Name: isolation and characterization of intact phycobilisome in cyanobacteria
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The authors thank Technology Commons, College of Life Science, National Taiwan University for the convenient use of the ultracentrifuge. The cyanobacterial strains Synechocystis sp. PCC 6803 and Leptolyngbya sp. JSC-1 was gifted from Dr. Chu, Hsiu-An at Academia Sinica, Taiwan, and Dr. Donald A. Bryant at Pennsylvania State University, USA, respectively. This work was funded by the Ministry of Science and Technology (Taiwan) (109-2636-B-002-013- and 110-2628-B-002-065-) and the Ministry of Education (Taiwan) Yushan Young Scholar Program (109V1102 and 110V1102).

The Synechocystis sp.&#160;PCC 6803, the model glucose-tolerant strain, was obtained from Dr. Chu, Hsiu-An at Academia Sinica, Taiwan. Leptolyngbya sp. JSC-1, the non-model filamentous,was obtained from Dr. Donald A. Bryant at Pennsylvania State University, USA.
1. Cell culture and harvesting
<ol>
	<li>Inoculate Syn6803 or JSC-1&#160;cells using a metallic inoculation loop into a 100 mL conical flask containing 50 mL of B-HEPES medium28</s...

<ol>
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This protocol describes a simple and standard method for isolating intact PBS in two types of cyanobacteria, unicellular model Syn6803,&#160;and filamentous non-model JSC-1. The critical steps of the protocol are cell homogenization and ultracentrifugation on a discontinuous density gradient of sucrose. Generally, the disruption of filamentous cells is more complicated than unicellular ones. Increasing the amount of the starting material (the wet weight of the cell pellet) and the repetition of bead-beating were helpful ...

Phycobilisome (PBS) is a huge water-soluble pigment-protein complex that attaches to the cytoplasmic side of the photosystems in the thylakoid membranes of cyanobacteria1. PBS is primarily composed of colored phycobiliproteins and colorless linker proteins1,2. The phycobiliproteins can be divided into four major groups: phycoerythrin, phycoerythrocyanin, phycocyanin, and allophycocyanin3. The four major groups absorb different wavelengths of light energy in the range of 490-650 nm, which chlorophylls absorbed inefficiently3. The PBS can serve as a light-harvesting antenna for collecting light energy and delivering it to Photosystem II and I4.
The structure and composition of PBS vary from species to species. Collectively, three shapes of phycobilisome (hemidiscodial, bundle-shaped, and rod-shaped) have been identified in different cyanobacterial species5. Even in the same species, the composition of PBS changes in response to the environment, such as light quality and nutrient depletion6,7,8,9,10,11. Therefore, the experimental procedure to isolate PBS from cyanobacteria has been instrumental in studying PBS12. Over several decades, many different protocols have isolated PBS and analyzed its structure, composition, and function6,7,8,12,13,14,15,16,17. The wide variety of methods for PBS isolation indeed provides flexibility in isolating the complex in different species with different reagents and instruments. However, it also makes choosing a suitable protocol more difficult for scientists unfamiliar with cyanobacteria and PBS. Therefore, a generalized and straightforward protocol is developed in this work for those interested in starting PBS isolation from cyanobacteria.
The methods for isolating PBS from previous publications are summarized here. Since PBS is a water-soluble protein complex and is readily dissociated, a high ionic strength phosphate buffer is required to stabilize PBS during extraction18. Several research articles that describe methods for the isolation of PBS from cyanobacterium have been published in the past. Most of the methods require a high concentration of phosphate buffer8,14,15,18,19. However, the procedures for mechanical disrupting of the cells vary, such as glass beads-assisted extraction, sonication20, and French press6,8,14. Different phycobiliproteins can be obtained by precipitation with ammonium sulfate20 and purified by HPLC21 or a chromatographic column22. On the other hand, intact PBS can be easily isolated by sucrose density gradient ultracentrifugation6,8,15.
In this protocol, one model cyanobacterium and one non-model cyanobacterium were used as the materials for PBS isolation. They are model unicellular&#160;glucose-tolerant Synechocystis sp.&#160;PCC 6803 (hereafter Syn6803) and non-model filamentous Leptolyngbya sp. JSC-1 (hereafter JSC-1), respectively7,23,24. The protocol begins by disruption of the unicellular and filamentous cyanobacteria in a high-ionic-strength phosphate buffer. After lysis, the supernatants are collected by centrifugation and then treated with a nonionic detergent (Triton X-100) to solubilize the water-soluble proteins from the thylakoid membranes. The total water-soluble proteins are applied to a discontinuous sucrose density gradient to fractionate the PBS. The discontinuous sucrose gradient in this protocol consists of four sucrose solutions and partitions the intact PBS in the lowest fractions of sucrose layer25. The integrity of PBS can be analyzed by SDS-PAGE, zinc-staining, and 77K fluorescence spectroscopy6,7,8,26. This method is suitable for scientists who aim to isolate intact PBS from cyanobacteria and study its spectral, structural, and compositional properties.
There are several advantages of this protocol. (1) This method is standardized and can be used for isolating intact PBS from both unicellular and filamentous cyanobacteria. Most of the articles describe the method that applied in either one type of cyanobacteria4,7,8,12,13,14,16,18. (2) This method is performed at room temperature, as PBS dissociates at low temperature19,27. (3) This method describes using a bead-beater to disrupt the cells; therefore, it is cheaper and safer than high-pressured French press and possible hearing damage from sonicator in other methods8,13,14,20. (4) This method isolates intact PBS by sucrose gradient ultra-centrifugation. In this way, intact PBS with different sizes and partially dissociated PBS can be separated based on sucrose concentration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1 mm glass beads</td>
			<td>BioSpec</td>
			<td>11079101</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>13 mL centrifugation tube</td>
			<td>Hitachi</td>
			<td>13PA</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>40 mL centrifugation tube</td>
			<td>Hitachi</td>
			<td>40PA</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>8.1875.2500</td>
			<td>for Coomassie Blue staining</td>
		</tr>
		<tr>
			<td>B-HEPES medium</td>
			<td></td>
			<td></td>
			<td>A modified cyanobacterial medium from BG-11 medium</td>
		</tr>
		<tr>
			<td>Brilliant Blue R-250</td>
			<td>Sigma</td>
			<td>B-0149</td>
			<td>for Coomassie Blue staining</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Wako pure chemical industries</td>
			<td>2-291</td>
			<td>protein loading buffer</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>Radwag</td>
			<td>WLC 2/A2/C/2</td>
			<td>for the wet weight measurement of cell pellets</td>
		</tr>
		<tr>
			<td>Fluorescence spectrophotometer</td>
			<td>Hitachi</td>
			<td>F-7000</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>BioShop</td>
			<td>Gly001.500</td>
			<td>protein loading buffer</td>
		</tr>
		<tr>
			<td>High-Speed refrigerated centrifuge</td>
			<td>Hitachi</td>
			<td>CR22N</td>
			<td>for buffer exchange</td>
		</tr>
		<tr>
			<td>Leptolyngbya sp. JSC-1</td>
			<td></td>
			<td></td>
			<td>from Dr. Donald A. Bryant at Pennsylvania State University, USA.</td>
		</tr>
		<tr>
			<td>Low temperature measurement accessory</td>
			<td>Hitachi</td>
			<td>5J0-0112</td>
			<td>The accessory includes a transparent Dewar container for 77K fluorescence spectra</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Merck</td>
			<td>1.07018,2511</td>
			<td>for Coomassie Blue staining</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Thermo Fisher</td>
			<td>Pico 21</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Mini-Beadbeater-16</td>
			<td>BioSpec</td>
			<td>Model 607</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>PanReac AppliChem</td>
			<td>121512.121</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>PanReac AppliChem</td>
			<td>141509.121</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Screw cap vial</td>
			<td>BioSpec</td>
			<td>10832</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>SmartView Pro Imager</td>
			<td>Major Science</td>
			<td>UVCI-2300</td>
			<td>for Znic staining signal detection</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Zymeset</td>
			<td>BSD101</td>
			<td>protein loading buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Zymeset</td>
			<td>BSU101</td>
			<td>for PBS isolation</td>
		</tr>
		<tr>
			<td>Synechocystis sp. PCC 6803</td>
			<td></td>
			<td></td>
			<td>glucose-tolerant strain from Dr. Chu, Hsiu-An at Academia Sinica, Taiwan</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>BioShop</td>
			<td>TRS 011.1</td>
			<td>protein loading buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BioShop</td>
			<td>TRX 506.500</td>
			<td>for PBS extraction</td>
		</tr>
		<tr>
			<td>Ultra 10 K membrane centrifugal filter</td>
			<td>Millipore</td>
			<td>UFC901024</td>
			<td>for buffer exchange</td>
		</tr>
		<tr>
			<td>Ultra 3 K membrane centrifugal filter</td>
			<td>Millipore</td>
			<td>UFC500324</td>
			<td>for buffer exchange</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Hitachi</td>
			<td>CP80WX</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>UV/Vis spectrophotometer</td>
			<td>Agilent</td>
			<td>Cary 60</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td>PanReac AppliChem</td>
			<td>131787.121</td>
			<td>for Znic staining</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>BioBasic</td>
			<td>MB0338</td>
			<td>protein loading buffer</td>
		</tr>
	</tbody>
</table>

isolation and characterization of intact phycobilisome in cyanobacteria

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

The Syn6803 and JSC-1 cells were cultivated in conical flasks with constant stirring in B-HEPES medium at 30 &#176;C, under a LED white light (50 &#181;mol photons m-2s-1) in a growth chamber filled with 1% (v/v) CO2. At the exponential growth phase (OD750 = ~0.5), the cells were subcultured into fresh medium with a final optical density OD750 = ~0.2. After reaching the late exponential growth phase (OD750 = 0.6-0.8), the cultures were collected and centri...

Watch this Scientific Journal Video about Isolation and Characterization of Intact Phycobilisome in Cyanobacteria at JoVE.com

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

It is a simple and a fast protocol that allow people unfamiliar with cyanobacteria to isolate phycobilisome at ease. The advantage of this technique is that it is adapted to a small scale and it is easy to perform. It is a simple, reliable, and efficient method for isolating intact phycobilisomes of model and non-model cynobacteria.Begin by transferring the cell culture of 0.8 OD at 750 nanometers into a one liter flask and dilute it in 500 milliliters of B-HEPES medium to a achieve the OD of 0.2 at 750 nanometers. Grow the culture with constant stirring at 30 degree Celsius until the OD reaches 0.8 to 1, indicating a late exponential growth phase. Centrifuge the culture at a speed of 10, 000 times g for 20 minutes at room temperature and discard the supernatant.Suspend the cell pellets and washed twice with 0.75 molar potassium phosphate buffer, pH seven. Pellet the cells in a 50 milliliter centrifuge tube by centrification at room temperature. Discard the supernatant and resuspend the cells with 0.75 molar potassium phosphate buffer.Measure the wet weight of the pellet by an electronic balance and resuspend the one gram wet weight of the pellet in five milliliters of the buffer. Add one milliliter of cell suspension and 0.4 to 0.6 grams of 0.1 millimeter glass beads into a two milliliter screw cap vial. Break the cells for 30 seconds by a bead beater.Allow the tubes to cool in a room temperature water bath for two minutes. After lysis, centrifuge the vials for 10 seconds at room temperature at a speed of 500 times g. Collect the supernatant with a pipette into a 15 milliliter centrifuge tube.Wash the beads with 0.5 milliliters of 0.75 molar potassium phosphate buffer one time, then transferred to the same centrifuge tube. Add Triton X-100 to the lysed cell suspension and incubate on a rocking shaker at room temperature and 40 RPM for 20 minutes or until the solution becomes homogenous. Centrifuge the tubes for 20 minutes at 17, 210 times g and store the supernatant without the upper Triton micelle layer at room temperature for up to one hour.Make four concentrations of sucrose in 0.75 molar potassium phosphate buffer at pH seven. Place 2.8 milliliters of two molar sucrose buffer at the bottom of the 40 milliliter centrifuge tube and overlay with three layers of sucrose solutions. Finally, add three milliliters of PBS containing the supernatant fraction and centrifuge the resulting gradients.Upon ultracentrification, condense the fractionated PBS and phycobiliproteins between the layers and observe the blue bands in Syn6803 and interfaces. Remove the sucrose by repeatedly concentrating and diluting the fractions with 0.75 molar potassium phosphate buffer and membrane the centrifugal filter units. For measuring PBS fluorescence dilute the concentrated PBS sample with 0.75 molar potassium phosphate buffer to make up to 500 microliters volume.Add the diluted PBS sample to a transparent glass tube and freeze the tube in liquid nitrogen until it is entirely frozen. Move the frozen tube to a transparent dewar container pre-filled with liquid nitrogen. Complete cell lysis shows the supernatant and dark blue-green color before centrification.The sucrose density gradient separation of isolated PBS shows several fractions of dissociated phycobiliproteins and the lowest fraction represents the intact PBS. The absorption spectra from the dissociated and intact PBS of JSC one and Syn6803 have the same peak at 622 nanometers, corresponding to the phycocyanin. Moreover, the phycoerythrin absorption peak at 571 nanometers in both dissociated and intact PBS of JSC-1.The emission spectra of the dissociated PBS have one strong peak at approximately 650 nanometers in Syn6803 and JSC-1, representing the emission from phycocyanin. The fluorescent emission maximal peaks at 651 nanometers and 684 nanometers reveal that the energy harvested by phycocyanin was transferred efficiently to allophycocyanin and terminal emitters, which are ApcD and ApcE, in the core. The figure shows zinc stained SDS-PAGE under UV irradiation where the phycobiliprotein subunits are strongly fluorescent and the ApcE showed weak fluorescence, indicated with the arrow.Kumasi blue staining shows that the upper fractions contain other non-PBS water soluble proteins and the intact PBS and Syn6803 shows a prominent ApcE band, indicated with an arrow, lacking in the dissociated PBS fractions. Ultracentrifugation demonstrates that the sucrose gradient prepared at a ratio of 1:3 displays wider bands than the gradient made at a ratio of 1:10. It is important to lysis the cells and solvolysis the supernatants completely to obtain more phycobilisomes.Other methods, like cryo-EM or mixed spectrometry, can be used to study the protein structure or the protein composition of phycobilisomes.

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