Team:HIT-Harbin/Experiments
From 2013.igem.org
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+ | <h4 class="hashed"><span>Experiment Schedule</span></h4> | ||
<p></p>JuL. 10~15 Preparation of media, competent cells and experimental reagents | <p></p>JuL. 10~15 Preparation of media, competent cells and experimental reagents | ||
<p></p>JuL. 16~20 Preparation of parts from iGEM | <p></p>JuL. 16~20 Preparation of parts from iGEM | ||
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<p></p>Aug. 29~Sep. 15 Test of the device | <p></p>Aug. 29~Sep. 15 Test of the device | ||
<p></p>Sep. 16~25 Remaining experiments | <p></p>Sep. 16~25 Remaining experiments | ||
+ | <div align="center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/6/6b/HIT-Harbin_team_HIT.png"/> | ||
+ | <p>Fig: Agarose electrophoresis for our parts and maker forming "HIT"</p> | ||
+ | </div> | ||
+ | <p>Part A :Plasmid carrying Plac+RBS+hrpR+T</p> | ||
+ | <p>Part B :Plasmid carrying Ptet+RBS+hrpS+T</p> | ||
+ | <p>Part C :Plasmid carrying PhrpL+RBS+RFP+T</p> | ||
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+ | <h4 class="hashed"><span>Ligation for our device</span></h4> | ||
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<img src="https://static.igem.org/mediawiki/2013/4/45/Experiment2.png"/> | <img src="https://static.igem.org/mediawiki/2013/4/45/Experiment2.png"/> | ||
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- | <p> | + | <h4 class="hashed"><span>1.Test of device</span></h4> |
+ | <p></p>1)Preparation of IPTG solution: add 240mg IPTG powder into 10mL dd H2O. We filtrated the solution to sterilize it and broke it into EP tubes. The concentration is 24mg/mL (100mM/mL). then we stored them in -20℃. | ||
+ | <p></p>2)To test the four different combination according to the strength of promoters, we made different concentrations of IPTG for the bacteria. | ||
+ | <div align="center"> | ||
+ | <p> Table 1 IPTG formula</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/a/a9/Experiment4.png"/> | ||
+ | </div> | ||
+ | <p></p>We grew the IPTG-added culture in 37℃ at 120rpm, overnight. Unfortunately, we haven’t observed the expected color of RFP (Red Fluorescence Protein). | ||
+ | <div align="center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/c/c2/Experiment5.png"/> | ||
+ | <p>Fig 3. No red after night</p> | ||
+ | </div> | ||
+ | <p></p>3)After that, we tested the reporting sub-circuit of our device. We connect the constitutive promoter PLac with RBS+RFP+T, transformed the bacteria and grew it overnight. The red in the results proved that the reporting sub-circuit is working. | ||
+ | <div align="center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/1/10/Experiment6.png"/> | ||
+ | <p></p>Fig 4. Constitutive promoter expressing RFP | ||
+ | <p></p>(Left is 12h, right is 24h) | ||
+ | </div> | ||
+ | <p></p>4)We sent our whole device to companies for sequencing, but it failed. | ||
+ | <img src="https://static.igem.org/mediawiki/2013/7/71/HIT-Harbin_team.png"/> | ||
+ | <p>Fig: the happy moment when we saw the culture becoming red for the first time</p> | ||
+ | |||
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- | + | <h4 class="hashed"><span>2.Investigating the relationship between the concentration of RFP with that of IPTG</span></h4> | |
+ | <p></p>1)Measuring absorbance of RFP | ||
+ | <p></p>We grew bacteria without device and bacteria with our device in same volume until stationary phase. Taking bacteria without device as background, we measured the absorbance of bacteria with our device (the max absorption peak is 504nm).But absorbance in 504nm is higher than 1,which present a bad linear relation between absorbance and concentraton. RFP has absorption in 450nm,and absorbance is between 0.1 and 1(better linear relation).Occasionally, we find a RFP standard curve under 450nm on the web. it was very lucky compared with our failure in testing our device. | ||
+ | Before the mensuration, we diluted the two groups according to table2. We took the mean of two measures as the useful data. | ||
+ | <div align="center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/8/8f/Experiment8.png"/> | ||
+ | <p>Fig.5 RFP absorbance varying with wave length</p> | ||
+ | <p>Table 2 Dilution of Two groups of bacteria</p> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/2/21/Experiment7.png"/> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/7/74/Experiment9.png"/> | ||
+ | <p>Fig 6. The relationship between RFP concentration and absorbance(OD450)</p> | ||
+ | </div> | ||
+ | |||
+ | <p></p>2)The actual relationship between RFP concentration and absorbance | ||
+ | <div align="center"> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/8/88/Experiment10.png"/> | ||
+ | <p>Fig 7. RFP standard curve obtain from the web,<a href="http://www.cellbiolabs.com/sites/default/files/AKR-122-rfp-elisa-kit.pdf">Click here</a> | ||
+ | </div> | ||
+ | <p></p>Through the standard curve, we can convert the relative concentration to the absolute concentration, and finally get the relationship between IPTG concentration and RFP concentration. | ||
+ | <p></p>Compared to crushing cells to separate RFP, our method is simpler and easy to practice. Moreover, our relative concentration curve is credible. If the standard curve is reliable, our calculated result of RFP will be precise. | ||
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Latest revision as of 14:26, 27 September 2013
Project/experiments
Experiment Schedule
JuL. 10~15 Preparation of media, competent cells and experimental reagents JuL. 16~20 Preparation of parts from iGEM JuL. 21~31 Respective ligation of strong, intermediate and weak RBS with sub-circuit hrpR/hrpS/tet/RFP Aug. 1~10 Ligation of terminators Aug. 11~20 Successful ligation of the four sub-circuits Aug. 21~28 Combination of sub-circuits: the device Aug. 29~Sep. 15 Test of the device Sep. 16~25 Remaining experiments![](https://static.igem.org/mediawiki/2013/6/6b/HIT-Harbin_team_HIT.png)
Fig: Agarose electrophoresis for our parts and maker forming "HIT"
Part A :Plasmid carrying Plac+RBS+hrpR+T
Part B :Plasmid carrying Ptet+RBS+hrpS+T
Part C :Plasmid carrying PhrpL+RBS+RFP+T
Ligation for our device
![](https://static.igem.org/mediawiki/2013/4/45/Experiment2.png)
Fig 1. PCR resuLts of our own parts
(1: hrpL; 2: hrpS; 3: hrpR; 4: Ptet +strong RBS+hrpS+T; 5:Ptet+intermediate RBS+hrpS+T; 6: Ptet+weak RBS+hrpS+T; 7: PIPTG +strong RBS+hrpR+T; 8 is not needed; 9: PIPTG +weak RBS+hrpR+T; 10: PhrpL+strong RBS+tetR+T; 11: PhrpL+intermediate RBS+tetR+T; 12: PhrpL+weak RBS+tetR+T; 13: PhrpL+strong RBS+RFP+T; 14: PhrpL+weak RBS+RFP+T; 15: PhrpL+weak RBS+RFP+T)
![](https://static.igem.org/mediawiki/2013/c/ca/Experiment3.png)
Fig 1.EcoR1 and Pst1 double restriction enzyme cleavage for hrpL AND gate(BBa_K1014014) device and B-POM1(BBa_K1014999)
1:plasmid carrying BBa_K1014014; 2:double restriction enzyme cleavage for 1; 3:plasmid carrying BBa_K1014999; 4:double restriction enzyme cleavage for 3
1.Test of device
1)Preparation of IPTG solution: add 240mg IPTG powder into 10mL dd H2O. We filtrated the solution to sterilize it and broke it into EP tubes. The concentration is 24mg/mL (100mM/mL). then we stored them in -20℃. 2)To test the four different combination according to the strength of promoters, we made different concentrations of IPTG for the bacteria.Table 1 IPTG formula
![](https://static.igem.org/mediawiki/2013/a/a9/Experiment4.png)
![](https://static.igem.org/mediawiki/2013/c/c2/Experiment5.png)
Fig 3. No red after night
![](https://static.igem.org/mediawiki/2013/1/10/Experiment6.png)
![](https://static.igem.org/mediawiki/2013/7/71/HIT-Harbin_team.png)
Fig: the happy moment when we saw the culture becoming red for the first time
2.Investigating the relationship between the concentration of RFP with that of IPTG
1)Measuring absorbance of RFP We grew bacteria without device and bacteria with our device in same volume until stationary phase. Taking bacteria without device as background, we measured the absorbance of bacteria with our device (the max absorption peak is 504nm).But absorbance in 504nm is higher than 1,which present a bad linear relation between absorbance and concentraton. RFP has absorption in 450nm,and absorbance is between 0.1 and 1(better linear relation).Occasionally, we find a RFP standard curve under 450nm on the web. it was very lucky compared with our failure in testing our device. Before the mensuration, we diluted the two groups according to table2. We took the mean of two measures as the useful data.![](https://static.igem.org/mediawiki/2013/8/8f/Experiment8.png)
Fig.5 RFP absorbance varying with wave length
Table 2 Dilution of Two groups of bacteria
![](https://static.igem.org/mediawiki/2013/2/21/Experiment7.png)
![](https://static.igem.org/mediawiki/2013/7/74/Experiment9.png)
Fig 6. The relationship between RFP concentration and absorbance(OD450)
![](https://static.igem.org/mediawiki/2013/8/88/Experiment10.png)
Fig 7. RFP standard curve obtain from the web,Click here