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

Digestion (20μL):

pSB1C3: 5μL

EcoRI: 1μL

PstI: 1μL

Buffer (EcoRI Buffer): 2μL

ddH2O: 12μL

Electrophoresis

Gel Extraction

Digestion (20μL):

pbrR_MBP_STD (pSB1A2): 5μL

EcoRI: 1μL

PstI: 1μL

Buffer (EcoRI Buffer): 2μL

ddH2O: 12μL

Electrophoresis

Gel Extraction

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert (PbrR_MBP_STD digested with EcoRI and SpeI): 6μL

Vector (pSB1C3 backbone digested with EcoRI and SpeI): 2μL

Transformation: OmniMAX

9.2

Mini-Prep

Verification: Digestion (20μL):

Plasmid: 5μL

EcoRI: 1μL

PstI: 1μL

Buffer (EcoRI Buffer): 2μL

ddH2O: 12μL

Electrophoresis to verify

There should be one band, which is about 500bp

DNA Sequencing

To get the plasmid: pbrR-MBP (pSB1C3)

9.3

Construct the concentration of Hg(II) ions for induction

   Final Concentration/M Volume/μL Original Concentration/M

1. 5*10-5 25 10-3

2. 3*10-5 15 10-3

3. 10-5 5 10-3

4. 8*10-6 4 10-3

5. 5*10-6 2.5 10-3

6. 3*10-6 1.5 10-3

7. 10-6 0.5 10-3

8. 8*10-7 4 10-4

9. 5*10-7 2.5 10-4

10. 3*10-7 1.5 10-4

11. 10-7 0.5 10-4

12. 8*10-8 4 10-5

13. 5*10-8 2.5 10-5

14. 3*10-8 1.5 10-5

15. 10-8 0.5 10-5

16. 8*10-9 0.4 10-5

17. 5*10-9 25 10-7

18. 3*10-9 15 10-7

19. 10-9 5 10-7

20. 0 0 0

9.5

Learn the protocol for preparation of competent cells for transformation for bi-transformation

Pick up one colony from each agar plate: J23109 and J23112

Grow the culture overnight

9.6

Re-activate the culture of J23109 and J23112 in fresh LB with ampicilin and kanamyclin

Measure the value of OD600

Induced with Hg(II) ions of different concentration for 2hrs

Centrifugation at 5000r for 5min

Resuspension with PBS

Measure the value of OD600 and GFP with ELISA

9.7

Learn about Western Blot with Boxuan Zhao

9.15

Design the traffic light assay

LacZ full length (RBS: B0034) BBa_I1732017 2_3K 3093bp pSB1A2

LacZ α fragment BBa_I732006 1_23H 234bp pSB1AK3

Plasmid1: 1_18I MerR (pSB3K3)

Plasmid2: PmerT - LacZ full length / LacZ α fragment (pSB1C3)

X-GAL assay

Positive Transformation of 2_3K and 1_23H: TansMAX

9.17

Pick up 2 colonies from each agar plate: 2_3K and 1_23H

Grow the culture for 12hrs

Mini-Prep

Get the plasmids: 2_3K (LacZ full length_ pSB1A2)

1_23H (LacZ α fragment_ pSB1AK3)

9.18

Digestion (20μL):

LacZ full length: 5μL

SpeI: 1μL

PstI: 1μL

Buffer (NEBuffer4): 2μL

ddH2O: 12μL

Digestion (20μL):

LacZα fragment: 5μL

SpeI: 1μL

PstI: 1μL

Buffer (NEBuffer4): 2μL

ddH2O: 12μL

Electrophoresis

Gel Extraction

FAILED(T_T)

Wrong enzyme used

9.19

Digestion (20μL):

LacZ full length: 5μL

XbaI: 1μL

PstI: 1μL

Buffer (NEBuffer3): 2μL

ddH2O: 12μL

Digestion (20μL):

LacZ α fragment: 5μL

XbaI: 1μL

PstI: 1μL

Buffer (NEBuffer3): 2μL

ddH2O: 12μL

Electrophoresis

Gel Extraction

The band for LacZ full length is clear but not for LacZ α fragment

Change method

PCR from 1_23H by EasyPFU SuperMix (20μL)

2*EasyPFU SuperMix: 10μL

ddH2O: 7μL

Primer_Univ_For: 1.5μL

Primer_Univ_Rev:1.5μL

Template: 0.3μL

Electrophoresis

Gel Extraction

There should be one band for LacZ α fragment: 250bp

9.20

Digestion (20μL):

RBS_pSB1A2: 5μL

SpeI: 1μL

PstI: 1μL

Buffer (NEBuffer2): 2μL

ddH2O: 12μL

Electrophoresis

Gel Extraction

Get the RBS_pSB1A2 backbone digested with SpeI and PstI

Digestion (20μL):

PmerT_pSB1C3: 5μL

SpeI: 1μL

PstI: 1μL

Buffer (NEBuffer2): 2μL

ddH2O: 12μL

Electrophoresis

Gel Extraction

Get the PmerT_pSB1C3 backbone digested with SpeI and PstI

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert (LacZ α fragment digested with XbaI and PstI): 6μL

Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert (LacZ full length digested with XbaI and PstI): 6μL

Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL

Transformation of RBS-LacZ α fragment (pSB1A2)

9.21

No recognizable colonies grew on the agar plates (T_T)

FAILED (>.<||)

Forgot to digest the PCR product

Digestion (20μL):

1_23H PCR product: 5μL

XbaI: 1μL

PstI: 1μL

Buffer (NEBuffer3): 2μL

ddH2O: 12μL

Purification of the digested product

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert (LacZ α fragment digested with XbaI and PstI): 6μL

Vector (RBS_pSB1A2 backbone digested with SpeI and PstI): 2μL

Transformation of RBS-LacZ α fragment (pSB1A2)

Transformation of PmerT-LacZ full length (pSB1C3)

9.22

Pick up 6 colonies from each agar plate of RBS-LacZ α fragment (pSB1A2) and PmerT-LacZ full length (pSB1C3)

Grow the culture for 12hrs

Mini-prep of PmerT-LacZ full length (pSB1C3)

Digestion (20μL):

PmerT-LacZ full length (pSB1C3): 5μL

XbaI: 1μL

PstI: 1μL

Buffer (NEBuffer3): 2μL

ddH2O: 12μL

Electrophoresis to verify

Band No.1, 2, 3, 6 are of correct size

DNA Sequencing

To get the plasmid: PmerT-LacZ full length (pSB1C3)

Mini-prep of RBS-LacZ α fragment (pSB1A2)

Digestion (20μL):

RBS-LacZ α fragment (pSB1A2): 5μL

XbaI: 1μL

PstI: 1μL

Buffer (NEBuffer3): 2μL

ddH2O: 12μL

Electrophoresis to verify

Band No.1, 2, 3, 4, 5, 6 are all of correct size

9.23

PCR to get RBS-LacZ α fragment by FastPFU (50μL)

5*phusionHF buffer: 10μL

2.5mM dNTPs: 5μL

Polymerase: 1μL

Primer_Univ_For: 1.5μL

Primer_Univ_Rev:1.5μL

Template: 0.2μL

ddH2O: 32μL

Electrophoresis

Excise the band of 250bp

Gel Extraction

Digestion (20μL):

RBS-LacZ α fragment: 5μL

XbaI: 1μL

PstI: 1μL

Buffer (NEBuffer3): 2μL

ddH2O: 12μL

Purification of digested product

Ligation (10μL):

Ligase: 1μL

Ligase buffer: 1μL

Insert (LacZ α fragment digested with XbaI and PstI): 6μL

Vector (PmerT_pSB1C3 backbone digested with SpeI and PstI): 2μL

9.24

Transformation: Trans5α

Pick up 6 colonies from the agar plate: PmerT- RBS-LacZ α fragment (pSB1C3)

Grow the culture for overnight

9.25

Mini-prep of PmerT- RBS-LacZ α fragment (pSB1C3)

Digestion (20μL):

PmerT- RBS-LacZ α fragment (pSB1C3): 5μL

XbaI: 1μL

PstI: 1μL

Buffer (NEBuffer3): 2μL

ddH2O: 12μL

Electrophoresis to verify

Band No.1, 2, 3, 4, 5, 6 are all of correct size

DNA Sequencing

To get the plasmid: PmerT- RBS-LacZ α fragment (pSB1C3)

9.26

Construct the mutant promoter P88 and P3 into the traffic light system

9.27

Annealing to form the promoter from designed primers (95℃ 5min):

Primer_For_P88: 1.5μL

Primer_Rev_P88:1.5μL

Phosphorylation (37℃ 30min):

[ADD]

PNK: 1μL

Ligase buffer: 1μL

ddH2O: 4μL

Ligation:

[ADD]

Ligase: 1μL

Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL

Transformation: Trans5α

Annealing to form the promoter from designed primers (95℃ 5min):

Primer_For_P3: 1.5μL

Primer_Rev_P3:1.5μL

Phosphorylation (37℃ 30min):

[ADD]

PNK: 1μL

Ligase buffer: 1μL

ddH2O: 4μL

Ligation:

[ADD]

Ligase: 1μL

Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 1μL

Transformation: Trans5α

Annealing to form the promoter from designed primers (95℃ 5min):

Primer_For_P88: 3μL

Primer_Rev_P88: 3μL

Phosphorylation (37℃ 30min):

[ADD]

PNK: 1μL

Ligase buffer: 1μL

ddH2O: 1μL

Ligation:

[ADD]

Ligase: 2μL

Ligase buffer: 1μL

Insert: (LacZ full length digested with XbaI and PstI): 6μL

Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL

Transformation: Trans5α

Annealing to form the promoter from designed primers (95℃ 5min):

Primer_For_P3: 3μL

Primer_Rev_P3: 3μL

Phosphorylation (37℃ 30min):

[ADD]

PNK: 1μL

Ligase buffer: 1μL

ddH2O: 1μL

Ligation:

[ADD]

Ligase: 2μL

Ligase buffer: 1μL

Insert: (LacZ full length digested with XbaI and PstI): 6μL

Vector (pSB1C3 backbone digested with EcoRI and PstI): 2μL

Transformation: Trans5α

9.28

No recognizable colonies grew on the agar plates (T_T)

REPEAT

9.29

Still no recognizable colonies grew on the agar plates (TAT)

Change the ratio of different components

Annealing to form the promoter from designed primers (95℃ 5min):

Primer_For_P88: 15μL

Primer_Rev_P88:15μL

Phosphorylation (37℃ 30min):

[ADD]

PNK: 1μL

Ligase buffer: 3.5μL

ddH2O: 1μL

Ligation:

[ADD]

Ligase: 5μL

Ligase buffer: 1.5μL

Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL

Transformation: Trans5α

Annealing to form the promoter from designed primers (95℃ 5min):

Primer_For_P3: 15μL

Primer_Rev_P3:15μL

Phosphorylation (37℃ 30min):

[ADD]

PNK: 1μL

Ligase buffer: 3.5μL

ddH2O: 1μL

Ligation:

[ADD]

Ligase: 5μL

Ligase buffer: 1.5μL

Vector (RBS-LacZ α fragment _pSB1A2 backbone digested with EcoRI and XbaI): 10μL

Transformation: Trans5α

Annealing to form the promoter from designed primers (95℃ 5min):

Primer_For_P88: 7μL

Primer_Rev_P88: 7μL

Phosphorylation (37℃ 30min):

[ADD]

PNK: 1μL

Ligase buffer: 1.7μL

ddH2O: 1μL

Ligation:

[ADD]

Ligase: 5μL

Ligase buffer: 3.3μL

Insert: (LacZ full length digested with XbaI and PstI): 6μL

Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL

ddH2O: 9μL

Transformation: Trans5α

Annealing to form the promoter from designed primers (95℃ 5min):

Primer_For_P3: 7μL

Primer_Rev_P3: 7μL

Phosphorylation (37℃ 30min):

[ADD]

PNK: 1μL

Ligase buffer: 1.7μL

ddH2O: 1μL

Ligation:

[ADD]

Ligase: 5μL

Ligase buffer: 3.3μL

Insert: (LacZ full length digested with XbaI and PstI): 6μL

Vector (pSB1C3 backbone digested with EcoRI and PstI): 10μL

ddH2O: 9μL

Transformation: Trans5α

9.30

Still no recognizable colonies grew on the agar plates (TT_TT)

Hand over this work to Ying Sheng

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]