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Experiment Design

Abstract  Resistance of Cyanobacteria (Synechococcus SP. PCC 7002) to Sulfide compound -- Sulfide-Quinone Reductase 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.

The special ”CO2” infused device for cyanobacteria incubation

Method  Resistance of Synechococcus SP. PCC 7002 to 3 - (3,4-dichlorophenyl) - 1,1 – dimethylurea (DCMU) From the previous research, we discovered that the concentration of 3 - (3,4 - dichlorophenyl) - 1,1 – dimethylurea (DCMU) must be adjusted to meet our requirement. Under certain DCMU concentration, the presence of sulfide would be extreme decisive condition which determines whether the colonies live or die. In this experiment, DCMU is diluted with A2 medium to explore the relationship between DCMU concentration and cell growth. Sodium sulfide is added to the experimental group and its initial concentration is controlled to 10 mM.

DCMU structure and its mechanism on photosynthesis( )

 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.

Measurement  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.

 DCMU concentration and cell growth 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.

 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.


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

The horizontal axis stands for day passed, while the vertical axis is the absorbance of an OD 730nm. According to the graph, sqr expressing strain grew much better than wild type strain when sulfide was added into the medium. This result suggests that sqr is not only expressed but functional in the cyanobacteria.

2.Standard curve

We established a H2S standard curve to quantify H2S concentration inside our experiments.

3. lab incubation with E.coli

(1) different concentration under 24hr

We used different H2S concentration challenge ptrc-sqr transformed E.coli and tested H2S consumption in 24hrs. The result shows that ptrc-sqr transformed E.coli consumed much more H2S compred to the blank control, ptrc-str transformed E.coli. In other words, we believe our sqr gene works!!

(2) different concentration under 48hr

We followed the experiments in 48hrs and analyzed the H2S consumption in different groups. As the same as our expected, in 48hrs, our ptrc-sqr transformed E.coli depleted much more H2S than 24hrs timepoint.

Then we focus on the difference between 24 hour or 48 hour under same concentration of H2S

(1) 500mM H2S

500mM Str SQR 24hr 0.72 0.63 48hr 0.57 0.31

If we analyze two group in the same H2S concentration, it’s easier to find out that our ptrc-sqr transformed E.coli consumed H2S dramatically.

(2) 250mM H2S

250mM Str SQR 24hr 0.57 0.52 48hr 0.26 0.19

The experiment result are the same as previous group, ptrc-sqr transformed E.coli can consume much more H2S.

(3) 125mM H2S

125mM Str SQR 24hr 0.5 0.44 48hr 0.32 0.22

This diagram illustrates that even at a concentration of 125mM of sodium sulfide, the difference of consumption rate is obvious between sqr expressing strain and wild type one. Reference 1. Facultative Anoxygenic Photosynthesis in the Cyanobacterium Oscillatoria limnetica YEHUDA COHEN,* ETANA PADAN, AND MOSHE SHILO Department of Microbiological Chemistry, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received for publication 9 May 1975 2. Sulfide oxidation in gram-negative bacteria by expression of the sulfide–quinone reductase gene of Rhodobacter capsulatus and by electron transport to ubiquinone Hiroomi Shibata and Shigeki Kobayashi 2001 3. Sulfur metabolism in Thiorhodoceae. I. Quantitative measurements on growing cells of Chromatium okenii. Antonie Leeuwenhoek, 30: 225–238 Trüper, H.G., and Schlegel, H.G. 1964.