Team:HIT-Harbin/Experiments

From 2013.igem.org

(Difference between revisions)
 
(23 intermediate revisions not shown)
Line 27: Line 27:
                 <div class="mainarea">
                 <div class="mainarea">
                     <div class="multi-columns">
                     <div class="multi-columns">
-
<h4 class="hashed"><span>The circuit</span></h4>
 
                         <div class="half">
                         <div class="half">
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
+
<h4 class="hashed"><span>Experiment Schedule</span></h4>
-
                        </div>
+
                          <p></p>JuL. 10~15 Preparation of media, competent cells and experimental reagents
-
                        <div class="half last">
+
<p></p>JuL. 16~20 Preparation of parts from iGEM
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
+
<p></p>JuL. 21~31 Respective ligation of strong, intermediate and weak RBS with sub-circuit hrpR/hrpS/tet/RFP
-
                        </div>
+
<p></p>Aug. 1~10 Ligation of terminators
-
                    </div>
+
<p></p>Aug. 11~20 Successful ligation of the four sub-circuits
 +
<p></p>Aug. 21~28 Combination of sub-circuits: the device
 +
<p></p>Aug. 29~Sep. 15 Test of the device
 +
<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>
-
                    <div class="multi-columns">
 
-
<h4 class="hashed"><span>The circuit</span></h4>
 
-
                        <div class="third">
 
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
 
-
                        </div>
 
-
                        <div class="third">
 
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
 
-
                        </div>
 
-
                        <div class="third last">
 
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
 
-
                        </div>
 
-
                    </div>
 
-
                    <div class="multi-columns">
 
-
<h4 class="hashed"><span>The circuit</span></h4>
 
-
 
-
                        <div class="fourth">
 
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
 
                         </div>
                         </div>
-
                         <div class="fourth">
+
                         <div class="half last">
-
                             <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
+
<h4 class="hashed"><span>Ligation for our device</span></h4>
-
                        </div>
+
                             <div align="center">
-
                        <div class="fourth">
+
                              <img src="https://static.igem.org/mediawiki/2013/4/45/Experiment2.png"/>
-
                             <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
+
                              <p>Fig 1. PCR resuLts of our own parts</p>
-
                        </div>
+
                            </div>
-
                        <div class="fourth last">
+
                            <p>(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)</p>
-
                             <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum.</p>
+
                            <div align="center">
 +
                             <img src="https://static.igem.org/mediawiki/2013/c/ca/Experiment3.png"/>
 +
                            <p>Fig 1.EcoR1 and Pst1 double restriction enzyme cleavage for hrpL AND gate(BBa_K1014014) device and B-POM1(BBa_K1014999) </p>
 +
                            </div>  
 +
                             <p>1:plasmid carrying BBa_K1014014; 2:double restriction enzyme cleavage for 1; 3:plasmid carrying BBa_K1014999; 4:double restriction enzyme cleavage for 3</p>
                         </div>
                         </div>
                     </div>
                     </div>
-
 
+
<div class="multi-columns">
-
                    <div class="multi-columns">
+
-
 
+
-
<h4 class="hashed"><span>The circuit</span></h4>
+
-
                        <div class="fourth">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
-
                        </div>
+
                         <div class="half">
                         <div class="half">
-
                             <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.</p>
+
<h4 class="hashed"><span>1.Test of device</span></h4>
-
                        </div>
+
                             <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℃.
-
                        <div class="fourth last">
+
<p></p>2)To test the four different combination according to the strength of promoters, we made different concentrations of IPTG for the bacteria.
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
<div align="center">
-
                        </div>
+
<p> Table 1 IPTG formula</p>
-
                    </div>
+
<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>
-
                    <div class="multi-columns">
 
-
                        <div class="half">
 
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.</p>
 
-
                        </div>
 
-
                        <div class="fourth">
 
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
 
-
                        </div>
 
-
                        <div class="fourth last">
 
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
 
-
                        </div>
 
-
                    </div>
 
-
 
-
                    <div class="multi-columns">
 
-
                        <div class="fourth">
 
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
 
-
                        </div>
 
-
                        <div class="fourth">
 
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
 
                         </div>
                         </div>
                         <div class="half last">
                         <div class="half last">
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum. Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.</p>
+
<h4 class="hashed"><span>2.Investigating the relationship between the concentration of RFP with that of IPTG</span></h4>
-
                        </div>
+
<p></p>1)Measuring absorbance of RFP
-
                    </div>
+
<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.
-
                    <div class="multi-columns">
+
                         
-
                        <div class="third">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua.</p>
+
-
                        </div>
+
-
                        <div class="twothirds last">
+
-
                            <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.</p>
+
                         </div>
                         </div>
                     </div>
                     </div>
 +
<div class="multi-columns">
-
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum</p>
 
-
<p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum</p>
 
-
                  <h4 class="hashed"><span>Image positioning</span></h4>
 
-
                    <div class="preload alignleft">
 
-
                        <img src="https://static.igem.org/mediawiki/2013/2/27/099.png" alt="" />
 
-
                    </div>
 
-
                    <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum</p>
 
-
                    <p>Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem.</p>
 
-
 
-
                    <div class="separator"></div>
 
-
 
-
                    <div class="preload alignright">
 
-
                        <img src="https://static.igem.org/mediawiki/2013/2/27/099.png" alt="" />
 
-
                    </div>
 
-
                    <p>Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum</p>
 
-
                    <p>Sed ut perspiciatis unde omnis iste natus error sit voluptatem accusantium doloremque laudantium, totam rem aperiam, eaque ipsa quae ab illo inventore veritatis et quasi architecto beatae vitae dicta sunt explicabo. Nemo enim ipsam voluptatem quia voluptas sit aspernatur aut odit aut fugit, sed quia consequuntur magni dolores eos qui ratione voluptatem sequi nesciunt. Neque porro quisquam est, qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit, sed quia non numquam eius modi tempora incidunt ut labore et dolore magnam aliquam quaerat voluptatem.</p>
 
</div>
</div>

Latest revision as of 14:26, 27 September 2013

HIT-Harbin

HIT-Harbin

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

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

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)

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

We grew the IPTG-added culture in 37℃ at 120rpm, overnight. Unfortunately, we haven’t observed the expected color of RFP (Red Fluorescence Protein).

Fig 3. No red after night

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.

Fig 4. Constitutive promoter expressing RFP

(Left is 12h, right is 24h)

4)We sent our whole device to companies for sequencing, but it failed.

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.

Fig.5 RFP absorbance varying with wave length

Table 2 Dilution of Two groups of bacteria

Fig 6. The relationship between RFP concentration and absorbance(OD450)

2)The actual relationship between RFP concentration and absorbance

Fig 7. RFP standard curve obtain from the web,Click here

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.

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.

Retrieved from "http://2013.igem.org/Team:HIT-Harbin/Experiments"