Team:SEU A/Experiment/proof2

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       <a href="https://2012.igem.org/Team:SEU_A/Experiment/proof4"><img src='https://static.igem.org/mediawiki/2012/8/8a/Seua_exp_death.png' width='200' height='200'></img></a>
       <a href="https://2012.igem.org/Team:SEU_A/Experiment/proof4"><img src='https://static.igem.org/mediawiki/2012/8/8a/Seua_exp_death.png' width='200' height='200'></img></a>
</center>
</center>
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       <h2>AAA</h2></br>
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       <h2>The confirmation experiment of Sweet gene.</h2></br>
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</html>[[Media:The confirmation experiment of Sweet gene.pdf |The confirmation experiment of Sweet gene.pdf]]<html>
      
      
       We get the Sweet gene groE which could help E-coli folding the protein correctly at high temperature from the genome of BL21. Caring this protein would make the host bacteria grow better at high temperature.</br>
       We get the Sweet gene groE which could help E-coli folding the protein correctly at high temperature from the genome of BL21. Caring this protein would make the host bacteria grow better at high temperature.</br>
 +
 +
<img src='https://static.igem.org/mediawiki/2012/8/8d/Sweet_gene1.jpg'></img>
   
   
       Experimental procedure:</br>
       Experimental procedure:</br>
Line 81: Line 88:
Step 4: add 5ul bacterial liquid which has been diluted  times to liquid Luria-Bertani medium .Set temperature control. After cultivating for 12 to 16h, measure the OD600 of bacterial liquid.</br></br>
Step 4: add 5ul bacterial liquid which has been diluted  times to liquid Luria-Bertani medium .Set temperature control. After cultivating for 12 to 16h, measure the OD600 of bacterial liquid.</br></br>
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<center></br><img src='https://static.igem.org/mediawiki/2012/thumb/f/f9/Sweet_gene_Fig.1.jpg/800px-Sweet_gene_Fig.1.jpg'></img></br></center>
   
   
Fig1. The experimental procedure of Sweet gene’s confirmatory experiment
Fig1. The experimental procedure of Sweet gene’s confirmatory experiment
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The data of Sweet gene’s confirmation experiment version 1.0</br>
The data of Sweet gene’s confirmation experiment version 1.0</br>
      
      
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      Cell
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<center></br><img src='https://static.igem.org/mediawiki/2012/thumb/b/bb/Sweet_gene.Fig2.jpg/800px-Sweet_gene.Fig2.jpg'></img></br></center>
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OD600 groE Blank
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0.038 0.018
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0.023 0.022
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0.026 0.040
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0.016 0.014
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0.042 0.023
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0.032 0.018
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0.030 0.031
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0.036 0.030
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0.018 0.027
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Average value 0.029 0.025
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Fig2. The initial OD600 of the groE and Blank
Fig2. The initial OD600 of the groE and Blank
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Tem
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<center></br><img src='https://static.igem.org/mediawiki/2012/thumb/2/2b/Sweet_gene.Fig3.jpg/800px-Sweet_gene.Fig3.jpg'></img></br></center>
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Cell
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37℃  
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42℃
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groE
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（IPTG-） 0.071/0.065/0.057 0.034/0.041/0.037
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groE
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(IPTG+) 0.054/0.079/0.065 0.041/0.050/0.049
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Blank
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（IPTG-） 0.078/0.093/0.062 0.036/0.048/0.038
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Blank
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(IPTG+) 0.056/0.070/0.072 0.034/0.040/0.040
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Fig3. The OD600 of the E.coli after 16h
Fig3. The OD600 of the E.coli after 16h
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Tem
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<center></br><img src='https://static.igem.org/mediawiki/2012/thumb/9/9a/Sweet_gene.Fig4.jpg/800px-Sweet_gene.Fig4.jpg'></img></br></center>
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Cell
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37℃  
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42℃
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groE
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（IPTG-） 0.064 0.037
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groE
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(IPTG+) 0.066 0.047
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Blank
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（IPTG-） 0.078 0.041
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Blank
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(IPTG+) 0.066 0.038
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Fig4. The average value of the OD600 of fig 2
Fig4. The average value of the OD600 of fig 2
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The data of Sweet gene’s confirmation experiment version 2.0
The data of Sweet gene’s confirmation experiment version 2.0
The initial OD600 of the g+p ( groE on the backbone of pET32a) and p( plasmid pET32a)
The initial OD600 of the g+p ( groE on the backbone of pET32a) and p( plasmid pET32a)
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Cell OD600
 
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      g+p 0.123/0.138/0.135
 
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      p 0.270/0.262/0.256
 
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<center></br><img src='https://static.igem.org/mediawiki/2012/c/ca/Seua_Sweet_gene.5.jpg'></img></br></center>
After 4 h, we measure the OD600 of each kind of cell liquid:</br>
After 4 h, we measure the OD600 of each kind of cell liquid:</br>
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Cell volume of  bacterial liquid /ul
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<center></br><img src='https://static.igem.org/mediawiki/2012/thumb/8/83/Sweet_gene.6.jpg/600px-Sweet_gene.6.jpg'></img></br></center>
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37℃
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42℃
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g+p
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（IPTG-） 5 0.094/0.139/0.061 0.061/0.077/0.046/0.049
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10 0.078/0.069/0.157 0.048/0.046/0.052
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15 0.059/0.057/0.091 0.075/0.051/0.049
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g+p
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(0.8mM) 5 0.020/0.024/0.009 0.017/0.029/0.020
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10 0.015/0.035/0.016 0.021/0.027/0.029
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15 0.032/0.044/0.029 0.025/0.026/0.033
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g+p
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(1.6mM) 5 0.019/0.019/0.014 0.020/0.025/0.013/0.018
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10 0.024/0.024/0.027 0.057/0.026/0.035
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15 0.019/0.016/0.024 0.015/0.019/0.024/0.025
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P
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(IPTG-) 5 0.064/0.080/0.064 0.030/0.042/0.038
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10 0.069/0.055/0.064 0.034/0.039/0.040
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15 0.067/0.053/0.053 0.039/0.035/0.051
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-
P
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(0.8mM) 5 0.054/0.059/0.054 0.036/0.030/0.028/0.043
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-
10 0.066/0.050/0.061 0.049/0.029/0.083/0.035
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-
15 0.063/0.067/0.061 0.040/0.033/0.026
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-
P
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(1.6mM) 5 0.082/0.055/0.048/0.057 0.044/0.052/0.052
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-
10 0.051/0.077/0.065 0.068/0.057/0.047
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-
15 0.061/0.053/0.062 0.063/0.053/0.055
+
Dealing with these data, we got the results as follows:
Dealing with these data, we got the results as follows:
 +
<center></br><img src='https://static.igem.org/mediawiki/2012/thumb/b/bd/Sweet_gene.7.jpg/569px-Sweet_gene.7.jpg'></img></br></center>
 +
Averaging the different volume, we have a more visual figure. The Sweet gene plays a powerful role in the temperature struggle.</br>
 +
<img src='https://static.igem.org/mediawiki/2012/7/77/Sweet_gene.8.jpg'></img>
 +
After 17 hours:</br>
 +
<center></br><img src='https://static.igem.org/mediawiki/2012/thumb/c/c0/Sweet_gene.9.jpg/600px-Sweet_gene.9.jpg'></img></br></center>
 +
The average of data after 17h:</br>
-
 
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<center></br><img src='https://static.igem.org/mediawiki/2012/thumb/b/bc/Sweet_gene.10.jpg/504px-Sweet_gene.10.jpg'></img></br></center>
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-
 
+
-
 
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Cell volume of  bacterial liquid  /ul 37℃ 42℃ 42℃/37℃
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-
g+p
+
-
（IPTG-） 5 0.098 0.058 59.18%
+
-
10 0.101 0.049 48.36%
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-
15 0.069 0.058 84.06%
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g+p
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(0.8mM) 5 0.018 0.022 124.53%
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10 0.022 0.026 118.18%
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15 0.035 0.028 80.00%
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g+p
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(1.6mM) 5 0.017 0.019 109.62%
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10 0.025 0.039 156.00%
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15 0.020 0.021 106.78%
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P
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(IPTG-) 5 0.069 0.037 53.37%
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-
10 0.063 0.038 60.64%
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15 0.058 0.042 72.83%
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-
P
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-
(0.8mM) 5 0.056 0.034 61.08%
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-
10 0.059 0.049 83.05%
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15 0.064 0.033 51.83%
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P
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(1.6mM) 5 0.053 0.049 91.88%
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-
10 0.064 0.057 88.60%
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-
15 0.059 0.057 97.16%
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-
 
+
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+
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-
 
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-
 
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-
 
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-
 
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-
 
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-
 
+
-
 
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-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
Averaging the different volume, we have a more visual figure. The Sweet gene plays a powerful role in the temperature struggle。
+
-
 
+
-
+
-
 
+
-
 
+
-
After 17 hours:
+
-
 
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Cell volume of  bacterial liquid/ul
+
-
37℃
+
-
42℃
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g+p
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-
（IPTG-） 5 0.054/0.060/0.125 0.055/0.049/0.059
+
-
10 0.110/0.089/0.076 0.028/0.037/0.102/0.039
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-
15 0.124/0.156/0.144 0.285/0.248.0.241/0.272
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-
g+p
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-
(0.8mM) 5 0.048/0.053/0.100 0.028/0.061/0.047
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-
10 0.045/0.043/0.038 0.066/0.074/0.035
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-
15 0.070/0.086/0.065 0.043/0.043/0.074
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-
g+p
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-
(1.6mM) 5 0.080/0.160/0.091 0.048/0.045/0.060
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-
10 0.174/0.110/0.087 0.045/0.050/0.082/0.057
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-
15 0.225/0.114/0.103 0.066/0.057/0.062
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-
P
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-
(IPTG-) 5 0.086/0.126/0.092 0.125/0.059/0.061
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-
10 0.095/0.079/0.081/0.078 0.068/0.061/0.074
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-
15 0.098/0.092/0.076 0.072/0.133/0.079
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-
P
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-
(0.8mM) 5 0.138/0.138/0.138 0.078/0.067/0.062
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-
10 0.111/0.093/0.085 0.143/0.071/0.070
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-
15 0.072/0.076/0.062 0.092/0.058/0.049
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-
P
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-
(1.6mM) 5 0.126/0.082/0.078 0.062/0.065/0.079
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-
10 0.118/0.094/0.103 0.061/0.072/0.073
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-
15 0.086/0.059/0.043 0.065/0.097/0.061
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-
 
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-
The average of data after 17h:
+
-
 
+
-
 
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-
Cell volume of  bacterial liquid/ul 37℃ 42℃ 42℃/37℃
+
-
g+p(IPTG-) 5 0.08 0.054 67.50%
+
-
10 0.092 0.035 38.04%
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-
15 0.141 0.262 185.82%
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-
g+p(0.8mM) 5 0.067 0.045 67.16%
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-
10 0.042 0.058 138.10%
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-
15 0.074 0.053 71.62%
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-
g+p(1.6mM) 5 0.11 0.051 46.36%
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-
10 0.124 0.051 41.13%
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15 0.147 0.062 42.18%
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p(IPTG-) 5 0.101 0.082 81.19%
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10 0.083 0.068 81.93%
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-
15 0.089 0.095 106.74%
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p(0.8mM) 5 0.138 0.069 50.00%
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10 0.096 0.095 98.96%
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15 0.07 0.066 94.29%
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p(1.6mM) 5 0.095 0.069 72.63%
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-
10 0.105 0.069 65.71%
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-
15 0.063 0.074 117.46%
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After 17h, we found a strange phenomenon. The g+p lives better at 42℃ without IPTG, and we don’t know the reason. Besides, the Sweet gene still works in a temperature struggle.
After 17h, we found a strange phenomenon. The g+p lives better at 42℃ without IPTG, and we don’t know the reason. Besides, the Sweet gene still works in a temperature struggle.
-
   
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  <center></br><img src='https://static.igem.org/mediawiki/2012/4/43/Sweet_gene.11.jpg'></img></br></center>
After 45h：
After 45h：
-
Cell volume of  bacterial liquid /ul
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<center></br> <img src='https://static.igem.org/mediawiki/2012/thumb/b/ba/Sweet_gene.12.jpg/605px-Sweet_gene.12.jpg'></img></br></center>
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37℃  
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42℃
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g+p
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-
（ IPTG-） 5 0.036/0.070/0.048 0.086/0.078/0.083
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-
10 0.072/0.049/0.047 0.039/0.067/0.050
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-
15 0.061/0.055/0.060 0.061/0.052/0.060
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g+p
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-
(0.8mM) 5 0.080/0.069/0.058 0.039/0.044/0.033
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-
10 0.067/0.067/0.052 0.037/0.056/0.043
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-
15 0.115/0.048/0.073 0.048/0.044/0.049
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g+p
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-
(1.6mM) 5 0.072/0.061/0.070 0.041/0.059/0.039
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-
10 0.048/0.053/0.059 0.071/0.051/0.046
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-
15 0.104/0.032/0.042 0.038/0.037/0.036
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P
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( IPTG-) 5 0.068/0.097/0.082 0.034/0.051/0.045
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10 0.059/0.131/0.106 0.042/0.023/0.032
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-
15 0.078/0.075/0.144 0.046/0.043/0.046
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P
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(0.8mM) 5 0.049/0.066/0.080 0.060/0.039/0.041
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-
10 0.056/0.056/0.060 0.042/0.050/0.040
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-
15 0.039/0.144/0.050 0.040/0.037/0.042
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P
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-
(1.6mM) 5 0.050/0.102/0.085 0.051/0.032/0.035
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-
10 0.050/0.097/0.173/0.051 0.030/0.027/0.035
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15 0.066/0.066/0.065 0.033/0.061/0.042
+
 +
The average of data after 45h:
-
The average of data after 45h:
 
 +
<center></br><img src='https://static.igem.org/mediawiki/2012/thumb/d/d4/Sweet_gene.13.jpg/542px-Sweet_gene.13.jpg'></img></br></center>
-
Cell volume of  bacterial liquid/ul
+
After 45h, the strange phenomenon became stranger. There was no regularity anymore. The Sweet gene was no longer performing its function.  
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37℃
+
-
42℃ 42℃/37℃
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-
g+p(IPTG-) 5 0.051 0.054 106.54%
+
-
10 0.056 0.052 91.96%
+
-
15 0.059 0.262 443.22%
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-
g+p(0.8mM) 5 0.069 0.045 65.70%
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-
10 0.062 0.058 94.09%
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-
15 0.079 0.053 67.51%
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-
g+p(1.6mM) 5 0.068 0.051 75.00%
+
-
10 0.053 0.059 110.38%
+
-
15 0.059 0.062 104.52%
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-
p(IPTG-) 5 0.082 0.082 99.59%
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-
10 0.099 0.068 68.35%
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-
15 0.099 0.095 95.62%
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-
p(0.8mM) 5 0.065 0.069 106.15%
+
-
10 0.057 0.095 166.08%
+
-
15 0.078 0.066 85.04%
+
-
p(1.6mM) 5 0.079 0.069 86.92%
+
-
10 0.093 0.069 73.84%
+
-
15 0.066 0.074 112.63%
+
-
After 45h, the strange phenomenon became stranger. There was no regularity anymore. The Sweet gene was no longer performing its function.  
+
<center></br><img src='https://static.igem.org/mediawiki/2012/b/b0/Sweet_gene.14.jpg'></img></br></center>
   
   
We still compared the OD600 of one type of bacteria while time elapses.
We still compared the OD600 of one type of bacteria while time elapses.
Line 359: Line 152:
The volume of bacterial liquid is 5 ul.  
The volume of bacterial liquid is 5 ul.  
-
   
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  <center></br><img src='https://static.igem.org/mediawiki/2012/3/34/Sweet_gene.15.jpg'></img></br></center>
The volume of bacterial liquid is 10 ul.
The volume of bacterial liquid is 10 ul.
-
   
+
  <center></br><img src='https://static.igem.org/mediawiki/2012/e/e8/Sweet_gene.16.jpg'></img></br></center>
The volume of bacterial liquid is 15 ul.
The volume of bacterial liquid is 15 ul.
                  
                  
 +
<center></br><img src='https://static.igem.org/mediawiki/2012/d/db/Sweet_gene.17.jpg'></img></br></center>
 +
From these figures, we still got the same result as the data above.
From these figures, we still got the same result as the data above.

Latest revision as of 03:48, 27 September 2012

iGEM 2012 SEU_A Human Practice


Sweet gene

The confirmation experiment of Sweet gene.

The confirmation experiment of Sweet gene.


The confirmation experiment of Sweet gene.pdf We get the Sweet gene groE which could help E-coli folding the protein correctly at high temperature from the genome of BL21. Caring this protein would make the host bacteria grow better at high temperature.
Experimental procedure:
Step 1: recover groE and Blank ( E.coli without sweet gene),and shake them at 37℃ for 12 to 16h at 150rpm.
Step 2: measure the OD600 of bacterial liquid .
Step 3: use 50ul bacterial liquid which has been diluted times to coat plates. Set temperature and induced factor (IPTG, 1 mM) control.
Step 4: add 5ul bacterial liquid which has been diluted times to liquid Luria-Bertani medium .Set temperature control. After cultivating for 12 to 16h, measure the OD600 of bacterial liquid.



Fig1. The experimental procedure of Sweet gene’s confirmatory experiment The data of Sweet gene’s confirmation experiment version 1.0


Fig2. The initial OD600 of the groE and Blank


Fig3. The OD600 of the E.coli after 16h


Fig4. The average value of the OD600 of fig 2 The data of Sweet gene’s confirmation experiment version 2.0 The initial OD600 of the g+p ( groE on the backbone of pET32a) and p( plasmid pET32a)


After 4 h, we measure the OD600 of each kind of cell liquid:


Dealing with these data, we got the results as follows:


Averaging the different volume, we have a more visual figure. The Sweet gene plays a powerful role in the temperature struggle.
After 17 hours:


The average of data after 17h:


After 17h, we found a strange phenomenon. The g+p lives better at 42℃ without IPTG, and we don’t know the reason. Besides, the Sweet gene still works in a temperature struggle.


After 45h：


The average of data after 45h:


After 45h, the strange phenomenon became stranger. There was no regularity anymore. The Sweet gene was no longer performing its function.


We still compared the OD600 of one type of bacteria while time elapses. The volume of bacterial liquid is 5 ul.


The volume of bacterial liquid is 10 ul.


The volume of bacterial liquid is 15 ul.


From these figures, we still got the same result as the data above.
iGEM 2012 Edit Top



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