D2D

Overview of the Annealing Step

The annealing step follows the initial process and involves binding oligonucleotides (oligos) to single stranded DNA (ssDNA) that contains a specific gene (the bagel bee gene).

Definition: Plasmids are small, circular extrachromosomal pieces of DNA found in bacteria.
Natural Occurrence: Bacteria naturally produce plasmids, which are separate from their chromosomal DNA.
Function: Plasmids often carry what are termed "luxury genes," which may confer advantages such as antibiotic resistance, the ability to metabolize unusual substrates like sugars, or traits that increase virulence, such as toxin production or capsule formation.
Role in Biotechnology: In molecular biology, plasmids serve as vectors for transferring genes between different species.
Application in the Class: The class is conducting an experiment where they will generate a mutated plasmid vector containing the bagel bee gene.

Phosphorylation of Oligos: The oligos are first phosphorylated as part of the initial steps to prepare for annealing.
Binding Process: The oligos are laid down onto the ssDNA; this ssDNA is unique because it contains uracil instead of thymine (T), a characteristic typically found in RNA.
Source of ssDNA: The ssDNA plasmids are produced through a specialized procedure, utilizing a virus for construction, acquired from UC Davis.

Mutation Induction: The goal is to introduce a specific mutation into the bagel bee gene on the plasmid.
E. Coli Utilization: Once the plasmid is prepared with the oligo, it will be introduced into E. coli bacteria, which may incorporate the plasmid into its genome.
Expectation of Outcomes: After the transformation, some plasmids may carry the intended mutation while others may remain unchanged.

Uncertainty in Results: Unlike controlled labs where outcomes are known, these experiments represent real scientific inquiry with unpredictable results; the effectiveness of the mutation is uncertain until sequencing occurs.
Success Rates in Previous Trials: Historical success rates vary; previous trials had about 30% success, while others had up to 60%, indicating variability.
Importance of Data: Both successful and non-successful outcomes provide valuable data, as failures can inform future experiments about the functions of mutations.

Meticulous Pipetting: Emphasis on precision in handling small volumes of reagents to minimize experimental errors.
Enzyme Activity: Ensuring that enzymes (like polymerases and ligases) function correctly and that the ssDNA remains intact during the process is crucial.

Potential Outcomes in E. coli: E. coli may replace uracils in the dna with thymines, or it could potentially recognize the viral DNA as foreign and degrade it.
Unpredictable Results: There is a possibility that bacteria will produce plasmids without the desired mutation, and outcomes are primarily reliant on chance.

Real Science: The experiment is an exploration into molecular biology, showcasing the complexities and unpredictabilities of genetic manipulation.
Participant Engagement: Student participation involves selecting bacterial colonies for DNA extraction and sequencing, with an understanding of the potential for variability in results.