🧪Synthetic Biology Unit 2 – Molecular Biology & Genetics Basics

Key Concepts and Definitions

• Molecular biology studies the structure, function, and interactions of biological macromolecules (DNA, RNA, proteins)
• Genetics investigates the inheritance and variation of traits from parents to offspring
  • Includes the study of genes, alleles, and chromosomes
• Central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins
• Genome refers to the complete set of genetic material in an organism
  • Consists of DNA organized into chromosomes
• Gene is a segment of DNA that encodes instructions for making a specific protein or functional RNA molecule
• Genotype represents an organism's genetic makeup while phenotype is the observable characteristics resulting from gene expression
• Alleles are alternative forms of a gene that can result in different phenotypes
• Mutations are changes in the DNA sequence that can lead to genetic variation and potentially altered phenotypes

DNA Structure and Function

• DNA (deoxyribonucleic acid) is the hereditary material in nearly all living organisms
• DNA is composed of four nucleotide bases: adenine (A), thymine (T), guanine (G), and cytosine (C)
  • Bases pair through hydrogen bonds: A with T and G with C
• DNA has a double helix structure with two complementary strands running antiparallel to each other
• The sugar-phosphate backbone provides structural stability and allows for DNA replication
• DNA serves as a template for its own replication during cell division ensuring genetic information is passed to daughter cells
• DNA also acts as a template for transcription the process of producing RNA molecules
• Genetic information stored in DNA is used to guide the synthesis of proteins through the process of translation

Gene Expression and Regulation

• Gene expression is the process by which genetic information is used to synthesize functional gene products (proteins or RNA)
• Transcription is the first step of gene expression where DNA is used as a template to produce complementary RNA molecules
  • Transcription is catalyzed by RNA polymerase enzymes
• Translation is the second step of gene expression where the genetic code carried by mRNA is used to synthesize proteins
  • Occurs at ribosomes with the help of tRNA molecules
• Gene regulation controls when where and to what extent genes are expressed
• Promoters are DNA sequences upstream of genes that initiate transcription and serve as binding sites for transcription factors
• Enhancers are regulatory DNA sequences that can increase gene expression even when located far from the gene
• Silencers are regulatory DNA sequences that can decrease or suppress gene expression
• Epigenetic modifications (DNA methylation histone modifications) can alter gene expression without changing the DNA sequence

Mutations and Genetic Variation

• Mutations are permanent changes in the DNA sequence that can arise from errors during DNA replication exposure to mutagens or viral infections
• Point mutations involve the change of a single nucleotide base and can be classified as silent missense or nonsense mutations
  • Silent mutations do not change the amino acid sequence of the protein
  • Missense mutations result in a different amino acid being incorporated into the protein
  • Nonsense mutations introduce a premature stop codon leading to a truncated protein
• Insertions and deletions (indels) are mutations that add or remove nucleotides from the DNA sequence
  • Can cause frameshift mutations if the number of nucleotides added or removed is not a multiple of three
• Chromosomal mutations involve large-scale changes in the structure or number of chromosomes (translocations duplications deletions)
• Genetic variation refers to the differences in DNA sequences among individuals in a population
• Sources of genetic variation include mutations recombination during meiosis and independent assortment of chromosomes
• Genetic variation is essential for evolution as it provides the raw material for natural selection to act upon

Inheritance Patterns

• Mendelian inheritance describes the passing of traits from parents to offspring following the laws of segregation and independent assortment
  • Law of segregation states that alleles segregate during gamete formation and each gamete carries only one allele for each gene
  • Law of independent assortment states that alleles for different genes assort independently during gamete formation
• Punnett squares are diagrams used to predict the probability of offspring genotypes and phenotypes based on the genotypes of the parents
• Autosomal dominant inheritance occurs when a single dominant allele is sufficient to express the trait
  • Affected individuals typically have one affected parent
• Autosomal recessive inheritance requires two copies of the recessive allele for the trait to be expressed
  • Affected individuals often have unaffected parents who are carriers of the recessive allele
• X-linked inheritance involves genes located on the X chromosome and typically affects males more frequently than females
  • X-linked dominant traits are expressed when one copy of the dominant allele is present on the X chromosome
  • X-linked recessive traits require the presence of the recessive allele on the X chromosome in males or two copies in females
• Codominance occurs when both alleles are expressed equally in the phenotype (ABO blood group system)
• Incomplete dominance results in a phenotype that is intermediate between the two homozygous phenotypes (pink flowers in snapdragons)

Molecular Biology Techniques

• Polymerase Chain Reaction (PCR) is a technique used to amplify specific DNA sequences by using primers DNA polymerase and thermal cycling
  • Allows for the rapid generation of millions of copies of a target DNA sequence
• DNA sequencing determines the precise order of nucleotides in a DNA molecule
  • Sanger sequencing is a traditional method that uses dideoxynucleotides (ddNTPs) to terminate DNA synthesis at specific bases
  • Next-generation sequencing (NGS) technologies enable high-throughput parallel sequencing of multiple DNA fragments
• Gel electrophoresis separates DNA RNA or protein molecules based on their size and charge by applying an electric field to a gel matrix
  • Agarose gel electrophoresis is commonly used for DNA and RNA separation
  • SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is used for protein separation
• Restriction enzymes are endonucleases that recognize and cleave specific DNA sequences enabling the cutting and manipulation of DNA fragments
• DNA ligation joins DNA fragments together by forming phosphodiester bonds between the 3' hydroxyl and 5' phosphate groups of adjacent nucleotides
  • Catalyzed by DNA ligase enzymes
• Plasmids are small circular DNA molecules that can be used as vectors for cloning and expressing genes in bacteria
• Bacterial transformation introduces foreign DNA (such as plasmids) into bacterial cells enabling the amplification and expression of the introduced genes

Applications in Synthetic Biology

• Synthetic biology applies engineering principles to design and construct novel biological systems or redesign existing ones
• Metabolic engineering involves modifying metabolic pathways in organisms to produce desired compounds (biofuels pharmaceuticals)
  • Can be achieved by introducing new enzymes or optimizing existing pathways
• Genome editing techniques (CRISPR-Cas9 TALENs zinc finger nucleases) allow for precise modification of DNA sequences
  • Enable targeted gene knockouts insertions replacements and regulation
• Biosensors are engineered biological systems that detect and respond to specific stimuli (small molecules environmental conditions)
  • Can be used for environmental monitoring disease diagnostics and drug discovery
• Synthetic gene circuits are designed to perform specific functions in living cells (toggle switches oscillators logic gates)
  • Rely on the precise regulation of gene expression and protein-protein interactions
• Xenobiology explores the creation of novel biological systems based on non-natural molecules or altered genetic codes
  • Aims to expand the capabilities of living systems and create new forms of life
• Bioremediation uses engineered microorganisms to degrade or neutralize pollutants in the environment
  • Can be applied to clean up oil spills heavy metals and other contaminants

Common Challenges and Future Directions

• Standardization and modularity are essential for the efficient design and assembly of synthetic biological systems
  • Requires the development of standardized genetic parts (BioBricks) and assembly methods
• Biological complexity and context-dependence can make it difficult to predict the behavior of engineered biological systems
  • Requires a better understanding of gene regulation metabolic networks and cellular interactions
• Biosafety and biosecurity concerns arise from the potential misuse of synthetic biology technologies
  • Necessitates the development of robust safety measures and regulations
• Ethical and societal implications of synthetic biology need to be addressed through public engagement and policy discussions
  • Includes issues related to intellectual property environmental impact and the creation of novel life forms
• Scaling up and commercialization of synthetic biology applications require overcoming technical and economic barriers
  • Involves optimizing production processes reducing costs and ensuring product safety and efficacy
• Integration of computational tools (modeling simulation machine learning) is crucial for the design and optimization of synthetic biological systems
• Expansion of the genetic code and the development of orthogonal biological systems offer new opportunities for creating novel functions and materials
• Collaboration between disciplines (biology engineering computer science social sciences) is essential for advancing the field of synthetic biology and addressing its complex challenges