Team:NYMU-Taipei/ymiq3.html

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<div class="title">Experiments</div>
<div class="title">Experiments</div>
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   <p><span class="subtitle">The effect of sodium sulfide on Synechococcus SP. PCC 7942 growth rate</span></p>
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   <p><span class="subtitle">Resistance of Cyanobacteria (Synechococcus SP. PCC 7002) to Sulfide compound</span></p>
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   <p>After thoroughly examined the ability of sqr in Synechococcus SP. PCC 7002, we planned to conduct a series of similar experiments on Synechococcus SP. PCC 7942. Except for the cultivation medium, other growing conditions remained the same. Instinctively, the strain expressing sqr should grow better than the wile type strain.<br />
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   <p>Synechococcus sp. PCC7002 was cultured in the medium A2, a modification of Stevens et al. (1973) on a rotary shaker (100 rpm). The initial concentration of cells is controlled to an OD730 of 0.1 in fresh medium. Different concentration of sodium sulfide is added, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was put into experimental group. Cells are grown in the six-well-plates and monitored under OD730 every 24 hour.<br />
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     <p><span class="subtitle">DCMU concentration and cell growth</span></p>
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     <p><span class="subtitle">Resistance of Synechococcus SP. PCC 7002 to 3 - (3,4-dichlorophenyl) - 1,1 - dimethylurea</span></p>
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     <p>This experiment is similar to the second one of Synechococcus SP. PCC 7002 testing series. The main idea was to find the suitable DCMU concentration for Synechococcus SP. PCC 7942. As a matter of fact, both wild type and sqr expressing strain are used in the experiment.  <br />
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     <p>Synechococcus sp. PCC7002 was cultured in the medium A2, rotary shaker and initial cell concentration are the same as the last experiment. DCMU is diluted with A2 medium into different concentration, and 10 mM of sodium sulfide is added to the experimental group. The measurement method and frequency of cell growth has been described previously.  <br />
       <br />
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     <div align="left">
     <div align="left">
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       <p><span class="subtitle">Sulfide concentration and the growth of sqr expressing strain Synechococcus SP. PCC 7942</span></p>
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       <p><span class="subtitle">Sodium sulfide concentration and cell growth</span></p>
</div>
</div>
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     <p>It was expected that  SQR expressing strain and wild type counterpart would have different growth  rate under the presence of sulfide compounds. Though sulfide is naturally toxic  to <em>Synechococcus SP. PCC 7942</em>, the  strain with sqr should be able to metabolize sulfide and therefore prosper. As  the result, we analyze H2S amount to detect whether sqr gene work or not. Therefore, we perform Chemical microvolume  turbidimetry method to detect H2S concentration (see <strong>Sulfur Oxide Terminator part</strong>)<br />
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     <p>With the result of previous experiment, the concentration of both sodium sulfide and DCMU is adjusted. Sodium sulfide is diluted from 40mM to 2.5mM, while DCMU 0.5 μM is added into the experimental group.<br />
       <br />
       <br />
     </p>
     </p>
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       <p><span class="subtitle"> Sulfide oxidation in Escherichia coli expressing sulfide-quinone reductase gene</span></p>
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       <p><span class="subtitle">The effect of sodium sulfide on Synechococcus SP. PCC 7942 growth rate</span></p>
</div>
</div>
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Repots have it that Escherichia coli can express functional sulfide-quinone reductase (SQR). Therefore, we slightly adjusted the previous experiment and applied to the SQR gene from Synechococcus SP. PCC 7002. With methylene blue method, we would test the efficiency of SQR sulfide oxidation. Since such method involved in measurement of optical density, it is more appropriate to perform such experiment on colorless bacteria instead of engineered cyanobacteria strain.  
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In this experiment, we aimed to examine whether the sqr Synechococcus SP. PCC 7942 expressed is functional. Synechococcus SP. PCC 7942 with sqr and wild type was cultivated in BG-11 medium (Allen M.M. 1968) on the same rotary shaker described in the first experiment. Initial cell concentration is fixed to an OD730 of 0.1 in the medium. 2.5mM of sodium sulfide is added into the experimental group, and DCMU is diluted from 1μM to 0.5μM.  
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<div align="left">
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      <p><span class="subtitle">DCMU concentration and cell growth</span></p>
 +
</div>
 +
    <p>From the last experiment, we found it necessary to perform another independent experiment to find the optimal DCMU concentration of Synechococcus SP. PCC 7942. In addition, the initial cell concentration was adjusted to an OD730 of 0.2 in BG-11 medium. DCMU is diluted from 1μM to 0.125μM. Except of cell concentration, all condition remained the same for both sqr and wild type strain of Synechococcus SP. PCC 7942.<br />
 +
      <br />
 +
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<div align="left">
 +
      <p><span class="subtitle">Sulfide concentration and the growth of sqr expressing strain Synechococcus SP. PCC 7942</span></p>
 +
</div>
 +
    <p>In this experiment, the BG-11 medium is used and the initial cell concentration is adjusted to an OD 730 of 0.2 as the last test. In addition, the sodium sulfide is diluted from 5mM to 5/16mM, while DCMU 0.25μM is added to every well.<br />
 +
      <br />
 +
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<div align="left">
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      <p><span class="subtitle">Sulfide oxidation in Escherichia coli expressing sulfide-quinone reductase gene</span></p>
 +
</div>
 +
    <p>Both wild type and sqr expressing Escherichia coli was cultivated in LB medium with streptomycin. The initial cell concentration was controlled to an OD600 of 0.2 and grew in the test tube inside incubator of 37 degrees Celsius and 200 rpm. Sodium sulfide was diluted from 200μM to 125μM and added into test tube. The consumption of sulfide was measured every 90 minutes by the methylene blue method (Trüper and Schlegel 1964).<br />
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                 <li><a title="Methods" href="https://2012.igem.org/Team:NYMU-Taipei/ymiq2.html">Methods</a></li>
                 <li><a title="Methods" href="https://2012.igem.org/Team:NYMU-Taipei/ymiq2.html">Methods</a></li>
                 <li><a title="Experiments" href="https://2012.igem.org/Team:NYMU-Taipei/ymiq3.html">Experiments</a></li>
                 <li><a title="Experiments" href="https://2012.igem.org/Team:NYMU-Taipei/ymiq3.html">Experiments</a></li>
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                 <li><a title="Results & References" href="https://2012.igem.org/Team:NYMU-Taipei/ymiq4.html">Results &amp; References</a></li>
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                 <li><a title="Results & References" href="https://2012.igem.org/Team:NYMU-Taipei/ymiq4.html">Results &amp; References</a></li><li><a title="Further Experiments after Asia Jamboree" href="https://2012.igem.org/Team:NYMU-Taipei/ymiq5.html">Further Experiments after Asia Jamboree</a></li>
             </ul>
             </ul>
         </li>
         </li>

Latest revision as of 01:48, 27 October 2012

NYMU iGEM

Experiments

Resistance of Cyanobacteria (Synechococcus SP. PCC 7002) to Sulfide compound

Synechococcus sp. PCC7002 was cultured in the medium A2, a modification of Stevens et al. (1973) on a rotary shaker (100 rpm). The initial concentration of cells is controlled to an OD730 of 0.1 in fresh medium. Different concentration of sodium sulfide is added, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was put into experimental group. Cells are grown in the six-well-plates and monitored under OD730 every 24 hour.

Resistance of Synechococcus SP. PCC 7002 to 3 - (3,4-dichlorophenyl) - 1,1 - dimethylurea

Synechococcus sp. PCC7002 was cultured in the medium A2, rotary shaker and initial cell concentration are the same as the last experiment. DCMU is diluted with A2 medium into different concentration, and 10 mM of sodium sulfide is added to the experimental group. The measurement method and frequency of cell growth has been described previously.

Sodium sulfide concentration and cell growth

With the result of previous experiment, the concentration of both sodium sulfide and DCMU is adjusted. Sodium sulfide is diluted from 40mM to 2.5mM, while DCMU 0.5 μM is added into the experimental group.

The effect of sodium sulfide on Synechococcus SP. PCC 7942 growth rate

In this experiment, we aimed to examine whether the sqr Synechococcus SP. PCC 7942 expressed is functional. Synechococcus SP. PCC 7942 with sqr and wild type was cultivated in BG-11 medium (Allen M.M. 1968) on the same rotary shaker described in the first experiment. Initial cell concentration is fixed to an OD730 of 0.1 in the medium. 2.5mM of sodium sulfide is added into the experimental group, and DCMU is diluted from 1μM to 0.5μM.

DCMU concentration and cell growth

From the last experiment, we found it necessary to perform another independent experiment to find the optimal DCMU concentration of Synechococcus SP. PCC 7942. In addition, the initial cell concentration was adjusted to an OD730 of 0.2 in BG-11 medium. DCMU is diluted from 1μM to 0.125μM. Except of cell concentration, all condition remained the same for both sqr and wild type strain of Synechococcus SP. PCC 7942.

Sulfide concentration and the growth of sqr expressing strain Synechococcus SP. PCC 7942

In this experiment, the BG-11 medium is used and the initial cell concentration is adjusted to an OD 730 of 0.2 as the last test. In addition, the sodium sulfide is diluted from 5mM to 5/16mM, while DCMU 0.25μM is added to every well.

Sulfide oxidation in Escherichia coli expressing sulfide-quinone reductase gene

Both wild type and sqr expressing Escherichia coli was cultivated in LB medium with streptomycin. The initial cell concentration was controlled to an OD600 of 0.2 and grew in the test tube inside incubator of 37 degrees Celsius and 200 rpm. Sodium sulfide was diluted from 200μM to 125μM and added into test tube. The consumption of sulfide was measured every 90 minutes by the methylene blue method (Trüper and Schlegel 1964).


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