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                 <li><a title="Methods" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq2.html">Methods</a></li>
                 <li><a title="Methods" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq2.html">Methods</a></li>
                 <li><a title="Experiments" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq3.html">Experiments</a></li>
                 <li><a title="Experiments" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq3.html">Experiments</a></li>
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                 <li><a title="Results & References" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq4.html">Results &amp; References</a></li>
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                 <li><a title="Results & References" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq4.html">Results &amp; References</a></li><li><a title="Further Experiments after Asia Jamboree" href="http://2012.igem.org/Team:NYMU-Taipei/ymiq5.html">Further Experiments after Asia Jamboree</a></li>
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Latest revision as of 01:48, 27 October 2012

NYMU iGEM

Methods

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

Several Cyanobacteria have Sulfide-Quinone Reductase (sqr) and thus the ability to deprive electron from sulfide compound. According to both databases of NCBI and KEGG, the sqr in Synechococcus SP. PCC 7002 shared great similarity with that of Oscillatoria limnetica, which is reported to exhibit anoxygenic photosynthesis by consumed sulfide anion. Since we planned to express sqr from Synechococcus SP. PCC 7002 in Synechococcus SP. PCC 7942 and Escherichia coli, the experiment was designed to testify the property of the sqr. DCMU was added in the medium to inhibit photosystem II, and therefore only sodium sulfide in the medium can provide electron for carbon photoassimilation. By creating different dilution of sodium sulfide, we expected that the more sodium sulfide was present, the better the cell grew.


DCMU structure and its mechanism on photosynthesis
http://en.wikipedia.org/wiki/File:Diuron.png

Sodium sulfide concentration and cell growth

From the previous studies, it is suggested that Synechococcus SP. PCC 7002 is able to metabolize sulfide compounds. We took advantage of the results in our last experiment and adjusted the concentration of DCMU to an appropriate degree. Since sulfide would become the main reducing energy for photoassimilation under the effect of DCMU, we believe the more sulfide concentration in the wells, the better cell growth would be observed.



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

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. Though sulfide is naturally toxic to Synechococcus SP. PCC 7942, the strain with sqr should be able to metabolize sulfide and therefore prosper.


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

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 Synechococcus SP. PCC 7942, 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 Sulfur Oxide Terminator part)


Sulfide oxidation in Escherichia coli expressing sulfide-quinone reductase gene

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.