Team:Peking/Notebook/XTeng

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Contents

• June, 2010

• July, 2010

• August, 2010

• September, 2010

• October, 2010

June

6.7-6.13

Primer design

• For expression test

pET21a-MBP-His

pET 39b DsbA-MBP

pMAL p4X-MBP-MBP

pET21a-MerR-His

pET 39b DsbA-MerR

pMAL p4X-MBP-MerR

• For standard part

MBP_SD

DsbA-MBP_SD

MBP-MBP_SD

MerR_SD

DsbA-MerR_SD

MBP-MerR_SD

6.14-6.20

Literature Review & Experimental Design for Periplasmic MBP

July

[TOP]

7.5-7.11

DsbA-MerR (Periplasmic MerR) construction

Successful clones have been verified by sequencing.

Standardization for DsbA-MerR

Double digest shows the right result. Need to be sequenced.

MBP-His (cytosol MBP for expression test) construction

Double digest result shows that clone have failed. No band of right molecular weight can be detected.

MBP (cytosol MBP for mercury binding test) construction

Clones have been constructed, but need to be verified by double digestion.

Mercury Semiquantative Detection

Solution Preparation

1 g/L Dithizone Cloroform solution

1.038×10-3 mol/L HgCl2

Dithizone Eluant

Na2S detoxic solution

1 mol/L NaOH

Concentrated HNO3

Standard Mercury Concentration Determination

Mercury concentrated 25 times

Color change can be easily seen, but out of linear range of the standard curve.

7.12-7.18

DsbA-MBP (periplasmic MBP for expression test) construction

Band of expected molecular weight can be detected by double digest. Need to be sequenced.

Re-design the construction strategy for DsbA-MerR(MBP)

MBP-His & MBP construction

Failed.

Mercury Semiquantative Determination

Standard solution concentrated 10 times.

There is no significant difference between detected mercury concentration prepared in LB or water. But the absolute value is too low.

7.19-7.25

DsbA-MerR construction. Need to be verified.

DsbA-MBP construction. Need to be verified.

SDS-PAGE & Western Blot

Buffer preparation

5 × SDS-PAGE buffer

Tris – HCl pH 6.8

Tris – HCl pH 8.8

30% Acr / Bis-Acr

5 × TBS

5 × Transfer Buffer

1 × TTBS

SDS-PAGE result of DsbA-MerR

Overexpression band can be detected at the expected molecular weight (40 kD).

7.26-7.28 & 8.3-8.8

Double digest verification for DsbA-MBP, MBP-His & MBP.

Failed.

Check the template of MBP by sequencing.

Re-design for MBP & DsbA-MBP construction.

August

[TOP]

8.9-8.15

MBP-His construction

Sequencing.

MBP construction

Sequencing.

DsbA-MBP construction

strategy I

Sequencing.

Strategy II

Sequencing.

Standardization for DsbA-MerR

Sequencing.

Standardization for DsbA-MBP

DsbA-MerR sample preparation for SDS-PAGE

8.16-8.22

DsbA-MerR standardization finished.

Re-construct DsbA-MBP

Expression condition optimization for DsbA-MerR

8.23-8.29

SDS-PAGE & Western Blot for DsbA-MerR

SDS-PAGE & Western Blot for DsbA-MBP

The ratio of the primary antigen to secondary antigen should be optimized to get better figures.

8.30-9.5

SDS-PAGE for T3 polymerase

SDS-PAGE & Western Blot for MBP

The ratio of the primary antigen to secondary antigen should be optimized to get better figures.

September

[TOP]

9.6-9.12

Western Blot for MBP

Western Blot for DsbA-MBP

9.13-9.19

SDS-PAGE & Western Blot for lead DsbA-MBP

SDS-PAGE for lead MBP

The molecular weight of the overexpression bands does not meet our expectation. Check the plasmid.

Scan the spectrum of Crt EBI

9.20-9.26

Characterize the mutation of PmerT.

9.27-10.3

1-18I-pag, 2-2M-pag, 1-18E-pag construction

October

[TOP]

10.4-10.10

western blot for mercury Lpp-OmpA-MBP

10.11-10.17

Cigarette digestion for our biokit.

Failed.