C’-R):2μL

Template (DsbA-MBP): 0.2μL

=>RBS is prefixed to DsbA-MBP

Electrophoresis

FAILED!!!(TAT)

REPEAT-PCR with Gradient

Gradient: T=60℃ G=5

Electrophoresis

Gel extraction

8.11

2nd Round of Nest PCR by EasyPFU SuperMix (50μL)

2*EasyPFU SuperMix: 25μL

ddH2O: 20.8μL

Primer_For (T7-RBS-DsbA-F2): 2μL

Primer_Rev (PbrR-MBP-C’-R):2μL

Template (1st Round of Nest PCR product (RBS-DsbA-MBP)): 0.2μL

=>T7 is prefixed to 1st Round of Nest PCR product (RBS-DsbA-MBP)

Electrophoresis

Gel extraction

8.12

Digestion (20μL):

2nd Round of Nest PCR product (T7-RBS-DsbA-MBP): 5μL

EcoRI: 1μL

SpeI: 1μL

Buffer (EcoRI Buffer): 2μL

ddH2O: 12μL

Purification of digested product

Digestion (20μL):

pSB1C3: 5μL

EcoRI: 1μL

SpeI: 1μL

Buffer (EcoRI Buffer): 2μL

ddH2O: 12μL

Electrophoresis

Gel Extraction

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert (T7-RBS-DsbA-MBP digested with EcoRI and SpeI): 6μL

Vector (pSB1K3 backbone digested with EcoRI and SpeI): 2μL

Transformation: OmniMAX

8.13

Pick up 5 colonies from each agar plate

Grow the culture overnight

8.14

Mini-Prep

Verification: Digestion (20μL):

Plasmid: 5μL

PstI: 1μL

Buffer (NEBuffer4): 2μL

ddH2O: 12μL

Electrophoresis to verify

There should be two bands; one is about 3000bp and the other is 600bp

Verification: PCR by EasyTaq SuperMix

Template: 0.2μL

Primer_Univ_For: 1μL

Primer_Univ_Rev: 1μL

2*EasyTaq SuperMix: 5μL

ddH2O:3μL

Electrophoresis to verify

There should be one band, which is about 1200bp

DNA Sequencing

To get the plasmid: DsbA-MBP (pET39b)

8.16

FAILED (T~T)

Why?

Because the reverse primer cannot specifically distinguish the C terminal from the N terminal of the MBP, new primer including the sequence of His-tag is designed in order to recognize the C terminal only

Hand over this work to Donghai Liang

[8.17-8.31] Physical Training

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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