Biotechnology: Principles & Processes Flashcards

Principles of Biotechnology

• Definition of Biotechnology (European Federation of Biotechnology - EFB):

  • "The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services."

• Modern biotechnology rests on two primary foundation techniques:

  • Genetic Engineering: Techniques to alter the chemistry of genetic material ($DNA$/$RNA$) to introduce into host organisms, thus changing the phenotype of the host cell.
  • Bioprocess Engineering: Maintenance of sterile (microbial contamination-free) conditions to enable growth of desired microbes/eukaryotic cells in large quantities for commercial production.

Tools of Recombinant DNA Technology

Restriction Enzymes ("Molecular Scissors")

• Belong to a larger class of enzymes called Nucleases.
  • Exonucleases: Remove nucleotides from the ends of the $DNA$
  • Endonucleases: Cut at specific positions within the $DNA$ strand.
• Palindromic Recognition Sequences:
  • Endonucleases inspect $DNA$ length and cut specifically at palindromic sequences where the sequence reads the same in the $5' \rightarrow 3'$ direction on both complementary strands.
• Action of EcoRI Enzyme:
  • Palindromic sequence:
  • $5'\text{- G A A T T C -}3'$
  • $3'\text{- C T T A A G -}5'$
  • $EcoRI$ cuts specifically between bases $G$ and $A$ on both strands.
  • Leaves sticky ends: --A-A-T-T-C / G--.

Naming of Restriction Enzymes

• Standard nomenclature rules for restriction enzymes:
  • First letter: Derived from the genus name (Capitalized).
  • Next two letters: Derived from the species name (Lowercase).
  • Next letter: Indicates the specific strain.
  • Roman numeral: Indicates the order of isolation from that strain.
• Example ($EcoRI$):
  • $E$ $ ightarrow$ Escherichia
  • $co$ $ ightarrow$ coli
  • $R$ $ ightarrow$ Strain $RY13$
  • $I$ $ ightarrow$ $1^{\text{st}}$ enzyme isolated

Cloning Vectors ("Vehicle DNA")

• Plasmids and Bacteriophages have the ability to replicate within bacterial cells independent of chromosomal control.
• Key features required for cloning into a vector:
  • Origin of Replication ($ori$): Sequence where replication starts; controls copy number of linked $DNA$
  • Selectable Marker: Genes encoding resistance to antibiotics (e.g., $amp^R$, $tet^R$ in $pBR322$) used to identify and select transformants from non-transformants.
  • Cloning Sites: Unique recognition sites for restriction enzymes. Insertion of a foreign gene into a marker gene inactivates it (Insertional Inactivation).

Vector Plasmid pBR322 Structure

• Total Size: $4361\text{ bp}$
• Selectable Markers & Restriction Sites:
  • $amp^R$ (Ampicillin resistance gene): Contains recognition sites for $PstI$ and $PvuI$
  • $tet^R$ (Tetracycline resistance gene): Contains recognition sites for $BamHI$ and $SalI$
  • Additional Unique Restriction Sites: $EcoRI$, $ClaI$, $HindIII$
• Functional Regions:
  • $ori$ (Origin of replication): Site initiating $DNA$ replication and controlling vector copy number.
  • $rop$: Codes for proteins involved in plasmid replication.

• Insertional Inactivation Example:
  • Insertion of recombinant $DNA$ within the coding sequence of the $\beta\text{-galactosidase}$ enzyme inactivates the enzyme.
  • Recombinant colonies produce white colonies, whereas non-recombinants produce blue colonies in the presence of a chromogenic substrate.

Competent Host Formation (Transformation)

• $DNA$ is a hydrophilic molecule and cannot pass through cell membranes directly.
• Host cells are rendered competent via specific transformation protocols:
  • Chemical Method:
  • Target Organism: Bacteria (E. coli)
  • Mechanism / Process: Treatment with divalent cation ($Ca^{2+}$), followed by heat shock ($42^\circ\text{C}$) and placement back on ice.
  • Micro-injection:
  • Target Organism: Animal Cells
  • Mechanism / Process: Recombinant $DNA$ is directly injected into the nucleus using a glass micropipette.
  • Biolistics / Gene Gun:
  • Target Organism: Plant Cells
  • Mechanism / Process: Cells bombarded with high-velocity microparticles of gold or tungsten coated with $DNA$
  • Disarmed Pathogens:
  • Target Organism: Plants & Animals
  • Mechanism / Process: Modified Agrobacterium tumefaciens (plants) or Retroviruses (animals) used to infect cells safely.

Steps in Recombinant DNA Technology

Step 1: Isolation of Genetic Material (DNA)

• Cell walls are digested using specific lytic enzymes:
  • Lysozyme: Digests bacterial cell walls.
  • Cellulase: Digests plant cell walls.
  • Chitinase: Digests fungal cell walls.
• $RNA$ is removed by treatment with Ribonuclease.
• Proteins are removed by treatment with Protease.
• Purified $DNA$ precipitates out as fine threads upon adding Chilled Ethanol.

Step 2: Gel Electrophoresis (Separation & Isolation)

• Negatively charged $DNA$ fragments move toward the Anode (+) under an electric field through an agarose matrix.
• Smaller $DNA$ fragments move faster and travel farther across the gel.
• $DNA$ is stained with Ethidium Bromide (EtBr) and exposed to UV light to visualize bright orange-colored bands.
• Elution: The process of cutting out and extracting $DNA$ bands from the agarose gel matrix.

Step 3: Polymerase Chain Reaction (PCR)

• Amplifies a specific gene segment in vitro into billions of copies using three sequential steps:
  • 1. Denaturation ($\sim 94^\circ\text{C}$):
  • High temperature breaks hydrogen bonds, separating double-stranded $DNA$ into two single strands.
  • 2. Annealing ($\sim 54^\circ\text{C}$):
  • Two sets of oligonucleotide primers bind to complementary regions at the $3'$ ends of single strands.
  • 3. Extension ($\sim 72^\circ\text{C}$):
  • Thermostable Taq Polymerase (isolated from the bacterium Thermus aquaticus) extends primers using $dNTPs$. It remains active at high temperatures.

Bioreactors & Downstream Processing

Bioreactors

• Large vessels ($100\text{--}1000\text{ liters}$) providing optimal growth parameters (temperature, $pH$, substrate, salts, vitamins, oxygen) to transform raw materials into specific recombinant products biologically.
• Simple Stirred-tank Bioreactor:
  • Equipped with an impeller for continuous mixing and air availability throughout the culture.
  • Has foam controller, temperature, and $pH$ control systems.
• Sparged Stirred-tank Bioreactor:
  • Sterile air bubbles are sparged (bubbled) through the reactor chamber, dramatically increasing surface area for gas exchange.

Downstream Processing

• Refers to all collective operations after the biosynthetic stage is completed:
  • Separation & Purification: Product is isolated and purified from the culture broth.
  • Formulation: Product is formulated with suitable preservatives.
  • Quality Control & Clinical Trials: Strict clinical testing is performed prior to commercial distribution.

Summary of Key Enzymes in Biotechnology

• Restriction Endonuclease:
  • Source / Origin: Bacterial cells
  • Primary Biological Function: Cuts double-stranded $DNA$ at specific recognition sequences.
• DNA Ligase:
  • Source / Origin: Bacteriophage / E. coli
  • Primary Biological Function: Joins sticky/blunt ends by forming phosphodiester bonds.
• Taq Polymerase:
  • Source / Origin: Thermus aquaticus
  • Primary Biological Function: Thermostable $DNA$ polymerase used in the PCR extension step.
• Lysozyme / Chitinase:
  • Source / Origin: Egg white / Fungi
  • Primary Biological Function: Breaks bacterial/fungal cell walls during $DNA$ extraction.
• Alkaline Phosphatase:
  • Source / Origin: Calf intestine / Bacteria
  • Primary Biological Function: Prevents self-ligation of vector by removing the $5'$ phosphate group.