[MUSIC] Hi, my name is Jesse Traller. I'm a senior phycologist at Global Algae Innovations where we grow microalgae large-scale outdoors. Today I'm going to speak to you about genetic engineering and the tools that have developed in a major class in microalgae diatoms. So diatoms are key primary producers in the ocean. They're a major class of eukaryotic microalgae belonging to the SAR subgroup. So SAR stands for stramenopiles, aveolates, and rhizaria, of which diatoms are part of the stramenopile lineage. They're responsible for roughly 45% of the ocean's primary productivity. And if we look at that on the global scale, 20% of the oxygen we breathe is credited to diatoms, so one in every five breaths. They come in two distinct shapes, centric and pennate. And the centric diatoms are cylindrical and shaped like a can, an example of which is shown in the red box on the right. And the pennate, shaped like a almond or a penne pasta shown in the blue box. They are thought of as jewels of the sea due to their silica cell wall. They require silicon as an essential macronutrient to grow their cell wall, which are these intricately designed structures that come in all different shapes and sizes, as you can see in both of the images on the right. And these cell walls are also investigated for nanomaterials. So there have been several genetic tools that have been developed in diatoms. Generally, diatoms are diploid. They do undergo sexual cycle, but the vast majority of their life cycle is in diploid state. They have been amenable to nuclear transformation. So vectors get incorporated within the nuclear genome and the transformants maintain a high stability. There are two different model species that are focused on often in diatom research, eccentric and pennate, that have been used for genetic studies. So thalassiosira pseudonana is eccentric diatom and phaeodactylum tricornutum, down at the bottom, is a pennate with two different morphotypes shown there. They both have had their genome sequenced, and they're relatively on the small side at 34 and 28 megabase pair. So these constructs developed in these organisms have been used across other diatom species, and there have been other species that have been focused on for genetic tools. There's a wide array of molecular tools that have been developed. Some examples are vectors that can encode for varying levels of mRNA expression, including inducible promoters. There's also been numerous studies on protein localization using fluorescent markers for visualization. And then, there have also been gene knockdown in gene silencing strategies such as RNAi, antisense, TALEN, and CRISPR CAS9 systems. So to transform a diatom, traditionally what has been used is a biolistic particle bombardment. But there have also been studies that have used electroporation and bacterial conjugation more recently. For biolistic particle bombardment, you use what's called a gene gun, shown off to the right there, and your vector of interest is coded to a tungsten bead, which is then shot onto a plate of diatom cells, and that vector then gets incorporated into the nuclear genome of the diatom. There have also been studies that have been using DNA insertion through conjugation. These conjugation based methods directly transfer design plasmids called episomes from E Coli bacteria into the diatoms. There's an image shown on the right there. These episomes are maintained in the diatom genomes as closed circles with the copy number that is the same order of the native chromosomes. They also replicate within the diatom. There are also numerous selectable markers that have been used, particularly those for antibiotic resistance. Examples are the ShBle cassette for zeocin resistance in nptII for neomycin. Vectors are often constructed using traditional technologies such as Gateway or Gibson cloning. So here's an example of a diatom expression vector off on the right-hand side. This vector here was for investigating carotenoid synthesis and in phaeodactylum. The Ptpsy blue arrow is the gene of interest. And it is fused with a GFP fusion, and the GFP is used to screen for positive transformants and also visualize where this protein is located in the cell. It's flanked by a promoter and a terminator, and these are from phugo xanthine chlorophyll protein, FCP, that have come from diatom DNA. And this promoter cassette is one example here of over expression vector, but there are other examples such as RPL41 and ACCase that are used within diatoms that have varying levels of mRNA. These over-expression constructs are super helpful for linking physiological processes to gene functions. So here's an example on the left that shows the mRNA level of wild-type and then the over-expression constructs. And you can compare and contrast these transformants with your wild-type. You can also create constructs of native promoters to understand what's naturally occurring with mRNA levels within the cell. So nitrate reductase is an example of an inducible promoter, and it's been very helpful in diatom research. This gives the ability to turn on and off gene expression for your gene of Interest. So here's two examples up here with eccentric and a pennate diatom. In phaeodactylum, you have the cells grown under nitrate NO3. And here, this is driving expression of GFP fluorescence shown in green. But when you have the cells under nitrogen free media without nitrogen, you have decrease in the GFP fluorescence, as you can't see the green. Similarly, with cyclotella cryptica on the right, you have growth under nitrate and visualization of GFP and green. And then, once you place the cells in a media with ammonia, a different nitrogen source, you have a decrease in the mRNA levels of your gene of interest, this being GFP fluorescence. So as I mention, there are strategies to do gene silencing in diatoms, and here's a study of this. This is using homozygous gene knockout of a native nitrate reductase gene using TALEN. So TALEN stands for transcription activator-like effector nuclease. And this study used homologous recombination to interrupt the target site and insert an antibiotic resistance marker, ShBle. So the zeocin resistance gene is flanked by the FCP promoter and terminator to drive expression of that, and it's within this nitrate reductase which has been knocked out. Positive knockouts were screened for growth on zeocin, and then lack of growth under nitrate. So in the culture plate on the left-hand side, you see the knockout without growth under NO3. But you can see recovery of growth in the presence of ammonia. And these knockouts have helped to understand nitrogen metabolism in diatoms, and particularly nutrient uptake. Here's another great example of research using vector constructs. So in this case, GFP is fused to a gene of interest, and the fluorescence from green fluorescent protein can be used to identify where the protein is located in the cell. So you can use GFP, but also there have been studies that have used YFP and CFP and others. So on the right-hand side, you have different transformants expressing carbonic anhydrase enzymes, and they're located in different areas of the cell. So in the orange box, you have carbonic anhydrase which has localized to the chloroplast. As you can see, it aligns with the red chlorophyll autofluorescence and the GFP as well. Additionally, you have another carbonic anhydrase in the blue box that localized to the plasma membrane. So this helps to understand cellular metabolism and organelle function and the processes that occur within the organelles. So in addition to understanding the biology of diatoms, there have been studies that have focused on biotechnological applications. So there have been studies that have focused on recombinant protein production, including producing therapeutic proteins in these diatom cells, and other work done on producing vaccines that can be used and fed to livestock. In addition to those, there have been studies which have utilized the silicon cell wall of diatoms to help deliver chemotherapeutic drugs to cancer cells. So in the study on the right, there is chemotherapeutic drug expressed on the cell surface of the diatom cell wall, and that had helped to drive delivery to cancer cells. Additionally, there have been genetic engineering studies which can be used to modify diatom metabolism to enhance native features that the cell has. So for instance, increasing the Omega-3 fatty acids or lipids in the cell by over-expression of those pathways. And additionally, increasing pigment production. One particular area of interest is focusing on carotenoids. So diatom molecular biology has been advanced and the opportunities are endless. Diatoms as microalgae are both ecologically and Industrial relevant organisms, and they have been amenable to sophisticated genetic engineering platforms. This broad molecular tool kit has been tested in a wide array of species and has really been used to understand critical cellular processes. Going forward, it's important for researchers to expand the toolkit into more industrially relevant species, and this will help to drive commercial production using diatoms. Additionally, there should be more refinement and optimization of tools such as improving efficiencies in transformation, and this will create a robust system that can be used to develop commercial production in diatoms.
