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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.
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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
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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 ( )

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 ( )


  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