Part:BBa_K896000:Experience

Function analysis of SQR gene:

Abstract 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.

Method and materials:

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. Detailed materials and methods click here.[http://2012.igem.org/Team:NYMU-Taipei/ymis4.html]

The same method used in Environmental Protection Administration Executive Yuan, Taiwan: [http://www.niea.gov.tw/niea/AIR/A40671A.htm 排放管道中硫化氫檢驗法－甲烯藍比色法]

The reaction formula and the compound of DPDA

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:

Figure 2

Figure 3

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 4A,4B) And SQR transformed E.coli consumed H2S dramatically.(figure 5)

Following were results.

Figure 4

(A)different concentration of H2S under 24 hrs

(B)different concentration of H2S under 48 hrs

Figure 5

Conclusion

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

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