Difference between revisions of "Part:BBa K896000"

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	'''Function analysis of SQR gene''':<p></p>		'''Function analysis of SQR gene''':<p></p>
−	'''Method:'''  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, the more sodium sulfide was present, the better the growth of cyanobacteria was.<p></p>	+	'''Method:'''
		+	1. Resistance of Synechococcus SP. PCC 7002 to 3 - (3,4-dichlorophenyl) - 1,1 – dimethylurea (DCMU)<p></p>
		+	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. <p></p>
		+
		+	2. Sodium sulfide concentration and cell growth<p></p>
		+	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.<p></p>
		+
		+	3. The effect of sodium sulfide on Synechococcus SP. PCC 7942 growth rate<p></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.<p></p>
		+
		+	4. DCMU concentration and cell growth<p></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.<p></p>
		+
		+	5. Sulfide concentration and the growth of sqr expressing strain Synechococcus SP. PCC 7942<p></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.<p></p>
		+
		+	6. Sulfide oxidation in Escherichia coli expressing sulfide-quinone reductase gene<p></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.<p></p>
		+
		+
	'''Results:'''		'''Results:'''

---

Revision as of 14:15, 27 September 2012

SQR(sulfide quinone reductase),from Synechococcus sp. PCC 7002 plasmid pAQ7

SQR introduction:

Sulfide-dependent anoxygenic photosynthesis, driven by photosystem I (PS/I) alone, among cyanobacteria was first described for Oscillatoria limnetica from Solar Lake. Later photosynthetic sulfide oxidation in O. limnetica led to the discovery of sulfide-quinone reductase (SQR; E.C.1.8.5.′), a novel enzyme that transfers electrons from sulfide into the quinone pool.

Above are sulfide-induced sulfide-Quinone Reductase with the electron transport system.

(Cohen, Y., E. Padan, and M. Shilo, Facultative anoxygenic photosynthesis in the cyanobacterium Oscillatoria limnetica. J Bacteriol, 1975. 123(3): p. 855-61.)

Cloning of SQR gene:

SQR gene came from Synechococcus elongatus PCC7002 because the hypersaline strain S. elongatus PCC 7002, which is already sequenced, is 96% similar to O.limnetica SQR gene.

Figure 1

Function analysis of SQR gene:

Method:

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

Results:

After establishing a H2S standard curve to quantify H2S concentration, different H2S concentration challenge SQR transformed E.coli and H2S consumption in 24hrs or 48hrs were tested. The result showed that SQR transformed E.coli consumed much more H2S compred to the blank control, Str transformed E.coli. Our SQR transformed E.coli depleted much more H2S in 48 hours than in 24 hours timepoint.(figure 2A,2B) And SQR transformed E.coli consumed H2S dramatically.(figure 3)

Following were results in our experiment.

Figure 2

(A)different concentration of H2S under 24 hrs

(B)different concentration of H2S under 48 hrs

Figure 3

Conclusion

Our cloning product SQR(sulfide quinone reductase),from Synechococcus sp. PCC 7002 plasmid pAQ7, could transfer electrons from sulfide into the quinone pool.

References

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.

Sequence and Features