Synthetic Biology Lecture Notes PDF

Synthetic Biology -redesign life How to generate transgenic or genetically modified organism? General process 1. Isolation/amplification of selected genes 2. Fusion o...

Synthetic Biology -redesign life How to generate transgenic or genetically modified organism? General process 1. Isolation/amplification of selected genes 2. Fusion of selected gene with selected vector (recombine DNA) 3. Delivery of recombinant DNA into a host cell. 4. Selection of genetically modified host 5. Breeding for offspring with desired genetic modification DNA cloning Recombinant protein production, desirable phenotype  Genetic engineering, from one molecule to an entire pathway Transfer of an entire pathway from its natural host to a production host. Applications: Production of new chemicals, e.g. alcohol, solvents, biofuels, food additives, dyes, and antibiotics, etc. Degradation of agricultural products/wastes and environmental pollutants, etc. 一  growing palm trees For bioFuel contlicts with Farm Land needed to sustain Food Supps. https://www.eia.gov/energyexplained/biofuels/ Engineering the xylose pathway for biofuel production Cellulose Engineer a cell that can break down xylose into simplien components partially degradecellulose majority 1 Of Biomass Cannot sed. , 1 transporter transporter ( Ixpon 1 ) - ( (mport ) https://doi.org/10.1016/j.copbio.2019.01.006  From xylose waste to alcohol From plant carbohydrate polymers to Pathway Engineering for Xylose Utilization alcohol Xylulose 5-P transaldolase Xylose xylose isomerase waste Transketolase xylulose kinase Fructose 6-P Glyceraldehyde 3P Alcohol Xylose, a five-carbon sugar, is produced abundantly during breakdown of plant polysaccharide polymers but is not used in fermentation to alcohol. Introducing four genes in the biosynthesis pathway in Zymomonas (a bacteria naturally found in alcoholic drinks) into yeast to increase the alcohol yield. Sequential (or simultaneous) transfer of multiple genes in the pathway The impossible meat The impossible meat Compared to a beef patty, the Impossible Burger requires 96% less land and 89% fewer greenhouse gases. But plant proteins don’t taste like meat! The impossible meat Expression Constructs and Methods of Genetically Engineering Methylotrophic Yeast Other examples Microbial production of shark squalene (fish oil), stevia, milk whey where plant/animal pathway was moved en mass into a microbe. Useful strategies to improve production (using squalene as an example): 1. Upregulation of the sterol pathway. 2. Knockout of competing pathways. 3. Regeneration of co-factor NADPH. 4. Compartmentalizing the pathway into the mitochondria, where substrates/cofactors/enzymes can reach higher concentration; also reduces potential cytotoxicity. 5. Storage of squalene in lipid droplets. https://link.springer.com/article/10.1007/s11274-022-03273-w Redesigned cells as products Immune cell therapies https://doi.org/10.1016/j.cell.2020.03.001 Redesigned cells as products PROVEN, nitrogen-fixing bacteria that work for corns https://www.pivotbio.com/our-technology Redesigned cells as products PROVEN, nitrogen-fixing bacteria that work for corns https://www.pivotbio.com/our-technology Synthetic biology– an example https://2021.igem.org/Team:NUS_Singapore https://2021.igem.org/Team:NUS_Singapore https://2021.igem.org/Team:NUS_Singapore https://2021.igem.org/Team:NUS_Singapore  Shinebothlight☆ Yeabie. A broad range anti-microbial protein ㉑ Completely hactivatebeforeDiBposal. Building regulatory circuits in yeast https://2021.igem.org/Team:NUS_Singapore Building regulatory circuits in yeast https://2021.igem.org/Team:NUS_Singapore Culture Yeast in the Dark Defencin Flocculate kill https://2021.igem.org/Team:NUS_Singapore Synthetic biology Seeks interchangeable parts from natural biology to assemble into systems that act unnaturally. Uses unnatural molecules to reproduce emergent behaviours from natural biology. synthetic unnatural molecules synthetic unnatural organisms New letters in genetic alphabet a, Chemical structure of the d5SICS–dNaM UBP compared to the natural dG–dC base pair. Nature volume 509, pages385–388 (2014) It sounds perfect! But how to enable the functions of unnatural nucleotides as genetic material? they need to pair with Eah other introduced intothecell they need to be Polymerase l tRVtall need to be I RNA DNA Polymerase Engineered.  Unnatural amino acids- Amino acids that are not one of the 20 naturally-occurring ones. Function and biophysical properties of a protein – determined by the collective biophysical properties of the amino acids found in the protein Making proteins with new, unnatural amino acids- Translating stop codons to unnatural amino acids- Biochem (Lond) Volume 45 Issue 1 2023 2-6 10.1042/bio_2023_102 TAG Engineered https://doi.org/10.1042/bio_2023_102 Translating stop codons to unnatural amino acids- Biochem (Lond) Volume 45 Issue 1 2023 2-6 10.1042/bio_2023_102 https://doi.org/10.1042/bio_2023_102 Unnatural amino acids- Function and biophysical/biochemical properties of a protein – determined by the collective biophysical properties of the amino acids found in the protein  Synthetic biology Seeks interchangeable parts from natural biology to assemble into systems that act unnaturally. Uses unnatural molecules to reproduce emergent behaviours from natural biology. synthetic unnatural molecules synthetic unnatural organisms 一  Synthetic bacteria and yeast 2008 Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome (0.58 mb) (Science 319:1215- 1220) 2010 Creation of a bacterial cell controlled by a chemically synthesized genome (M. mycoides, 1 mb) 2011 Yeast genome Sc2.0 project to synthesize entire yeast genome (12 Mb for Saccharomyces cerevisiae) 2011-2017, Nine strains of yeast in which 1 or 2 of the 16 chromosomes is replaced by synthetic DNA 2019, Total synthesis of Escherichia coli with a recoded genome (4 mb Syn61) 2021, designer bacteria: rewrite genetic code. 2008 DOI: 10.1126/science.1151721 Gibson et al (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52-56. 2010 Creation of a bacterial cell controlled by a chemically synthesized genome (M. mycoides, 1 mb) Synthesis of whole M. mycoides genome Transfer the synthetic genome to a closely related recipient M. capricolum The donor genome spontaneously replaced the host genome (by an unknow mechanism) The synthetic lineage is phenotypically indistinguishable from the native donor cells Gibson et al (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52-56. Recommended reading Cohan (2010) Current Biology 20(16):R675-7 Features of synthetic cells Easy manipulation of the synthetic genome for DNA insertion, deletion and targeted changes Inserted new genes to produce useful proteins Manipulate biological pathways to achieve designer bacteria Updates on synthetic chromosomes in yeast Dai et al (2020) Sc3.0 revamping and minimizing the yeast genome. BMC Genome Biology 21:205, https://doi.org/10.1186/s13059-020-02130-z  Scales of synthetic biology Minimal life Engineering of single genes Base pairs 102 103 104 105 106 107 108 109 Building a new pathway or a new regulatory circuit  Driving forces https://www.nature.com/articles/s41570-022-00456-9 Driving forces Challenges and concerns Safety and security concerns. Synthetic biology could pose a significant threat to national security if it were used for nefarious purposes, such as developing new biological or chemical weapons. Additionally, the computational tools used for synthetic biology could be vulnerable to cyberthreats such as automation hacking. For example, a bad actor could manipulate or steal information and use it to create drugs, weapons, or other harmful products. Environmental effects. Organisms made using synthetic biology and released into the environment could have unknown, unintended, and potentially irreversible effects on ecosystems. Such effects could be widespread if, for example, these organisms negatively affected food or water systems. Public acceptance and access. The public may hesitate to accept certain applications of synthetic biology due to concerns about interfering with nature and about unintended effects. In addition, some medical applications could be inaccessible for some patients due to cost or location of treatment centers. https://www.gao.gov/products/gao-23-106648 Future perspectives Synthetic biology market— 2021 $10 billion 2030 $37-100 billion