[MUSIC] I'm Anthony Berndt, a postdoctoral fellow at University of California at San Diego in Steve Mayfield's lab. I'll be presenting today on an introduction to molecular biology, synthetic biology and genetic engineering with specific focus on algae biotechnology. So, molecular biology is an approach for staying large-scale biological phenomenon at the scale of life's substituent macromolecules. This includes nucleic acids, proteins, lipids, carbohydrates and intermediary metabolites that make up these macromolecules. Molecular biology encompasses biochemical, genetic and informatics techniques for interrogating biological processes. Molecular biology has expanded to include more than just the study of single molecules. In the -Omics era, molecular biology includes genomics, proteomics and metabolomics amongst others that are the large-scale collective characterization and quantification of biological molecules that make up an organism or organisms. Large-scale databases are publicly accessible and can be rapidly searched and analyzed. Sequence genomes and transcriptomes have been a recent focal point because of the easy interconversion of DNA sequence and the primary amino acid sequence of proteins. Large DNA datasets can help us define gene structure. We can infer things such as promoters, regulatory regions and untranslated parts of transcripts. In the diagram presented here, I have aligned mRNA sequencing data with chromatin immunoprecipitation of a acetylated histone. This acetylated histone is often associated with open chromatin regions. When I plot the enrichment of this acetylated histone against the aligned transcripts, we can see a enriched peak just upstream of the promoter region marked in blue that potentially indicates actively regulated chromatin and the outer boundaries of this regulatory region. Taking the sequence of this gene, I can compare it to many other genes found in other algae species. And looking at the multiple sequence alignment presented in the top figure, we can see regions of high evolutionary conservation with the evolutionary conservation track plotted in yellow underneath, as well as regions where the conservation is not as strong. Highly conserved sequence may mean that these regions are functionally important. We can also use informatics methods to plot phylogenetic trees and infer evolutionary relationships of species based upon their DNA or protein sequence. Molecular biology has developed new tools for manipulating the genomes of organisms. A commonly utilized one is a loss of function mutation also known as a knock out. This uses DNA modification techniques to mutate or even delete a gene. Knock outs are often used for reverse genetic analysis. That is we have a gene sequence and then we look for a phenotype of a design mutant. Here we're showing the use of a targeting vector that integrates into the host genome through homologous recombination. So the host vector contains homology arms representing Exon1 and Exon3 of the targeted gene as well as a neomycin resistance cassette. So this gives resistance to the antibiotic neomycin. Upon integration and recombination into the host's genome, the second Exon is deleted, generating a non-functional transcript and the now mutated organisms can be selected for an neomycin containing media. Another method for manipulating the genomes of algae and other organisms is a gain of function mutation or a knock in. This is the addition of genetic material. It's often used to either restore the function of a mutant or generate new function. Here we're showing the integration of an expression cassette into the host's genome. In this case, it's randomly integrating into the genome in an untargeted manner and generating the expression of a recombinant protein. There are two common transformation methods used in algae, the first one being electroporation. This is usually used for manipulating the nuclear genome. In this case, DNA is mixed with the cells and an electrical potential applied across the cells. This opens up pores in the cell membranes and allows the entry of DNA into the host. Since DNA is negatively charged, it moves toward the positive electrode, the DNA can then exist as a extrachromosomal, episomal element or integrate into the host's genome. Another method used to transform algae is the use of biolistic delivery or a gene gun. This is generally used for chloroplast genome transformation. In this case, compressed helium is used to propel DNA coding gold beads at high speed into a lawn of algae. The DNA then integrates into the chloroplast genome and the transformed algae can be selected for on antibiotic containing media. Over the years, a new field of biology has developed called synthetic biology, synthetic biology is primarily focused on the design, modeling and tuning of biological pathways. If molecular biology answers the question, how does it work, synthetic biology answers the question of now that we know how it works, what can we build with it? Molecular biology provides the toolkit whereas synthetic biology uses these tools to build new systems. Here we're showing an expression vector that we have used to express the a high-value protein of interest. Going left to right, we can start with the AR1 promoter. So this is a semi-synthetic promoter that utilizes the promoters of two highly expressed genes, heat shock 70A and Rbcs2 to generate high recombinant protein expression. These promoters seem to act synergistically and give much higher expression than either single promoter by itself. In yellow are the five prime and three prime untranslated regions of Rbcs2 that stabilize the mRNA transcript. The integration of this transgene is selected for using the bleomycin resistance cassette. So, this antibiotic resistance gene stoichiometrically binds to bleomycin or zeocin antibiotics and leads to the selection of only very high expressing clones. This antibiotic resistance gene itself contains the first intron of Rbcs2, this intron itself acts as a transcriptional enhancer. Following this antibiotic resistance gene is a viral self-cleaving peptide. This peptide causes the production of two separate peptides, the antibiotic resistance gene and the gene of interest from a single mRNA. Finally we have the gene of interest shown in red, this gene of interest has been codon optimized generating high translational efficiency of our gene of interest. To date, multiple proteins have been made in algae, many of these have significant commercial or biomedical applications. For example, monoclonal antibodies had been produced in the algae Chlamydomonas reinhardtii and these proteins may have significant application to anti-cancer treatment. Additionally, colostrum proteins, colostrum is the milk that mammals produce in the first few days to weeks after giving birth have been produced. These include osteopontin which has been shown to have significant beneficial effects to gut health. We can also express recombinant proteins in algae to construct new biosynthetic pathways, one example of this is the production of patchouli which is the major component of patchouli oil. Thanks for viewing this overview of molecular biology and biotechnology in algae and I hope you enjoy the rest of the course.
