Team:Peking/Notebook/QZWu

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<font color=#FFFFFF><font size=4><b>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
<font color=#FFFFFF><font size=4><b>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Haoqian Zhang</b></font></font>-->
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Haoqian Zhang</b></font></font>-->
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To characterize the regulating role of MerR on PmerT, promoters from parts-registry constutitive promoter library with different strength were prefixed before MerR coding sequence to exogenously maintain MerR expression at different intensity. GFP expression response to mercury concentrations was measured by microplate reader.
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<img src="http://2010.igem.org/wiki/images/9/91/PKU_Adobe_Reader_Logo.jpg" width=20>
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<a href="http://2010.igem.org/wiki/images/6/69/Note-bamboo.pdf"><font color=#FFFFFF>download her notes</font></a>
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=='''Contents'''==
 +
* <span style="font-size:4mm;">[[Team:Peking/Notebook/QZWu#July| July, 2010]]</span>
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/QZWu#August| August, 2010]]</span>
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/QZWu#September| September, 2010]]</span>
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/QZWu#October| October, 2010]]</span>
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==July==
 +
==July==
 +
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"
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|-
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|style="text-align:center"| Mon
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|style="text-align:center"| Tue
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|style="text-align:center"| Wed
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|style="text-align:center"| Thu
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|style="text-align:center"| Fri
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|style="text-align:center"| Sat
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|style="text-align:center"| Sun
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|-
 +
|style="text-align:center"| -
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|style="text-align:center"| -
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|style="text-align:center"| -
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|style="text-align:center"| -
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|style="text-align:center"| 1
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.2|2]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.3|3]]
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|-
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.4|4]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.5|5]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.6|6]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.7|7]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.8|8]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.9|9]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.10|10]]
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|-
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.11|11]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.12|12]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|13]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|14]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|15]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|16]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|17]]
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|-
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|18]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|19]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|20]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|21]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.13-7.22|22]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.23|23]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.24|24]]
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|-
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.25|25]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.26|26]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.27 |27]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.27-7.28|28]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.29|29]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.30|30]]
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|style="text-align:center"| [[Team:Peking/Notebook/QZWu#7.31|31]]
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|-
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|style="text-align:center"| -
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|style="text-align:center"| -
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|style="text-align:center"| -
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|style="text-align:center"| -
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|style="text-align:center"| -
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|style="text-align:center"| -
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|style="text-align:center"| -
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|}
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[[http://2010.igem.org/Team:Peking/Notebook/QZWu TOP]]
 +
===7.2===
 +
 +
1. Dissolve J23101, J23116, J23103 parts from the well and transformed them into trans 5α.
 +
 +
 +
 +
===7.3===
 +
 +
1. Anneal PmerT with Forward and reverse primers.
 +
 +
2. Ligase PmerT with E0840.
 +
 +
3. Transform.
 +
 +
 +
 +
===7.4===
 +
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1. Miniprep pc plasmids and PmerT-E0840.
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 +
 +
 +
===7.5===
 +
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1. Ligase RBS and merR, then transform.
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 +
2. Digest pc plasmids with EcoRI and SpeI.
 +
 +
 +
 +
===7.6===
 +
 +
1. Miniprep RBS-merR plasmid.
 +
 +
2. Digest the plasmid with XbaI and PstI.
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 +
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===7.7===
 +
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1. Ligase pc with RBS-merR, then transform.
 +
 +
2. Confirm the product by PCR.
 +
 +
 +
 +
===7.8===
 +
 +
1. Miniprep pc-RBS-merR plasmid.
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 +
 +
 +
===7.9===
 +
 +
1. Confirm the product by PCR.
 +
 +
 +
 +
===7.10===
 +
 +
1. Digest Psb3K3 backbone with EcoRI and PstI.
 +
 +
2. Help ZTZ with his Pbad-E0840.
 +
 +
 +
 +
===7.11===
 +
 +
1. Miniprep pc-RBS-merR plasmid and digest them with EcoRI and PstI.
 +
 +
 +
 +
===7.12===
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1. Ligase pc-RBS-merR with Psb3K3 backbone.
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 +
2. Transform.
 +
 +
3. For I’ve been confused about all the plasmids I can only PCR them all to test which ones are correct.
 +
 +
 +
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===7.13-7.22===
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Keep on the construction of ps-RBS-merR~
 +
 +
A little frustrated, but things will be fine!
 +
 +
 +
 +
===7.23===
 +
 +
Get one clone, J23103-RBS-merR.
 +
 +
Strategy change.
 +
 +
Order primers that consist of pc and RBS sequences.
 +
 +
Use J23103 –RBS-merR as template.
 +
 +
Try to get other clones by PCR.
 +
 +
 +
 +
===7.24===
 +
 +
1. Purify PCR products.
 +
 +
2. Digest them with EcoRI and PstI.
 +
 +
 +
 +
===7.25===
 +
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1. Ligase pc-RBS-merR with Psb1A2 and Psb3K3 backbone.
 +
 +
2. Transform.
 +
 +
 +
 +
===7.26===
 +
 +
1. Miniprep plasmids.
 +
 +
2. Send them for sequencing.
 +
 +
 +
 +
===7.27===
 +
 +
Cannot wait for the sequencing results, start function test.
 +
 +
1. Prepare cells with PmerT-E0840 into competent cells.
 +
 +
2. Transform pc-RBS-merR plasmids with different resistance into them.
 +
 +
 +
 +
===7.28===
 +
 +
Test GFP expression level.
 +
 +
For the first time, there are only 5 Hg concentration gradients and no repeats.
 +
 +
Get data, sort of weird.
 +
 +
No significantly differences.
 +
 +
Happy anyway.
 +
 +
 +
 +
===7.29===
 +
 +
This time 10 gradients and 3 repeats.
 +
 +
The GFP of J23103 is extraordinarily higher than others. And no differences can be observed between the others.
 +
 +
 +
 +
===7.30===
 +
 +
Try again~
 +
 +
 +
 +
===7.31===
 +
 +
Sequencing results come. Bad news: It seems that the products were polluted by merR.
-
<p><font color=#FFFFFF>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
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<a href="http://2010.igem.org/Team:Peking/Bioabsorbenthome"><font size=4><b><font color=#FFFFFF>----contents----</font></font></b></a>
 
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<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
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<font size=3><font color=#FFFFFF>*Personal Notes</font></font>
 
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<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
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<a href="http://2010.igem.org/Team:Peking/Protocols"><font size=3><font color=#FFFFFF>*Protocols</font></font></a>
 
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<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
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<font size=3><font color=#FFFFFF>*Others</font></font>
 
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<div id="notebook home">
 
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<div id="titlegreen">
 
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<br><br>
 
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<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
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<a href=""><font size=5><b><font color=#74DF00>SUBTITLE</font></font></b></a>
 
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<div id="project description bottom">
<div id="project description bottom">

Revision as of 04:02, 27 October 2010




   Qianzhu Wu's Notes
                                                                                                                                                goto her page
To characterize the regulating role of MerR on PmerT, promoters from parts-registry constutitive promoter library with different strength were prefixed before MerR coding sequence to exogenously maintain MerR expression at different intensity. GFP expression response to mercury concentrations was measured by microplate reader.


download her notes

Contents


July

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]

7.2

1. Dissolve J23101, J23116, J23103 parts from the well and transformed them into trans 5α.


7.3

1. Anneal PmerT with Forward and reverse primers.

2. Ligase PmerT with E0840.

3. Transform.


7.4

1. Miniprep pc plasmids and PmerT-E0840.


7.5

1. Ligase RBS and merR, then transform.

2. Digest pc plasmids with EcoRI and SpeI.


7.6

1. Miniprep RBS-merR plasmid.

2. Digest the plasmid with XbaI and PstI.


7.7

1. Ligase pc with RBS-merR, then transform.

2. Confirm the product by PCR.


7.8

1. Miniprep pc-RBS-merR plasmid.


7.9

1. Confirm the product by PCR.


7.10

1. Digest Psb3K3 backbone with EcoRI and PstI.

2. Help ZTZ with his Pbad-E0840.


7.11

1. Miniprep pc-RBS-merR plasmid and digest them with EcoRI and PstI.


7.12

1. Ligase pc-RBS-merR with Psb3K3 backbone.

2. Transform.

3. For I’ve been confused about all the plasmids I can only PCR them all to test which ones are correct.


7.13-7.22

Keep on the construction of ps-RBS-merR~

A little frustrated, but things will be fine!


7.23

Get one clone, J23103-RBS-merR.

Strategy change.

Order primers that consist of pc and RBS sequences.

Use J23103 –RBS-merR as template.

Try to get other clones by PCR.


7.24

1. Purify PCR products.

2. Digest them with EcoRI and PstI.


7.25

1. Ligase pc-RBS-merR with Psb1A2 and Psb3K3 backbone.

2. Transform.


7.26

1. Miniprep plasmids.

2. Send them for sequencing.


7.27

Cannot wait for the sequencing results, start function test.

1. Prepare cells with PmerT-E0840 into competent cells.

2. Transform pc-RBS-merR plasmids with different resistance into them.


7.28

Test GFP expression level.

For the first time, there are only 5 Hg concentration gradients and no repeats.

Get data, sort of weird.

No significantly differences.

Happy anyway.


7.29

This time 10 gradients and 3 repeats.

The GFP of J23103 is extraordinarily higher than others. And no differences can be observed between the others.


7.30

Try again~


7.31

Sequencing results come. Bad news: It seems that the products were polluted by merR.




<a href="http://2010.igem.org/Team:Peking/Team/QZWu">==go to her page==</a>        

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