Colony Phenotypic and Genotypic Analysis

AraC Inducible Promoter

• AraC is a chemically inducible promoter that can act as both a positive and negative regulator; its activity is modulated by the presence or absence of arabinose.

• AraC forms a dimer, which can either repress or activate transcription depending on its conformation and the presence of arabinose.

• The addition of arabinose activates the pBad promoter, leading to increased gene expression by altering the conformation of the AraC dimer.

• CAP (Catabolite Activator Protein) can also stimulate AraC to bind to specific DNA sequences (I1 and I2), enhancing the activation of the pBad promoter in the presence of arabinose. The binding of CAP is influenced by glucose levels; low glucose levels increase cAMP, which binds to CAP, promoting its binding to DNA.

Insert Orientation

• To determine the orientation of an insert, asymmetric restriction enzymes or primer placement is required to ensure correct gene expression.

• Example plasmid:

  • GFP (Green Fluorescent Protein): A reporter gene that emits green light when expressed.

  • Amp (Ampicillin resistance gene): Allows for selection of bacteria that contain the plasmid.

  • Kan (Kanamycin resistance gene): An alternative selection marker to Amp.

  • Restriction enzyme sites: BamHI, NotI, EcoRI: Common restriction sites used for cloning.

GFP Localization

• GFP expressed from a histone promoter: When using BamHI and EcoRI, it will localize throughout the cytosol, providing a general indication of gene expression.

  • Components:

    • Histone ORF (Open Reading Frame): Contains the genetic information for the histone protein.

    • Amp (Ampicillin resistance gene): Allows for selection of bacteria that contain the plasmid.

    • Histone Promoter: Drives the expression of the GFP gene.

    • Restriction enzyme sites: BamHI, EcoRI, NotI: Common restriction sites used for cloning.

• GFP fused to the histone protein: When using NotI and BamHI and keeping the construct in frame, it will localize to nucleosomes, allowing visualization of chromatin structure and dynamics.

  • Components:

    • Histone ORF (Open Reading Frame): Contains the genetic information for the histone protein.

    • Amp (Ampicillin resistance gene): Allows for selection of bacteria that contain the plasmid.

    • Histone Promoter: Drives the expression of the GFP gene.

    • Restriction enzyme sites: BamHI, EcoRI, NotI: Common restriction sites used for cloning.

Fluorescence

• Jablonski Diagram: Explains the physical process of fluorescence, detailing the energy transitions in a molecule.

  • Absorption: Molecule absorbs a photon and transitions to a higher energy state (S₁), where S₁ represents the first excited singlet state.

  • Non-radiative transition: Energy is lost as heat; the molecule moves to the lowest vibrational state of S₁, which is a rapid process known as internal conversion.

  • Fluorescence: Molecule returns to the ground state (S₀) by emitting a photon, typically at a longer wavelength than the absorbed photon. The emitted photon's energy corresponds to the energy difference between the lowest vibrational state of S₁ and a vibrational level in S₀.

Fluorescent Molecules

• Ethidium Bromide:

  • Chemical formula: C₂₁H₂₀BrN₃: A DNA intercalating agent widely used for visualizing DNA bands in electrophoresis.

• Green Fluorescent Protein (GFP):

  • Beta-barrel structure (~40 Å): Provides stability and protects the chromophore.

  • Alpha-helix (~30 Å): Important for the overall structure and function of GFP.

  • Chromophore formed by Ser65, Tyr66, and Gly67 residues: Responsible for fluorescence, with modifications altering its spectral properties.

History of GFP

• Doug Prasher identified and cloned the gene that encoded GFP but initially struggled to achieve expression in other organisms; this challenge was later overcome by Martin Chalfie.

• He did not receive the Nobel Prize for its discovery; the 2008 Nobel Prize in Chemistry was awarded to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien for the discovery and development of GFP.