The overall goal of this procedure is to determine the glycogen content in cyanobacteria using an enzyme-based selective hydrolysis and assay. This method can help answer a key question in the cyanobacteria field, such as physiology, molecular genetics, and bioengineering of different cyanobacteria strain under investigation. The main advantage of this technique is that it's adapted to a small scale, it's easy to perform, and highly sensitive and specific to glycogen.
Though this method can provide insight into cyanobacteria, it can also be applied to other microorganisms that accumulate glycogen or starch, such as E.Coli, yeast, microalgae, and various heterotrophic and phototrophic microorganisms. Begin this protocol with preparation of cyanobacterial cultures as described in the text protocol. Transfer one milliliter of cyanobacterial culture or cell suspension to a 1.5 milliliter tube.
Then centrifuge at 6, 000 times g and four degrees Celsius for 10 minutes. Discard the supernatant before resuspending the pellet in one milliliter of 50 millimolar Tris-HCL pH eight. Centrifuge the resuspended pellet as before.
Discard the supernatant, and resuspend the cell pellet a second time in the Tris-HCL buffer. Centrifuge the tubes as before and discard the supernatant. Then thoroughly resuspend the pellet in 500 microliters of 50 millimolar Tris-HCL buffer pH eight.
It is crucial to have the pellet well-suspended for an efficient lysis. Keep the resuspension in ice. Lyse the resuspended cells at four degrees Celsius by 30 cycles of ultrasonication with each cycle consisting of 30 seconds at a frequency of 20 kilohertz with a maximum amplitude, followed by 90 seconds without.
Centrifuge the tube containing the lysate at 6, 000 times g and four degrees Celsius for 10 minutes. Following centrifugation, the pellet should be mainly large cell debris, and the supernatant is used for further analysis. At this point, determine the protein concentration by using the protein assay kit.
Remove chlorophyll a from the cell lysate by mixing 900 microliters of ethanol and 100 microliters of the obtained supernatant in a 1.5 milliliter screw cap tube. After closing the cap, heat the tube at 90 degrees Celsius for 10 minutes, using a standard laboratory heating block. Then, incubate the tube in ice for 30 minutes.
Next, centrifuge the tube at 20, 000 times g and four degrees Celsius for 30 minutes. Following centrifugation, carefully remove the supernatant. The pellet contains glycogen.
Lightly dry the pellet in air to remove excess ethanol. Do not excessively dry the pellet to avoid difficult dissolving it. Measure the absorbance at 666 nanometers of the obtained supernatant to determine the chlorophyll a content.
The value can be used to normalize the glycogen content. Dissolve the pellet in 100 microliters of 50 millimolar sodium acetate pH five. Mix these materials well, using a vortex.
Mixing by pipetting is not recommended because the mixture is viscous. Also add 50 microliters of eight units per milliliter amyloglucosidase and 50 microliters of two units per milliliter of alpha-amylase. Next, incubate the mixture at 60 degrees Celsius in a heating block for two hours to enable the digestion of glycogen into glucose molecules.
Following incubation, centrifuge the sample at 20, 000 times g for five minutes and transfer the supernatant to a new 1.5 milliliter tube. Measure the concentration of glucose in the supernatant following enzymatic hydrolysis, using the GOD-POD reagent. Transfer 100 microliters of the supernatant from the glycogen precipitation step to a well in a 96 well plate.
As a negative control, use 100 microliters of 50 millimolar sodium acetate pH five. For generating the calibration curve, also measure the glucose standard solutions. Add 150 microliters of GOD-POD reagent to each sample and quickly mix by pipetting.
Incubate the plate at 25 degrees Celsius for 30 minutes. Then, record the absorbance value at 510 nanometers, using a plate reader. Calculate the amount of glycogen as the glucose equivalent, using a calibration curve obtained from the glucose standards.
The glycogen contents in synechocystis are shown here. The two mutant strains, delta pmg A and delta pmg R, which are known to hyperaccumulate glycogen, were compared to the wild type strain. As expected, the mutant strains showed elevated levels of glycogen.
Conversely, a mutant that lacks glycogen synthase did not accumulate glycogen. The strains used here have been engineered to produce mannitol. Notably, the glycogen synthase mutant produced more mannitol than the control strain, which suggests that the carbohydrate synthesized by photosynthesis is redirected to mannitol in the mutant strain lacking the ability to synthesize glycogen.
After watching this video, you should be able to quantitatively determine the glycogen content in cyanobacteria in five hours. When performing this procedure, it's important to remember to thoroughly resuspend the cells and solubilize glycogen pellets to obtain reliable results. Following this procedure, other methods like metabolic enzyme activity assays can be performed using the remaining cell lysates in order to answer additional questions, like how carbohydrate metabolism is regulated in cyanobacteria.
