Gene Expression and CRISPR Mechanisms in Bacteria

Tertiary Structure and Enzymatic Function

• Incorrect tertiary structure does not turn X gal blue
• Caspase nine: an enzyme that can cut DNA
  • Its action is conditional, being able to cut DNA under specific circumstances.

Gene Expression in Bacteria

• X Gene: When expressed, it produces caspase nine, which can then cut DNA.
• Important to understand the role of promoters:
  • Site where RNA polymerase binds to initiate transcription.
  • Some promoters work under specific conditions.

Arabinose Inducible Promoter

• Arabinose: A sugar classified as a monosaccharide.
  • The arabinose-inducible promoter requires arabinose to function;
  • When present, RNA polymerase binds and activates transcription.
  • Without arabinose, RNA polymerase cannot bind, thus the bacteria cannot repair DNA damage.

Functional Genes in Genetic Experiments

• LacZ Gene: Codes for the enzyme beta-galactosidase.
  • When functional, it breaks down X gal into a blue compound.
  • If repaired improperly (e.g., after a cut), it may produce non-functional proteins.

Transformation in Bacteria

• Transformation: The process where bacteria uptake DNA.
  • Bacteria will grow with different plasmids; spectinomycin is used to ensure only transformed bacteria survive:
  • If transformed, they resist the antibiotic.

Overview of Plasmids Used in Experiment

• Both plasmids used contain:
  • A gene for resistance to spectinomycin (SPT gene)
  • A section for donor DNA that can repair DNA.

Detailed Steps of Transformation

1. Transformation with p donor guide:
   • Contains the SPT gene and short guide RNA gene which guides caspase nine to the target.
2. Caspase nine is guided to the LacZ gene for cutting.
3. Cutting Action:
   • Introducing DNA break is essential for the next steps.
   • Only effective if arabinose is present to enable repair.
   • If unregulated, bacteria will die from the cut without fix (if arabinose not present).

Role of Short Guide RNA (sgRNA)

• sgRNA binds to caspase nine, directing it specifically to the LacZ gene.
• Critical for ensuring that cuts happen precisely in targeted gene areas.

Mechanism of Gene Editing Using CRISPR

• After cutting with caspase nine:
  • Donor DNA fills in the gap left; can change the gene.
  • Desired modifications made by inserting particular sequences into LacZ gene.

Nonsense Mutation Example

• A stop codon was inserted into the LacZ coding sequence:
  • Results in a dysfunctional beta-galactosidase protein.
  • Bacteria expressing this edited LacZ gene will appear white (lack ability to turn X gal blue).

Summary of Experimental Outcomes

• If there is no arabinose present during transformations:
  • The bacteria cannot repair DNA cuts and will die.
• If arabinose is available, transformation could lead to successful modifications being expressed, altering color of colonies due to faulty beta-galactosidase.

Conclusion of Gene Editing Process

• CRISPR allows precise targeting of genes for modification.
• Understanding regulation (like arabinose's role) is critical in determining the success of genetic transformations and resulting outputs.