Sterile Technique and Bacterial Transformation October 7, 2015

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

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 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 https://upload.wikimedia.org/wikipedia/commons/4/42/Plasmid_replication_%28english%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 (CaCl2) + 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 cdc http://www.niaid.nih.gov/topics/antimicrobialResistance/Understanding/Pages/mutation.aspx

Antibiotic resistance in medicine Medicine.net http://www.niaid.nih.gov/topics/antimicrobialResistance/Understanding/Pages/mutation.aspx

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

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 Medicine.net

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

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