Team:Peking/Notebook/TZZhu

From 2010.igem.org

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<img src="https://static.igem.org/mediawiki/2010/9/91/PKU_Adobe_Reader_Logo.jpg" width=20>
<img src="https://static.igem.org/mediawiki/2010/9/91/PKU_Adobe_Reader_Logo.jpg" width=20>
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<a href="https://static.igem.org/mediawiki/2010/5/50/Note_PH.pdf"><font color=#FFFFFF>download his notes</font></a>
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<a href="https://static.igem.org/mediawiki/2010/5/5e/TZZ.pdf"><font color=#FFFFFF>download his notes</font></a>
</html>
</html>
=='''Contents'''==
=='''Contents'''==
-
* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#July| July, 2010]]</span>
+
* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 1st| Week 1st]]</span>
-
* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#August| August, 2010]]</span>
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 2nd| Week 2nd]]</span>
-
* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#September| September, 2010]]</span>
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 3rd| Week 3rd]]</span>
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#October| October, 2010]]</span>
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 4th| Week 4th]]</span>
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==July==
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 5th| Week 5th]]</span>
-
{| 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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|-
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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"| 2
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.3|3]]
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|-
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.4|4]]
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|style="text-align:center"|[[Team:Peking/Notebook/HPan#7.5|5]]
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-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.6|6]]
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.7|7]]
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.8|8]]
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-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.9|9]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.10|10]]
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-
|-
+
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.11|11]]
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-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#7.12|12]]
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-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.13|13]]
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-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#7.14|14]]
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-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.15|15]]
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-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.16|16]]
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-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#7.17|17]]
+
-
|-
+
-
|style="text-align:center"|18
+
-
|style="text-align:center"| 19
+
-
|style="text-align:center"| 20
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-
|style="text-align:center"|21
+
-
|style="text-align:center"| 22
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-
|style="text-align:center"| 23
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-
|style="text-align:center"| 24
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-
|-
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* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 6th| Week 6th]]</span>
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.25|25]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.26|26]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#7.27|27]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.28|28]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.29|29]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.30|30]]
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-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#7.31|31]]
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-
|-
+
-
|}
+
-
[[https://2010.igem.org/Team:Peking/Notebook/HPan TOP]]
+
-
===7.3===
+
-
Transformation of plasmid: Terminator(B0015) and T7 promoter(BBa_I719005)
+
-
===7.4===
+
-
PCR to standardize the MerT and MerP from plasmid NRI
+
-
Amplification of bacterium: Terminator(B0015) and T7 promoter(BBa_I719005)
+
* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 7th| Week 7th]]</span>
-
===7.5===
+
-
Miniprep: Terminator(B0015) and T7 promoter(BBa_I719005)
+
-
Digestion and identification by Electrophoresis
+
* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 8th| Week 8th]]</span>
-
Terminator(B0015)        EcoRI and XbaII
+
* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 9th| Week 9th]]</span>
-
T7 promoter(BBa_I719005)  SpeI and PstI
+
* <span style="font-size:4mm;">[[Team:Peking/Notebook/HPan#Week 10th| Week 10th]]</span>
-
phiR73 delta(BBa_I746352)  EcoRI and SpeI
+
===Week 1st===
-
PO promoter(BBa_I746361)  EcoRI and XbaI
+
Get the vector with Psal promoter and double digest it with X,P.  
-
===7.6===
+
-
Ligation of phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
+
-
Transformation of ligation mixture of phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
+
Get the insert of GFP gene and double digest it with S,P
-
===7.7===
+
-
Amplification of bacterium: phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
+
-
Miniprep: phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
+
Ligase the two part together in order to cnstruct the plasmid of Psal promoter and GFP,  
-
===7.8===
+
-
Digestion and identification by Electrophoresis, but the result had been failed.
+
-
phiR73 delta+Terminator  XbaI and PstI
+
Transfer the plasmid into trans 5a bacteria
-
RBS(B0032)            SpeI and PstI
+
Get the vector with Pbad promoter and double digest it with X,P.
-
Digestion and identification by Electrophoresis again
+
Get the insert of GFP gene and double digest it with S,P
-
===7.9===
+
-
Ligation of phiR73 delta+Terminator(insert) and RBS(B0032)(vector)
+
-
Transformation of ligation mixture of phiR73 delta+Terminator(insert) and RBS(B0032)(vector)
+
Ligase the two part together in order to cnstruct the plasmid of Pbad promoter and GFP,
-
Digestion and identification by Electrophoresis
+
Transfer the plasmid into trans 5a bacteria
-
Terminator(B0015)            XbaI and PstI
 
-
AraC                        XbaI and PstI
 
-
Miniprep: plasmid of backbone PSB1C3
+
===Week 2nd===
-
===7.10===
+
Get the vector with T7 promoter and double digest it with X,P.  
-
Amplification of RBS+phiR73 delta+Terminator
+
-
===7.11===
+
-
The amplification had failed and amplification again.
+
-
===7.12===
+
-
Miniprep: phiR73 RBS+delta+Terminator
+
-
Digestion and identification by Electrophoresis
+
Get the insert of GFP gene and double digest it with S,P
-
RBS+phiR73 delta+Terminator    XbaI and PstI
+
Ligase the two part together in order to cnstruct the plasmid of T7 promoter and GFP,
-
===7.13===
+
Transfer the plasmid into trans 5a bacteria
-
Ligation of RBS+phiR73 delta+terminator(insert) and T7 promoter((BBa_I719005))(vector)
+
-
Transformation of ligation mixture of RBS+phiR73 delta+Terminator and T7 promoter((BBa_I719005))(vector)
 
-
===7.14===
 
-
Amplification of T7 promoter+RBS+phiR73 delta+Terminator
 
-
Miniprep: T7 promoter+RBS+phiR73 delta+Terminator
 
-
Digestion and identification by Electrophoresis
+
===Week 3rd===
-
T7 promoter+RBS+phiR73 delta+Terminator    EcoRI and SpeI
+
Make the different types of protection media with gradient of trehalose PVP.
-
===7.15===
+
-
Ligation of T7 promoter+RBS+phiR73 delta+Terminator(insert) and PO promoter(BBa_I746361)(vector)
+
-
Transformation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter
 
-
===7.16===
 
-
Amplification of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter
 
-
Miniprep: T7 promoter+RBS+phiR73 delta+Terminator+PO promoter
 
-
===7.17===
 
-
Digestion and identification by Electrophoresis
 
-
T7 promoter+RBS+phiR73 delta+Terminator+PO promoter  EcoRI and SpeI
+
===Week 4th===
-
===7.25===
+
-
Digestion and identification by Electrophoresis
+
-
GFP generator(E0840)  EcoRI and Xbal
+
Use IPTG to induce the fresh cell containing plasmid of T7 promoter and GFP gene to express GFP.]
-
Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter(insert) and GFP generato T7 promoter+RBS+phiR73
+
Detect it with flow cytometry assay and enzyme-linked immunoadsordent assay.
-
delta+Terminator+PO promoter+GFP generator r(E0840)(vector)
+
-
Transformation of Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
+
Determine the respone curve of the cell, and the intensity of GFP expression level.
-
===7.26===
+
-
Amplification of Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
+
-
===7.27===
+
-
Miniprep: T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
+
-
Digestion and identification by Electrophoresis
 
-
T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
 
-
===7.28===
 
-
Positive transformation T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator into strain BL21a
 
-
===7.29===
 
-
PbrR PCR, 50 uL system with Easy Pfu DNA Polymerase
 
-
Induce strain BL21a(consist of plasmid T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator) by iptg to gain the expression of GFP, but there were no obvious difference between strain induced and uninduced.
+
===Week 5th===
-
===7.30===
+
-
Retrieve the PCR product and identification by Electrophoresis
+
-
Digestion:
+
Transfer the cells of T7 promoter and GFP, Mer promoter and GFP into protection media and dry them in the water pump. Store in fringe in 4°C.
-
PbrR  EcoRI and PstI/XbaI and PstI
 
-
Ligation of PbrR(insert) and PSB1K3(vector)
 
-
Transformation of PbrR(PSB1K3)
+
===Week 6th===
-
===7.31===
+
-
Ligation of PbrR(insert) and RBS(B0034)
+
-
Transformation of RBS+PbrR
+
ShangHai EXPO
-
Amplification of PbrR(PSB1K3)
 
-
==August==
 
-
{| 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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|-
 
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.1|1]]
 
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.2|2]]
 
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.3|3]]
 
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.4|4]]
 
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.5|5]]
 
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.6|6]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.7|7]]
 
-
|-
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.8|8]]
 
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#8.9|9]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.10|10]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.11|11]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.12|12]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.13|13]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.14|14]]
 
-
|-
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.15|15]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.16|16]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.17|17]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.18|18]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.19|19]]
 
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|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.20|20]]
 
-
|style="text-align:center"| 21
 
-
|-
 
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|style="text-align:center"| 22
 
-
|style="text-align:center"| 23
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.24|24]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.25|25]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.26|26]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.27|27]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.28|28]]
 
-
|-
 
-
|style="text-align:center"| 29
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.30|30]]
 
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#8.31|31]]
 
-
|style="text-align:center"| -
 
-
|style="text-align:center"| -
 
-
|style="text-align:center"| -
 
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|style="text-align:center"| -
 
-
|}
 
-
[[https://2010.igem.org/Team:Peking/Notebook/HPan TOP]]
 
-
===8.1===
 
-
Miniprep:PbrR(PSB1K3)
 
-
Amplification of RBS+PbrR
 
-
===8.2===
 
-
Miniprep:RBS+PbrR
 
-
Digestion and identification by Electrophoresis
+
===Week 7th===
-
RBS+PbrR    XbaI and PstI
+
Recover the stored cells and use cytometry assay and enzyme-linked immunoadsordent assay, the same methods used in fresh cells to determine the same parameter.
-
Positive Transformation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator again into strain BL21
 
-
(DE3)
 
-
Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)
 
-
===8.3===
 
-
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
 
-
===8.4===
 
-
Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 promoter+RBS+phiR73 delta+Terminator+PO
 
-
promoter+GFP generator, but there is no colony of Pc promoter(1-18C)+RBS+PbrR
 
-
Digestion and identification by Electrophoresis, but the result had been failed.
+
===Week 8th===
-
N_B/B_X/B_SD/SD_B/PET21A/RBS+PbrR
+
Recover the stored cells and use cytometry assay and enzyme-linked immunoadsordent assay, the same methods used in fresh cells to determine the same parameter.
-
Ligation of Pc promoter(1-18C) and RBS+PbrR again
 
-
Induce strain BL21(DE3)(consist of plasmid T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator) by iptg
 
-
again to gain the expression of GFP, but there were still no obvious difference between strain induced and uninduced.
 
-
===8.5===
 
-
Bacterial colonies PCR
 
-
Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
===Week 9th===
-
Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
Recover the stored cells and use cytometry assay and enzyme-linked immunoadsordent assay, the same methods used in fresh cells to determine the same parameter.
-
Transformation of Pc promoter(1-18C)+RBS+PbrR again.
 
-
Digestion and identification by Electrophoresis again
 
-
N_B/B_X/B_SD/SD_B/PET21A
+
===Week 10th===
-
Digestion and identification by Electrophoresis, but the result had been failed.
+
Collect the figure and data achieved in the previous three weeks to see how the condition and vigor of cells change.  
-
Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
Determine which kind of protection media is the best.
-
Ligation of N_B, B_X(insert) and PET21A, B_SD, SD_B,PSB1K3
 
-
Transformation of PET21A-NX and PSB3K3-BSD
 
-
Amplification of Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again
+
<html>
-
===8.6===
+
-
Amplification of Pc promoter(1-18C)+RBS+PbrR again
+
-
 
+
-
Bacterial colonies PCR again, but the result had been failed.
+
-
 
+
-
Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
-
 
+
-
Miniprep: Pc promoter(1-18C)+RBS+PbrR
+
-
 
+
-
Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again
+
-
 
+
-
Digestion and identification by Electrophoresis, but the result had been failed again.
+
-
 
+
-
Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
-
===8.7===
+
-
PbrR PCR, 50 uL system with Easy Pfu DNA Polymerase for the second time
+
-
 
+
-
Digestion and identification by Electrophoresis, but the result had been failed again.
+
-
 
+
-
PbrR        XbaI and PstI
+
-
 
+
-
RBS(B0034)  SpeI and PstI
+
-
 
+
-
Ligation of PbrR(insert) and RBS(B0034)(vector)
+
-
===8.8===
+
-
Bacterial colonies PCR again, but the result had been failed.
+
-
 
+
-
PbrR+RBS
+
-
 
+
-
Amplification of RBS(B0034)
+
-
===8.9===
+
-
Digestion and identification by Electrophoresis
+
-
 
+
-
PbrR(PSB1K3)        XbaI and PstI
+
-
 
+
-
RBS(B0034)          SpeI and PstI
+
-
 
+
-
Ligation of PbrR(insert) and RBS(vector) for the second time
+
-
 
+
-
Transformation of RBS+PbrR again
+
-
 
+
-
===8.10===
+
-
Digestion and identification by Electrophoresis
+
-
 
+
-
PbrR(PCR product)        XbaI and PstI
+
-
 
+
-
Ligation of PbrR(insert) and PSB1K3(vector)
+
-
 
+
-
Transformation fo PbrR(PSB1K3)
+
-
===8.11===
+
-
Bacterial colonies PCR , PbrR(PSB1K3)
+
-
 
+
-
PbrD/PbrT PCR, 50 uL system with Easy Pfu DNA Polymerase
+
-
 
+
-
Indentification by Electrophoresis, but the PbrT had been failed.
+
-
 
+
-
PbrT PCR, 50 uL system with Easy Pfu DNA Polymerase again.
+
-
 
+
-
Digestion and identification by Electrophoresis: PbrD and PSB1C3(backbone)
+
-
===8.12===
+
-
Digestion and identification by Electrophoresis: PbrR(PSB1K3)/PbrT
+
-
 
+
-
Positive transformation of RBS(B0034)
+
-
 
+
-
Ligation of PbrD(insert) and PSB1C3(vector), PbrT(insert) and PSB1C3(vector)
+
-
 
+
-
Miniprep: RBS(B0034)
+
-
 
+
-
Digestion and identification by Electrophoresis: RBS(B0034)
+
-
 
+
-
===8.13===
+
-
Transformation of PbrD(PSB1C3), PbrT(PSB1C3), but there were no colony for them.
+
-
 
+
-
Ligation of PbrD/PbrT/PbrR(insert) and RBS(B0034)(vector)
+
-
===8.14===
+
-
PbrD/PbrT/PbrR PCR, 50 uL system with Easy Taq DNA Polymerase again, but the result of PbrT had been failed.
+
-
 
+
-
Ligation of PbrD(insert) and PSB1C3(vector), PbrT(insert) and PSB1C3(vector) again
+
-
===8.15===
+
-
Amplification of PbrT+RBS and PbrD+RBS
+
-
 
+
-
Digestion and identification by Electrophoresis: PbrD and PbrR again
+
-
 
+
-
===8.16===
+
-
Miniprep: RBS+PbrT and RBS+PbrD
+
-
 
+
-
Digestion and identification by Electrophoresis: RBS+PbrT and RBS+PbrD
+
-
===8.17===
+
-
Miniprep: RBS+PbrT, RBS+PbrR and RBS+PbrD
+
-
 
+
-
Digestion and identification by Electrophoresis: RBS+PbrT, RBS+PbrR and RBS+PbrD
+
-
 
+
-
Ligation of RBS+PbrT(insert) and Terminator(B0015), RBS+PbrD(insert) and T7 promoter(vector)
+
-
 
+
-
Transformation of RBS+PbrT+Terminator and T7 promoter+RBS+PbrD
+
-
===8.18===
+
-
Digestion and identification by Electrophoresis:RBS+PbrR
+
-
===8.19===
+
-
Miniprep: RBS+PbrT+Terminator and T7 promoter+RBS+PbrD
+
-
 
+
-
Digestion and identification by Electrophoresis: RBS+PbrT+Terminator and T7 promoter+RBS+PbrD
+
-
===8.20===
+
-
Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector)
+
-
===8.24===
+
-
Amplification of RBS+PbrR
+
-
 
+
-
Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) again
+
-
===8.25===
+
-
Miniprep: RBS+pbrR
+
-
 
+
-
Transformation of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
+
-
 
+
-
Digestion and identification by Electrophoresis: RBS+PbrR
+
-
===8.26===
+
-
Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)(vector)
+
-
===8.27===
+
-
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
-
===8.28===
+
-
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
-
 
+
-
Miniprep: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
+
-
===8.30===
+
-
Digestion and identification by Electrophoresis: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7
+
-
promoter+RBS+Terminator+RBS+PbrT+Terminator
+
-
===8.31===
+
-
Ligation of MerP/PpbrA(insert) and CrtebI(vector)
+
-
==September==
+
-
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"
+
-
|-
+
-
|style="text-align:center"| Mon
+
-
|style="text-align:center"| Tue
+
-
|style="text-align:center"| Wed
+
-
|style="text-align:center"| Thu
+
-
|style="text-align:center"| Fri
+
-
|style="text-align:center"| Sat
+
-
|style="text-align:center"| Sun
+
-
|-
+
-
|style="text-align:center"| -
+
-
|style="text-align:center"| -
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.1|1]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.2|2]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.3|3]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.4|4]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.5|5]]
+
-
|-
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.6|6]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.7|7]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.8|8]]
+
-
|style="text-align:center"| 9
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.10|10]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.11|11]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.12|12]]
+
-
|-
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.13|13]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.14|14]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.15|15]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.16|16]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|17]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|18]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|19]]
+
-
|-
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|20]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|21]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.22|22]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.23|23]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.24|24]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.25|25]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.26|26]]
+
-
|-
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.27|27]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#9.28|28]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#9.29|29]]
+
-
|style="text-align:center"| 30
+
-
|style="text-align:center"| -
+
-
|style="text-align:center"| -
+
-
|style="text-align:center"| -
+
-
|}
+
-
[[https://2010.igem.org/Team:Peking/Notebook/HPan TOP]]
+
-
===9.1===
+
-
Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into bacterium which consist of plasmid
+
-
PpbrA+RBS+GFP, but there is no expression of GFP
+
-
===9.2===
+
-
The sequencing results showed that the sequence of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7
+
-
promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect
+
-
 
+
-
PbrR PCR, 50 uL system with Easy Taq DNA Polymerase for the fourth time
+
-
 
+
-
Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third time
+
-
===9.3===
+
-
Retrieve the PCR product and identification by Electrophoresis
+
-
 
+
-
Transformation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third
+
-
 
+
-
Ligation of PbrR(insert) and RBS(vector)
+
-
===9.4===
+
-
Amplification of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
+
-
 
+
-
Transformation of RBS+PbrR
+
-
===9.5===
+
-
Miniprep: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
+
-
 
+
-
Amplification of RBS+PbrR
+
-
 
+
-
Digestion and identification by Electrophoresis: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
+
-
 
+
-
===9.6===
+
-
The sequencing results showed that the sequence of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect again
+
-
 
+
-
Ligation of RBS+PbrT(insert) and Terminator(B0015), RBS+PbrD(insert) and T7 promoter(vector)
+
-
 
+
-
Transformation of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
+
-
===9.7===
+
-
Amplification of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
+
-
===9.8===
+
-
Miniprep: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
+
-
 
+
-
Digestion and identification by Electrophoresis: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
+
-
===9.10===
+
-
Ligation of T7promoter+RBS+PbrD(insert) and RBS+PbrT+Terminator(vector).
+
-
 
+
-
Transformation of T7promoter+RBS+PbrD+RBS+PbrT+Terminator
+
-
===9.11===
+
-
Amplification of T7promoter+RBS+PbrD+RBS+PbrT+Terminator
+
-
 
+
-
Digestion and identification by Electrophoresis: RBS+PbrR
+
-
 
+
-
Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)(vector)
+
-
===9.12===
+
-
Miniprep: T7promoter+RBS+PbrD+RBS+PbrT+Terminator
+
-
 
+
-
Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
-
 
+
-
Digestion and identification by Electrophoresis: T7promoter+RBS+PbrD+RBS+PbrT+Terminator
+
-
===9.13===
+
-
The sequencing result of T7promoter+RBS+PbrD+RBS+PbrT+Terminator had failed again.
+
-
 
+
-
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
-
===9.14===
+
-
Digestion and identification by Electrophoresis: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
+
-
===9.15===
+
-
Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into PpbrA+GFP generator to detect the expression
+
-
level of GFP. But there were no expression of GFP.
+
-
===9.16===
+
-
The sequencing result showed than the sequence of PbrR was not intact.
+
-
===9.17-9.21===
+
-
Repeat the similar procedures to construct Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again.
+
-
===9.22===
+
-
Detect the expression level of GFP for the second time, but there were still no expression.
+
-
===9.23===
+
-
The sequencing result showed than the sequence of PbrR was not intact, which was similar to the sequence we had got before and Zhang Haoqian found that there was a PstI digest site in the sequence of PbrR
+
-
===9.24===
+
-
Transformation of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator into stain Trans 5a to detect the expression level of GFP
+
-
 
+
-
===9.25===
+
-
Ligation of Pc promoter+T7 polymerase(insert) and PSB3C5(backbone)
+
-
===9.26===
+
-
Miniprep: Pc promoter+T7 polymerase(PSB3C5)
+
-
 
+
-
Digestion and identification by Electrophoresis: Pc promoter+T7 polymerase(PSB3C5)
+
-
===9.27===
+
-
Transformation Pc promoter+T7 polymerase into bacterium with T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS
+
-
generator to detect the expression level of GFP
+
-
===9.28===
+
-
Amplification of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator and Pc promoter+T7 polymerase.
+
-
===9.29===
+
-
Detect the expression level of GFP
+
-
==October==
+
-
 
+
-
{| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff"
+
-
|-
+
-
|style="text-align:center"| Mon
+
-
|style="text-align:center"| Tue
+
-
|style="text-align:center"| Wed
+
-
|style="text-align:center"| Thu
+
-
|style="text-align:center"| Fri
+
-
|style="text-align:center"| Sat
+
-
|style="text-align:center"| Sun
+
-
|-
+
-
|style="text-align:center"| -
+
-
|style="text-align:center"| -
+
-
|style="text-align:center"|-
+
-
|style="text-align:center"|-
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.1|1]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.2|2]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.3|3]]
+
-
|-
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.4|4]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.5|5]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.6|6]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.7|7]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.8|8]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.9|9]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.10|10]]
+
-
|-
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.11|11]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.12-10.13|12]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.12-10.13|13]]
+
-
|style="text-align:center"|[[Team:Peking/Notebook/HPan#10.14|14]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.15|15]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.16|16]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.17|17]]
+
-
|-
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.18|18]]
+
-
|style="text-align:center"|19
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.20|20]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.21|21]]
+
-
|style="text-align:center"|22
+
-
|style="text-align:center"|23
+
-
|style="text-align:center"| 24
+
-
|-
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.25|25]]
+
-
|style="text-align:center"| [[Team:Peking/Notebook/HPan#10.26|26]]
+
-
|style="text-align:center"|27
+
-
|style="text-align:center"| -
+
-
|style="text-align:center"| -
+
-
|style="text-align:center"| -
+
-
|style="text-align:center"| -
+
-
|}
+
-
[[https://2010.igem.org/Team:Peking/Notebook/HPan TOP]]
+
-
===10.1===
+
-
PCR T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS with the forword primer of T3 promoter and the univ reverse primer, 50 uL system.
+
-
 
+
-
Retrieve the PCR product of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS
+
-
===10.2===
+
-
Ligation of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(insert) and PSB1C3 bacbone
+
-
 
+
-
Transformation of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3)
+
-
===10.3===
+
-
Amplification of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3)
+
-
 
+
-
PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR with eight different Pc promoter
+
-
forward primer and PbrR reverse primer
+
-
 
+
-
Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR
+
-
 
+
-
===10.4===
+
-
Miniprep: T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3)
+
-
 
+
-
Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3(backbone)
+
-
 
+
-
Digestion and identification by Electrophoresis: T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3)
+
-
===10.5===
+
-
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3
+
-
(backbone), but there were no colonies for Pc promoter(1-18C)+RBS+PbrR
+
-
===10.6===
+
-
Bacterial colonies PCR and Amplification of Pc promoter(1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB1C3)
+
-
 
+
-
Ligation of RBS+PbrR(insert) and Pc promoter(1-18C)(vector)
+
-
 
+
-
Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB3K3(backbone)
+
-
===10.7===
+
-
Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB1C3)
+
-
 
+
-
Transformation of Bacterial colonies PCR and Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB3K3), Pc promoter(1-18C)+RBS+PbrR(PSB1C3)
+
-
===10.8===
+
-
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB3K3), Pc promoter(1-18C)+RBS+PbrR(PSB1C3)
+
-
===10.9===
+
-
PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag with eight different Pc promoter
+
-
forward primer and Univ reverse primer
+
-
 
+
-
Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag, but the
+
-
product of Pc promoter(J23109) had been failed.
+
-
 
+
-
Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23112/J23114/J23117)+Pag(insert) and PSB1C3(backbone)
+
-
===10.10===
+
-
PCR Pc promoter(J23109)+Pag with Pc promoter(J23109) forward primer and Univ reverse primer
+
-
 
+
-
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23112/J23114/J23117)+Pag( PSB1C3)
+
-
 
+
-
Bacterial colonies PCR and Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag( PSB1C3)
+
-
 
+
-
Retrieve the PCR product of Pc promoter(J231097)+Pag
+
-
===10.11===
+
-
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag( PSB1C3)
+
-
 
+
-
Ligation of Pc promoter(J23109)+Pag(insert) and PSB1C3(backbone)
+
-
 
+
-
Transformation of Pc promoter(J23109)+Pag(PSB1C3)
+
-
===10.12-13===
+
-
Change the backbone of parts I had constructed from PSB1A3 to PSB1C3
+
-
===10.14===
+
-
PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR with eight different Pc promoter forward primer and PbrR reverse primer for the second time
+
-
 
+
-
Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR
+
-
 
+
-
Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3(backbone)
+
-
 
+
-
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3(backbone)
+
-
===10.15===
+
-
Bacterial colonies PCR and Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR( PSB1C3)
+
-
===10.16===
+
-
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR( PSB1C3)
+
-
 
+
-
PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J 23114)+Pag with six different Pc promoter forward primer and Univ reverse primer
+
-
 
+
-
Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag
+
-
 
+
-
Ligation of Pc promoter Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag (insert)+PSB1C3(backbone)
+
-
 
+
-
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3)
+
-
===10.17===
+
-
Bacterial colonies PCR and amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3), but
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the amplification had faild.
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===10.18===
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Bacterial colonies PCR and amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3), but the amplification had faild again.
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===10.20===
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Ligation of Pc promoter Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag (insert)+PSB1C3(backbone) again
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Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3)For the second time
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===10.21===
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Bacterial colonies PCR and amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3)
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===10.25===
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Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3)
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Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3) into strain with Psid-GFP to gain the expression of GFP
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===10.26===
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Detect the expression level of GFP in eight different strains
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Revision as of 04:45, 27 October 2010




   Tianze Zhu's Notes
                                                                                                                                                goto his page
My work is to find a way to achieve optimization of preservation of bioreporter bacteria. Utilizing preservation media and room temperature water pump to achieve a glassy form of bacteria sample and preserve in fridge.The other part of my work is to test and get the data of the dryed samples to see if the method has fulfilled our goal to preserve bacteria.

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Contents

Contents

Week 1st

Get the vector with Psal promoter and double digest it with X,P.

Get the insert of GFP gene and double digest it with S,P

Ligase the two part together in order to cnstruct the plasmid of Psal promoter and GFP,

Transfer the plasmid into trans 5a bacteria

Get the vector with Pbad promoter and double digest it with X,P.

Get the insert of GFP gene and double digest it with S,P

Ligase the two part together in order to cnstruct the plasmid of Pbad promoter and GFP,

Transfer the plasmid into trans 5a bacteria


Week 2nd

Get the vector with T7 promoter and double digest it with X,P.

Get the insert of GFP gene and double digest it with S,P

Ligase the two part together in order to cnstruct the plasmid of T7 promoter and GFP,

Transfer the plasmid into trans 5a bacteria


Week 3rd

Make the different types of protection media with gradient of trehalose PVP.


Week 4th

Use IPTG to induce the fresh cell containing plasmid of T7 promoter and GFP gene to express GFP.]

Detect it with flow cytometry assay and enzyme-linked immunoadsordent assay.

Determine the respone curve of the cell, and the intensity of GFP expression level.


Week 5th

Transfer the cells of T7 promoter and GFP, Mer promoter and GFP into protection media and dry them in the water pump. Store in fringe in 4°C.


Week 6th

ShangHai EXPO


Week 7th

Recover the stored cells and use cytometry assay and enzyme-linked immunoadsordent assay, the same methods used in fresh cells to determine the same parameter.


Week 8th

Recover the stored cells and use cytometry assay and enzyme-linked immunoadsordent assay, the same methods used in fresh cells to determine the same parameter.


Week 9th

Recover the stored cells and use cytometry assay and enzyme-linked immunoadsordent assay, the same methods used in fresh cells to determine the same parameter.


Week 10th

Collect the figure and data achieved in the previous three weeks to see how the condition and vigor of cells change.

Determine which kind of protection media is the best.