Team:Peking/Notebook/XTeng

From 2010.igem.org

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==July==
==July==
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|style="text-align:center"|[[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|27]]
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|style="text-align:center"| [[Team:Peking/Notebook/XTeng#7.26-7.28 & 8.3-8.8|28]]
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[[https://2010.igem.org/Team:Peking/Notebook/XTeng TOP]]
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===7.5-7.11===
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DsbA-MerR (Periplasmic MerR) construction
 +
 +
Successful clones have been verified by sequencing.
 +
 +
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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===
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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.
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Revision as of 07:38, 26 October 2010




   Xin Teng's Notes
                                                                                                                                                goto her page
After carefully studied the structure of MerR, I constructed MerR-MBP (Metal Binding Peptide). Additionaly, I helped to construct and standardize the periplasmic metal binding peptide – DsbA-MerR-MBP using a similar strategy as MerR-MBP. After inductive expression of MerR-MBP-His6, DsbA-PbrR-MBP-His6 and DsbA-MerR-MBP-His6, I use SDS-PAGE and western blot to confirm the expression and localization of MBP and DsbA-MBP.

There are many pictures and graphs in my notes, I strongly recommand you to download them for more precise reading.

download her notes


Contents

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

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

[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.