1. Sterile Technique and Bacterial Transformation
October 7, 2015

2. Molecular Cloning
Manipulation of DNA sequences to create recombinant DNA
Recombinant DNA (rDNA)- 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

3. How do we create recombinant DNA?
Amplify DNA sequences from original organism (ex. PCR)
Synthesize artificial sequences in the lab

4. 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

5. Bacterial use in rDNA amplification
Use bacteria as “factories” to rapidly and efficiently generate large amounts of DNA
Take advantage of bacterial growth rate: population doubles every ~20min
Isolate DNA from bacteria for subsequent analysis

6. 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 (CaCl2) + heat shock
Electroporation

7. 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?

8. 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

9. Antibiotic resistance in medicine
cdc

10. Antibiotic resistance in medicine
Medicine.net

11. How do we prevent contamination of our transformed bacteria?
Sterile Technique
Antibiotics will help too

12. 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

13. 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
Medicine.net

14. Our plasmids
Contain origin of replication sites, gene/sequence of interest, reporter gene, antibiotic resistance gene, promoters, enhancers, etc.

15. Next steps… Check growth of transformed cells in the morning
Confirm presence of cloned gene of interest in plasmid by restriction enzyme digest