Bacterial Transformation and Agarose Gel Electrophoresis

Genetic Transformation

The insertion of a gene into an organism in order to change the organism's trait. Literally means "change caused by genes".

Gene

A piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait.

Trait

A particular characteristic or feature of an organism that is determined by its genes. Also known as phenotype.

Plasmid

One or more small circular pieces of DNA that bacteria naturally contain in addition to one large chromosome. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival.

Green Fluorescent Protein (GFP)

A protein coded for by a gene from the bioluminescent jellyfish Aequorea victoria that causes the jellyfish to fluoresce and glow in the dark. When bacteria are transformed with the GFP gene, they produce the fluorescent protein and glow a brilliant green color under ultraviolet light.

Ampicillin Resistance

The ability of bacteria to survive and grow in the presence of the antibiotic ampicillin. The pGLO plasmid encodes a gene that provides resistance to ampicillin.

Arabinose

A sugar that can be added to a transformed cell's nutrient medium to switch on the gene for GFP expression in cells transformed with the pGLO plasmid.

Transformation Solution (CaCl2)

A solution containing calcium chloride used in the transformation procedure to help move the pGLO plasmid DNA through the E. coli cell membrane.

Heat Shock

A procedure in genetic transformation where cells are rapidly transferred from ice to a warm water bath (42°C) and back to ice. This step is intended to help introduce the plasmid DNA into the E. coli cells.

LB Agar Plates

Nutrient agar plates used to grow bacteria.

LB/amp Plates

LB nutrient agar plates that contain the antibiotic ampicillin. These plates are used for selection of bacteria that have been transformed with the pGLO plasmid, as only ampicillin-resistant bacteria can grow on them.

LB/amp/ara Plates

LB nutrient agar plates that contain both ampicillin and the sugar arabinose. On these plates, transformed bacteria with the pGLO plasmid will be ampicillin-resistant and will express the GFP gene in the presence of arabinose, causing them to appear fluorescent green under UV light.

Control Plate

A plate in an experiment that is used as a standard for comparison. For example, an LB plate without ampicillin can serve as a control to see if E.

Control Plate

A plate in an experiment that is used as a standard for comparison. For example, an LB plate without ampicillin can serve as a control to see if E. coli normally grows.

Phenotype

The observable traits or characteristics of an organism. Genetic transformation aims to change an organism's phenotype.

Agarose Gel Electrophoresis

A widely used technique to separate molecules based on charge, size, and shape. It is particularly useful for separating charged biomolecules such as DNA, RNA, and proteins.

Agarose Gel

A gel matrix made by dissolving agarose powder in a boiling buffer solution that is then cooled and solidified. The gel contains microscopic pores that act as a molecular sieve.

Electrophoresis Buffer

A buffer solution that the agarose gel is submerged in during electrophoresis. It serves as a conductor of electricity and helps to control the pH, which is important for the charge and stability of biological molecules.

Wells

Small indentations or pockets created in the agarose gel during casting where samples are loaded.

Electrodes

Conductors through which electricity enters and leaves the electrophoresis apparatus. The anode is the positive electrode, and negatively charged molecules migrate towards it. The cathode is the negative electrode, and positively charged molecules migrate towards it.

Migration

The movement of charged molecules through the agarose gel when an electric current is applied. Negatively charged molecules migrate towards the positive electrode, and positively charged molecules migrate towards the negative electrode. Smaller, more compact molecules generally move through the pores faster than larger or less compact ones.

Molecular Sieve

The microscopic pores within the agarose gel that separate molecules based on their size and shape. Smaller molecules can move through the pores more easily and thus migrate faster.

Charge, Size, Shape (affecting migration)

Factors that influence the mobility of molecules in agarose gels. Molecules with a greater net charge will migrate faster, and smaller, more compact molecules will move through the gel more quickly.

Loading Dye (Tracking Dye)

A colored dye that is mixed with a sample before loading it into the gel. It helps to color the sample for easier loading, makes the sample denser so it stays in the well, and migrates through the gel to help track the progress of electrophoresis.

DNA Stain

A pigmented or fluorescent molecule that interacts directly with DNA, allowing visualization of DNA fragments within the agarose gel after electrophoresis. DNA itself is colorless and invisible in the gel.