Using BioID to Understand Protein Expression in Microalgae

Jacob Oyler Author

07/27/2021 Added

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The development and implementation of a pulldown assay using a protein complex.

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[00:00:01.170]Welcome to this presentation on using bio ID to understand protein interactions
[00:00:05.850]in microalgae, let's get started.
[00:00:10.560]A lot of discussion and planning has occurred in recent years on how do you
[00:00:13.860]transition from oil based fuels to more environmentally sustainable source of
[00:00:18.000]biofuel. At first, it came in the form of first-generation biofuels,
[00:00:22.350]which consists of converting edible crops,
[00:00:24.360]such as corn and soy beans into ethanol.
[00:00:27.170]That is used as a traditional gasoline alternative. However,
[00:00:30.690]with the rising demand for these crops to be used as feed crops for animals,
[00:00:34.770]land for housing and urban development and their traditional use as direct food
[00:00:38.880]for the population,
[00:00:40.200]a new alternative has been sought after the most recent advancements in this
[00:00:44.280]area has been with third generation biofuels,
[00:00:46.860]which are based around the cultivation of lipids from algae species that can be
[00:00:50.790]converted into a biofuel. However,
[00:00:53.520]there are currently several factors that have prevented this from becoming
[00:00:56.220]feasible on the worldwide scale. Essentially,
[00:00:59.100]the amount of resources it takes to create algae-based biofuels makes it
[00:01:02.670]considerably more expensive than currently available gasoline an
[00:01:07.110]experimental approach towards tackling.
[00:01:08.850]This issue has been to increase the production of lipids coming from algae in
[00:01:13.350]order to execute this,
[00:01:14.730]the mechanism that concentrates carbon dioxide into the cell thylakoids ,
[00:01:18.600]within the chloroplast,
[00:01:20.010]which we call the CCM needs to be understood to a much greater extent.
[00:01:24.630]Our approach has been specifically focused on understanding how the gene
[00:01:28.140]expression of the three pumps on the cells.
[00:01:30.570]Three different membranes can be increased to produce more lipids in the algae
[00:01:34.590]species, Chlamydomonas
[00:01:37.050]seen in figure one is a representation of how the CCM works in Chlamydomonas
[00:01:40.950]First carbon dioxide is converted into bicarbonate.
[00:01:44.400]So it won't escape the cell
[00:01:45.240]Then it is transported across the cell membrane by the first pump,
[00:01:49.200]which is represented by the purple arrow.
[00:01:51.870]After that it is transferred across the chloroplast membrane by the second
[00:01:55.500]pump, and finally across the thylakoids membrane by the third pump.
[00:01:59.820]At this point, it is converted back into carbon dioxide and used in
[00:02:03.480]The typical photosynthetic reaction previous work has shown that the gene
[00:02:07.800]expression of three pumps is controlled by the gene VIG1.
[00:02:15.290]Although the VIG1 gene is known to be involved.
[00:02:17.960]It is not known what specific protein interactions occur with the VIG1 to
[00:02:22.820]control the pump expression. In order to determine this,
[00:02:25.910]a pull-down assay was designed by assembling a bio ID gene and
[00:02:30.890]the VIG1 gene into one protein complex bio ID is a protein that can be
[00:02:35.600]turned on and off by a process called biotinylating at its active site
[00:02:39.860]which is controlled by the biotynylating enzyme.
[00:02:42.770]Seen in figure two is a diagram of how the pull-down assay works.
[00:02:46.880]The turned off BioID protein.
[00:02:48.710]is first turned on by being biotynylated by the enzyme. Next,
[00:02:52.340]the now active bio ID is able to react and bind to the VIG1 protein
[00:02:57.260]Then the unknown proteins that normally interact with the VIG1 to control,
[00:03:01.090]the pumps will bind to the VIG1 protein
[00:03:04.090]These are represented by the black circles and these unknown proteins are also
[00:03:07.690]biotinylated. Now that the complex is completely bound together.
[00:03:11.890]Chemically induced affinity capture can be used with Streptavidin
[00:03:15.280]beads.
[00:03:16.240]This just means that the biotinylated proteins are chemically attracted to
[00:03:19.720]the Streptavidin
[00:03:20.770]So this can be used to isolate the complex and harvest the unknown proteins for
[00:03:24.580]analysis,
[00:03:26.050]the bio ID and VIG1 genes were transformed into Chlamydomonas via
[00:03:30.340]a plasmid
[00:03:34.500]A plasmid is a circular piece of DNA.
[00:03:36.810]It is often used for inserting new genes into a target source.
[00:03:40.470]Seen in figure three is the plasmid design that was used.
[00:03:43.630]The plasmid was 5,300 base pairs long and consists of four main parts.
[00:03:48.990]The translation start codon in the beginning of the genes that actually gets
[00:03:52.500]coded in the proteins. Once in the new cell,
[00:03:56.520]the first part is the blue color is the flag marker that allows the
[00:04:01.200]protein complex to be identified with an antibody during protein analysis.
[00:04:05.820]This was directly connected to the bio ID gene in orange and the VIG1 gene in
[00:04:10.500]green,
[00:04:11.280]further along the plasma and red is a Hygromycin antibiotic resistance gene.
[00:04:16.440]This resistance gene is used because when the transformation is performed,
[00:04:20.010]the algae cells will be placed on the growing plate that contains the antibolic
[00:04:23.550]Hygromycin.
[00:04:24.930]If the transformation is not successful and the plasmid is not taken up,
[00:04:28.950]then the algae cells will die. Instead of growing on the plate.
[00:04:35.520]After the algaes colonies grew up on the antibiotic plates,
[00:04:38.730]they were transferred to new growing plates that contain a 10 millimolar
[00:04:42.180]concentration of the chemical FI.
[00:04:44.790]If the algae colonies properly express the VIG1 protein,
[00:04:47.760]they will grow on that FI if not,
[00:04:49.770]then FI is toxic to the cells and the colonies will die.
[00:04:53.730]Seen in figures four and five is the same set of algae colonies on the
[00:04:57.660]tap plate,
[00:04:58.560]which is control plate to make sure they all grew on their normal growing media
[00:05:01.980]plate and the FI plate,
[00:05:04.320]all the colonies grew successfully on the tap plate,
[00:05:06.540]which was expected. For the FI plate,
[00:05:08.940]the positive control DF3 grew successfully and the negative control m70
[00:05:12.840]died like it was supposed to. For the numbered colonies.
[00:05:17.490]The different level of VIG1 expression was expected to be correlated to the
[00:05:21.330]amount they grew, dark green colonies, such as numbers, two, five,
[00:05:25.140]and nine should have had the most,
[00:05:27.240]VIG1 expression while other colonies like 10 and 14 had intermediate
[00:05:31.620]growth and expression and colonies like 11 and 12 had no expression and no
[00:05:35.790]growth. The colonies with the most,
[00:05:37.770]VIG1 expression were the best candidates for increased lipid
[00:05:40.500]production. So they were chosen for Western blot protein analysis,
[00:05:45.300]figure six is the Western blot.
[00:05:46.990]of the colonies that grew the most and had the greatest chance of showing expression
[00:05:50.610]of the protein complex.
[00:05:52.410]Seeing on the left are two black marks that are the protein marker at 67
[00:05:56.670]kilodalton and 170 kilodalton the higher, the kilodalton, the larger,
[00:06:01.130]the protein in question is. A flag antibody was used to locate the protein
[00:06:05.300]complex.
[00:06:06.110]That should have been around a hundred kilodaltons in size as can be seen
[00:06:10.160]inside the brackets.
[00:06:11.270]The protein does not appear to be present in any of the samples.
[00:06:14.570]So in the future, the experiment will have to be repeated.
[00:06:17.570]to find colonies that show clear expression of the protein complex
[00:06:23.780]going forward.
[00:06:24.590]Once the protein complex is clearly expressed and the other proteins in the VIG1
[00:06:28.430]interaction are collected.
[00:06:30.140]Mass spectroscopy will need to be conducted to enhance our understanding of
[00:06:34.220]exactly what other proteins are present in the complex and how they interact
[00:06:38.330]with VIG1 to control the gene expression of the membrane pumps.
[00:06:42.050]This will potentially be a large step towards engineering and economically
[00:06:45.710]feasible, algae biofuel.
[00:06:50.690]Special thanks to this Cerutti lab and NSF grant,
[00:06:53.780]2 0 5 0 5 7 4 for making this project possible.

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Using BioID to Understand Protein Expression in Microalgae

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