Team:NYMU-Taipei/ymiq5.html

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<div class="title">Methods</div>
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<div class="title">Further Experiments after Asia Jamboree</div>
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<br />
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  <p><span class="subtitle">Resistance of Cyanobacteria (Synechococcus SP. PCC 7002) to Sulfide compound</span></p>
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<div align="left">
<div align="left">
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  <p>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.  <br />
 
    
    
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   <div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw1.png" alt="" width="502" height="333" /><br />
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   <div class=out style='text-align:center'><img src="http://2012.igem.org/wiki/images/5/5e/Ymiq3.png" width="469" height="276" border="0" align="center"  /><br />
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     <p align="center">Sulfite quinine reductase, SQR use  hydrogen sulfide, H2S as electron resource to perform photosynthesis<br />
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     DCMU structure and its mechanism on photosynthesis<br />
+
  </p></div>
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    http://en.wikipedia.org/wiki/File:Diuron.png <br />
+
  In previous experiment, we’ve demonstrated that Sulfide-Quinone Reductase (sqr) expressed in Synechococcus sp. PCC7942 enhanced the growth of the unicellular cyanobacteria. Nevertheless, most of our observations were based on the difference of optical density between wild type and sqr expressing strain. Though it is quite common to estimate the growth of cyanobacteria by measuring the optical density under certain wavelength, such measurement only provides an approximate data for comparison, and various factors can influence the observation accuracy, With regard to that, we conduct another series of experiments via flow cytometer and plate count method to examine the exact cell concentration.
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  <br />
     <br />
     <br />
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  </div>
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       <div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw2.png" alt="" width="398" height="314" /><br />
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        Cyanobacteria contain different of cytochrome. We can detect them by different wavelength fluorescent light.<br />
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    <div align="left">
+
        ( http://www.masa.asn.au/phpBB3/viewtopic.php?t=233828&amp;p=960435 )<br />
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  <p><span class="subtitle">Sodium sulfide concentration and cell growth</span></p>
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        <br />
-
</div>
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    <p>From the previous  studies, it is suggested that <em>Synechococcus  SP. PCC 7002 </em>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.</p>
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       <div class=out style='text-align:center'><span class="out" style="text-align:center"><img src="http://2012.igem.org/wiki/images/2/23/Ymiq4-1.png" width="557" height="415" border="0" align="center" /></span><br />
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<br />
<br />
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      </div>
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      <br /><div align="left">
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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">Cyanobacteria Culture </span></p>
</div>
</div>
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      <p>Both wild type and sqr stain of <em>Synechococcus sp. PCC7942</em> were  cultivated in BG-11 medium (Allen M.M. 1968) [1]. The initial cell  concentration was adjusted to an OD730 of 0.2. The experiment was initialized  after the addition of unicellular blue-green algae, and the six-well plates  were placed on a 100 r.p.m. shaker.
-
    <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. Though sulfide is naturally toxic to Synechococcus SP. PCC 7942, the strain with sqr should be able to metabolize sulfide and therefore prosper.</p>
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      <br />
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       <div class=out style='text-align:center'><span class="out" style="text-align:center"><img src="http://2012.igem.org/wiki/images/5/57/Ymiq4-2.png" width="573" height="299" border="0" align="center"  /></span><br />
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        </p>
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  <div align="left">
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       <div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw3.png" alt="" width="434" height="334" /><br />
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      <p><span class="subtitle">Sulfide concentration and the growth of sqr expressing strain Synechococcus SP. PCC 7942</span></p>
+
        We culture Synechococcus sp. PCC7942 wildtype and sqr transformed one in BG-11 medium with suitable shaker.    <br />
 +
        <br />
 +
      </div>
 +
      <br /><div align="left">
 +
  <p><span class="subtitle">DCMU blocking assay</span></p>
</div>
</div>
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  <div align="left">
 
-
    <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 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)</div><br />
 
 +
 +
      <p>Freshly made sodium sulfide containing  medium was added into the experiment group. The concentration of sodium sulfide  was 2.5 mM, and additional 2.5mM of sodium sulfide was added every 24 hours. 3-(3,4-dichlorophenyl)-1,1-dimethylurea  (DCMU) [2], a chemical which can block photosystem II was also added into both  experiment and control group. A positive control group was established by  simply cultivated cyanobacteria in fresh medium. As followed, we analyze by  flow cytometry analysis, 730 nm absorption assay and plating of bacteria.
 +
<br /><div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw4.png" alt="" width="558" height="262" /><br />
 +
        DCMU specifically block photosynthesis system II<br />
 +
( http://5e.plantphys.net/article.php?ch=7&id=75 )<br />
 +
<br />
 +
        <br />
 +
      </div>
 +
<br />
<div align="left">
<div align="left">
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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">Flow cytometry analysis </span></p>
</div>
</div>
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  <div align="left">
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<div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw5.png" alt="" width="475" height="361" /><br />
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    <p>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.</div><br />
+
        We use flow cytometry with multiple wavelength fluorescent light to find out the differences between two groups.    <br />
-
      <br />
+
        <br />
-
 
+
</div>
 +
<p>The automation of viability analysis method  via flow cytometer was reported in previous research [3]. In our experiment, cells  were analyzed using a flow cytometer FACSCanto. The chlorophyll fluorescence  filter set (excitation: 435/40 nm; beam splitter: 510 nm; emission: 515 nm  long-pass) were used [3]. By using the automated counting function of FACSCanto, we examined the accurate number of cell. The green fluorescence of the cell  area was constrained at an intensity value of 50 to differentiate between red  (viable) and green (non-viable) cells [3]. The percentage of red and green  cells and the exact cell concentration was calculated.
 +
</p>
 +
<div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw6.png" alt="" width="558" height="385" /><br />
 +
        Different fluorescent dyes and its wavelength
 +
(http://www.epitomics.com/images/DyLight%20Spectra%20Chart.jpg )
 +
</div>
 +
<br />
 +
<br />
 +
<br />
 +
<div class="title">Results &amp; Discussion</div>
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<br />
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<div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw7.png" width="600" height="300" /><br />
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</div>
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<br /><div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw8.png" width="600" height="283" /><br />
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</div>
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<br /><div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw9.png" width="600" height="262" /><br />
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</div>
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<br /><div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw10.png" width="600" height="266" /><br />
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</div>
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<br /><div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw11.png" width="600" height="291" /><br />
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</div>
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<br /><div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw12.png" width="600" height="249" /><br />
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</div>
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<br /><div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw13.png" width="600" height="244" /><br />
 +
</div>
 +
<br />
 +
<ol>
 +
  <li>We kill wildtype cyanobacteria  with 4oC environment for 2 weeks</li>
 +
  <li>We find out that when adding  Na2S to our DCMU blocked SQR bacteria, they show more fluorescent light(higher  photosynthesis ability, live better)</li>
 +
  <li>We find out that when bacteria are  challenged with DCMU, SQR group can live better than Wldtype group, which means  SQR gene help cyanobacteria survive in the adversity(DCMU, Na2S)</li>
 +
  <li>It&rsquo;s very interesting when we  compare data:</li>
 +
</ol>
 +
<br />
 +
<div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw14.png" alt="" width="422" height="302" /><br />
 +
<p align="left">When DCMU challenge bacteria,  SQR transformed croup can produce more fluorescent light(higher photosynthesis  ability, live better)</div>
 +
<div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw15.png" alt="" width="405" height="299" />
 +
<p align="left">In SQR group, we use DCMU to challenge bacteria. Adding Na2S bacteria produce more fluorescent light. </div><div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw16.png" alt="" width="382" height="280" />
 +
  <p align="left">We survey several papers and hypothesize that the fluorescent comes from cytochrome b6f, which is an important role in the downstream of photosynthesis system II and the electron transport chain. (Erwin J. G. Peterman,Stephan-Olav Wenk et al, Fluorescence and Absorption Spectroscopy of the Weakly Fluorescent Chlorophyll a in Cytochrome b6f of Synechocystis PCC6803, Biophysical Journal Volume 75 July 1998 389–398)<br />
 +
    <br />
 +
    <br />
 +
</div>
 +
<div align="left">
 +
  <p><span class="subtitle">730 nm absorption</span></p>
 +
</div>
 +
<p>Based on the  flow cytometry result, we double check our bacteria amount by 730nm absorption:</p>
 +
<div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw17.png" alt="" width="559" height="394" /><br />
 +
</div>
 +
<ol>
 +
  <li>When challenge  bacteria with DCMU, SQR transformed cyanobacteria live better than wildtype(higher photosynthesis, better adversity resistance)</li>
 +
  <li>It&rsquo;s very  interesting that when add Na2S into two kinds of bacteria, it might produce  more difficulty for living(maybe the Na2S concentration is wrong? HS-  binds to Fe and generate worse environment for bacteria)<br />
 +
    <br />
 +
  </li>
 +
</ol>
 +
<div align="left">
 +
  <p><span class="subtitle">Plating of bacteria</span></p>
 +
</div>
 +
<p>Viable cell concentrations were also  determined by spreading 0.1 mL of culturing cyanobacteria on BG-11 agar plates.  Colony counts were conducted after these plates were incubated for 7 days at  room temperature. The plating was performed in duplicates for every sample.</p>
 +
<div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw18.png" width="509" height="557" /><br />
 +
  <p align="left" >We culture bacteria into solid plate. After incubation for about 10 days, we will count the colonies and compare each group. <br />
 +
    <br />
 +
    <br />
 +
</div>
 +
<div align="left">
 +
  <p><span class="subtitle">CO2 consumption analysis</span><br />
 +
</p>
 +
</div>
 +
<div class=out style='text-align:center'><img src="../../../Users/jaymes/Desktop/ymiqw19.png" alt="" width="407" height="482" /><br />
 +
  <p align="left" >We culture two group bacteria with stable lamina CO2 input and compare the CO2 for each group.<br />
   </div>
   </div>
 +
<p>It&rsquo;s very interesting that when adding Na2S  to both two bacteria group, we get the similar result. We hypothesize that Na2S  might generate adversity to bacteria. Thus, It&rsquo;s very important to analyze CO2  consumption. CO2 concentration and analysis were examined by the  infrared CO2 analyzer and FL22 Algal CO2 Package respectively  [4]. Cyanobacteria samples were cultured for 24 hours following CO2 analysis.  CO2 fixation rates were calculated in terms of CO2  exchange rate as the following equation: </p>
 +
<div class=out style='text-align:center'>
 +
  <p align="center" ><img src="../../../Users/jaymes/Desktop/ymiqw20.png" alt="" width="433" height="60" />  </div>
 +
<p>The HCO3 concentration in the  culture medium was also measured with Carbon Dioxide Enzymatic Assay of BQ Kits  (Bio-Quant, USA).</p>
 +
<div class=out style='text-align:center'>
 +
  <p align="center" ><img src="../../../Users/jaymes/Desktop/ymiqw21.png" alt="" width="558" height="320" />  <br />
 +
  <p align="left"> We will analyze CO2 consumption for SQR grop and wildtype cyanobacteria to find out the activity of them. Finally, We will use DCMU to challenge two groups of bacteria and perform this assay again ( http://www.quora.com/Carbon-Dioxide )    <br />
 +
    <br />
 +
<br />
 +
  </div><div align="left">
 +
  <p><span class="subtitle">References</span><br />
 +
</p>
 +
</div>
 +
  <ol>
 +
    <li><em>Allen MM (1968) Simple conditions for growth of unicellular  blue-green algae. J Phycol 4: 1-3</em></li>
 +
    <li><em>Adaptation to Hydrogen Sulfide of Oxygenic and Anoxygenic  Photosynthesis among Cyanobacteria YEHUDA COHEN,'* BO BARKER J0RGENSEN,2 NIELS  PETER REVSBECH,2 AND RICARDO POPLAWSKIl H. Steinitz Marine Biology Laboratory,  The Hebrew University of Jerusalem, Eilat 88103, Israel,1 and Institute of  Ecology and Genetics, University of Aarhus, Ny Munkegade, DK 8000, Aarhus C,  Denmark2 Received 1 July 1985/Accepted 22 October 1985</em></li>
 +
    <li><em>A simple viability analysis for unicellular cyanobacteria using a  new autofluorescence assay, automated microscopy, and ImageJ Katja Schulze1,2*,  Diana A López1, Ulrich M Tillich1 and Marcus Frohme1 BMC Biotechnology 2011,  11:118 doi:10.1186/1472-6750-11-118</em></li>
 +
    <li><em>Enhancing CO2 bio-mitigation by genetic engineering of  cyanobacteria† Pei-Hong Chen,a Hsien-Lin Liu,a Yin-Ju Chen,a Yi-Hsiang Cheng,a  Wei-Ling Lin,a Chien-Hung Yeha and Chuan-Hsiung Chang*ab Received 16th January  2012, Accepted 7th June 2012 DOI: 10.1039/c2ee21124f<br />
 +
    </em>  <br />
 +
</li>
 +
  </ol>
</div>
</div>
-
 
  </div>
  </div>
       </div>
       </div>

Revision as of 03:01, 27 October 2012

NYMU iGEM

Further Experiments after Asia Jamboree


Sulfite quinine reductase, SQR use hydrogen sulfide, H2S as electron resource to perform photosynthesis

In previous experiment, we’ve demonstrated that Sulfide-Quinone Reductase (sqr) expressed in Synechococcus sp. PCC7942 enhanced the growth of the unicellular cyanobacteria. Nevertheless, most of our observations were based on the difference of optical density between wild type and sqr expressing strain. Though it is quite common to estimate the growth of cyanobacteria by measuring the optical density under certain wavelength, such measurement only provides an approximate data for comparison, and various factors can influence the observation accuracy, With regard to that, we conduct another series of experiments via flow cytometer and plate count method to examine the exact cell concentration.


Cyanobacteria contain different of cytochrome. We can detect them by different wavelength fluorescent light.
( http://www.masa.asn.au/phpBB3/viewtopic.php?t=233828&p=960435 )



Cyanobacteria Culture

Both wild type and sqr stain of Synechococcus sp. PCC7942 were cultivated in BG-11 medium (Allen M.M. 1968) [1]. The initial cell concentration was adjusted to an OD730 of 0.2. The experiment was initialized after the addition of unicellular blue-green algae, and the six-well plates were placed on a 100 r.p.m. shaker.


We culture Synechococcus sp. PCC7942 wildtype and sqr transformed one in BG-11 medium with suitable shaker.


DCMU blocking assay

Freshly made sodium sulfide containing medium was added into the experiment group. The concentration of sodium sulfide was 2.5 mM, and additional 2.5mM of sodium sulfide was added every 24 hours. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) [2], a chemical which can block photosystem II was also added into both experiment and control group. A positive control group was established by simply cultivated cyanobacteria in fresh medium. As followed, we analyze by flow cytometry analysis, 730 nm absorption assay and plating of bacteria.


DCMU specifically block photosynthesis system II
( http://5e.plantphys.net/article.php?ch=7&id=75 )



Flow cytometry analysis


We use flow cytometry with multiple wavelength fluorescent light to find out the differences between two groups.

The automation of viability analysis method via flow cytometer was reported in previous research [3]. In our experiment, cells were analyzed using a flow cytometer FACSCanto. The chlorophyll fluorescence filter set (excitation: 435/40 nm; beam splitter: 510 nm; emission: 515 nm long-pass) were used [3]. By using the automated counting function of FACSCanto, we examined the accurate number of cell. The green fluorescence of the cell area was constrained at an intensity value of 50 to differentiate between red (viable) and green (non-viable) cells [3]. The percentage of red and green cells and the exact cell concentration was calculated.


Different fluorescent dyes and its wavelength (http://www.epitomics.com/images/DyLight%20Spectra%20Chart.jpg )



Results & Discussion















  1. We kill wildtype cyanobacteria with 4oC environment for 2 weeks
  2. We find out that when adding Na2S to our DCMU blocked SQR bacteria, they show more fluorescent light(higher photosynthesis ability, live better)
  3. We find out that when bacteria are challenged with DCMU, SQR group can live better than Wldtype group, which means SQR gene help cyanobacteria survive in the adversity(DCMU, Na2S)
  4. It’s very interesting when we compare data:


When DCMU challenge bacteria, SQR transformed croup can produce more fluorescent light(higher photosynthesis ability, live better)

In SQR group, we use DCMU to challenge bacteria. Adding Na2S bacteria produce more fluorescent light.

We survey several papers and hypothesize that the fluorescent comes from cytochrome b6f, which is an important role in the downstream of photosynthesis system II and the electron transport chain. (Erwin J. G. Peterman,Stephan-Olav Wenk et al, Fluorescence and Absorption Spectroscopy of the Weakly Fluorescent Chlorophyll a in Cytochrome b6f of Synechocystis PCC6803, Biophysical Journal Volume 75 July 1998 389–398)


730 nm absorption

Based on the flow cytometry result, we double check our bacteria amount by 730nm absorption:


  1. When challenge bacteria with DCMU, SQR transformed cyanobacteria live better than wildtype(higher photosynthesis, better adversity resistance)
  2. It’s very interesting that when add Na2S into two kinds of bacteria, it might produce more difficulty for living(maybe the Na2S concentration is wrong? HS- binds to Fe and generate worse environment for bacteria)

Plating of bacteria

Viable cell concentrations were also determined by spreading 0.1 mL of culturing cyanobacteria on BG-11 agar plates. Colony counts were conducted after these plates were incubated for 7 days at room temperature. The plating was performed in duplicates for every sample.


We culture bacteria into solid plate. After incubation for about 10 days, we will count the colonies and compare each group.


CO2 consumption analysis


We culture two group bacteria with stable lamina CO2 input and compare the CO2 for each group.

It’s very interesting that when adding Na2S to both two bacteria group, we get the similar result. We hypothesize that Na2S might generate adversity to bacteria. Thus, It’s very important to analyze CO2 consumption. CO2 concentration and analysis were examined by the infrared CO2 analyzer and FL22 Algal CO2 Package respectively [4]. Cyanobacteria samples were cultured for 24 hours following CO2 analysis. CO2 fixation rates were calculated in terms of CO2 exchange rate as the following equation:

The HCO3 concentration in the culture medium was also measured with Carbon Dioxide Enzymatic Assay of BQ Kits (Bio-Quant, USA).


We will analyze CO2 consumption for SQR grop and wildtype cyanobacteria to find out the activity of them. Finally, We will use DCMU to challenge two groups of bacteria and perform this assay again ( http://www.quora.com/Carbon-Dioxide )


References

  1. Allen MM (1968) Simple conditions for growth of unicellular blue-green algae. J Phycol 4: 1-3
  2. Adaptation to Hydrogen Sulfide of Oxygenic and Anoxygenic Photosynthesis among Cyanobacteria YEHUDA COHEN,'* BO BARKER J0RGENSEN,2 NIELS PETER REVSBECH,2 AND RICARDO POPLAWSKIl H. Steinitz Marine Biology Laboratory, The Hebrew University of Jerusalem, Eilat 88103, Israel,1 and Institute of Ecology and Genetics, University of Aarhus, Ny Munkegade, DK 8000, Aarhus C, Denmark2 Received 1 July 1985/Accepted 22 October 1985
  3. A simple viability analysis for unicellular cyanobacteria using a new autofluorescence assay, automated microscopy, and ImageJ Katja Schulze1,2*, Diana A López1, Ulrich M Tillich1 and Marcus Frohme1 BMC Biotechnology 2011, 11:118 doi:10.1186/1472-6750-11-118
  4. Enhancing CO2 bio-mitigation by genetic engineering of cyanobacteria† Pei-Hong Chen,a Hsien-Lin Liu,a Yin-Ju Chen,a Yi-Hsiang Cheng,a Wei-Ling Lin,a Chien-Hung Yeha and Chuan-Hsiung Chang*ab Received 16th January 2012, Accepted 7th June 2012 DOI: 10.1039/c2ee21124f