Sterile Technique and Bacterial Transformation October 7 2015
Sterile Technique and Bacterial Transformation October 7, 2015
Molecular Cloning • Manipulation of DNA sequences to create recombinant DNA • Recombinant DNA (r. DNA)- DNA constructed from multiple sequences in a laboratory setting and therefore do not exist in nature – Also referred to as DNA constructs • Used to amplify and study specific gene(s) of interest
How do we create recombinant DNA? • Amplify DNA sequences from original organism (ex. PCR) • Synthesize artificial sequences in the lab www. flmnh. ufl. edu
How do we create recombinant DNA? • Ligate (or paste) fragments of DNA together to connect sequences into single reading frame or region of gene transcription and expression • This typically generates circular DNA, also known as a plasmid, that contains all of the sequences required to transcribe and express your DNA of interest www. di. uq. edu. au
Bacterial use in r. DNA amplification • Use bacteria as “factories” to rapidly and efficiently generate large amounts of DNA • Take advantage of bacterial growth rate: population doubles every ~20 min • Isolate DNA from bacteria for subsequent analysis https: //upload. wikimedia. org/wikipedia/commons/4/42/Plas mid_replication_%28 english%29. svg
How do we get our plasmid DNA into the bacteria? • Bacterial Transformation- procedure to facilitate uptake of foreign DNA into bacteria • Use “competent” cells - treated to improve transfer of plasmid DNA – Chemically competent cells (Ca. Cl 2) + heat shock – Electroporation
Growing transformed cells • Luria (or lysogeny) broth (LB) or agar – Commonly used to grow bacteria – Contains tryptone (peptides), yeast extract (organic compounds) and sodium chloride (ions, osmotic balance) • However, these are requirements for any organism to grow • How do we selectively grow bacteria that contain our gene/plasmid of interest?
Antibiotic resistance • The plasmids with our gene also contain a sequence that code for naturally occurring genes that confer antibiotic resistance • Therefore, only bacteria containing plasmids with resistance gene will grow on a medium containing the antibiotic while other cells will die
Antibiotic resistance in medicine http: //www. niaid. nih. gov/topics/antimicrobial. Resistance/Understanding/Pages/mutation. aspx
Antibiotic resistance in medicine http: //www. niaid. nih. gov/topics/antimicrobial. Resistance/Understanding/Pages/mutation. aspx
How do we prevent contamination of our transformed bacteria? Sterile Technique
Sterile Technique • Use antimicrobial reagents to sterilize work area and equipment – 70% ethanol, 10% bleach, sometimes flame • Use clean, pre-sterilized reagents and plasticware – No open fires at JABSOM so will use all disposable plastics and filter tips for pipets • Minimize manipulation of cells – Don’t handle cells or plates unnecessarily • Minimize exposure to environment – Limit time tubes and plates are open – Conduct experiments in locations with minimum traffic • Properly dispose of all contaminated samples in biohazardous waste
Our experiment • Transform bacteria with two different plasmids containing different antibiotic resistance genes • Assess growth of transformed cells on different antibiotic-selective LB agar plates
Our plasmids
Next steps… • Check growth of transformed cells in the morning • Confirm presence of cloned gene of interest in plasmid by restriction enzyme digest
- Slides: 15