Biotech plasmids

Plasmid

A small, circular DNA molecule that is distinct from the chromosomal DNA in bacteria, often carrying genes that provide advantageous traits, such as antibiotic resistance.

Vector

A plasmid is considered a vector because it can carry foreign genetic material into a host cell.

ORI

The sequence of DNA that allows the plasmid to replicate within the host cell.

Selective Marker

A gene present on a plasmid that enables researchers to identify cells that have taken up the plasmid.

pGLO Transformation Lab Selective Marker

The antibiotic resistance gene for ampicillin (bla), which makes the bacteria resistant to ampicillin.

Competent Cells

Bacterial cells that are capable of taking up foreign DNA, such as plasmids.

Methods to Make Bacterial Cells Competent

Calcium chloride treatment (chemical transformation) and electroporation.

Genetic Transformation

The process by which foreign DNA is introduced into a cell, resulting in the alteration of the cell's genotype.

pGLO Plasmid Parts

ORI: Origin of replication; bla gene: Provides ampicillin resistance; GFP gene: Codes for GFP; araC gene: Regulates GFP expression; Promoter: Controls GFP expression; Operator: Regulates gene expression.

AraC Protein

Regulates the expression of the GFP gene in response to arabinose.

GFP

A protein that fluoresces green under UV light, used as a reporter to show whether the transformation was successful.

Steps of Genetic Transformation

Prepare competent cells, introduce plasmid DNA, heat shock or apply electroporation, plate the cells on selective media, incubate, and screen for colonies expressing the gene of interest.

Functions of CaCl₂ Solution

Prepares bacterial cells to become competent by increasing the permeability of the cell membrane.

Heat Shock

Temporarily increases the permeability of the bacterial cell membrane, allowing plasmid DNA to enter the cells.

LB (Luria Broth)

Provides nutrients for bacterial growth and recovery after the transformation process.

Arabinose

A sugar that acts as an inducer for the ara operon, allowing the expression of the GFP gene.

GFP Expression Regulation

In the absence of arabinose, AraC protein binds to the operator and prevents GFP gene transcription; in the presence of arabinose, it allows transcription.

Types of Media in pGLO Lab

LB agar (without antibiotics), LB agar + ampicillin, LB agar + ampicillin + arabinose.

Results on LB Plate

Growth of all bacteria (control group, no selection).

Results on LB + Ampicillin Plate

Growth only of transformed bacteria (those with the pGLO plasmid).

Results on LB + Ampicillin + Arabinose Plate

Growth of transformed bacteria with green fluorescence (only cells with the pGLO plasmid and arabinose exposure will express GFP).

Chromatography in Biotechnology

Used to separate and purify biomolecules based on size, charge, hydrophobicity, or affinity for specific materials.

Types of Column Chromatography

Ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography.

Column Chromatography Used in Class

Affinity chromatography to purify GFP by using a column that specifically binds to the GFP protein.

Lysozyme

An enzyme that breaks down bacterial cell walls by cleaving the peptidoglycan layer, helping to release intracellular contents (like GFP) during cell lysis.

Cell lysis

Breaks open bacterial cells to release proteins.

Binding to the column

GFP is captured by an affinity resin (e.g., His-tag or GFP-specific resin).

Washing

Removes unbound proteins and contaminants.

Elution

GFP is separated from the resin by adding an elution buffer (e.g., high salt or low pH).

Concentration

Concentrates the purified GFP for analysis.

Pellet

The solid material that is at the bottom of the tube after centrifugation, usually containing cell debris.

Supernatant

The liquid above the pellet, containing the soluble proteins, including GFP.

Micropipette

Typically has a digital readout that shows the volume in microliters (µL).

P1000

Can pipette between 100 and 1000 µL.

P20

Can pipette between 1 and 20 µL.

Bacterial transformation

The process by which bacterial cells take up foreign DNA from their environment.

bla gene

Encodes β-lactamase, an enzyme that breaks down the antibiotic ampicillin.

Selective marker

A gene that allows for the identification of bacteria that have taken up a plasmid, such as the bla gene on the pGLO plasmid.

Contamination

The introduction of non-transformed bacteria into a culture or plate.

-pGLO LB plate

Serves as a control to show the growth of non-transformed bacteria.

β-lactamase

The protein that confers resistance to ampicillin.

Charge neutralization

In plasmid transformation, calcium chloride (CaCl₂) is used to neutralize the negative charge of the DNA.

Recombinant DNA

DNA that has been artificially created by combining genetic material from different sources.

Order of buffers for GFP purification

1. Binding buffer: Stabilizes the GFP and allows it to bind to the affinity resin. 2. Washing buffer: Removes unbound proteins and contaminants while keeping GFP bound. 3. Elution buffer: Releases the GFP from the resin by altering conditions.

First major biotech-produced recombinant protein

Insulin, produced using recombinant DNA technology to treat diabetes.

Competent bacterium

A bacterium that has been treated to become able to take up foreign DNA.

Supernatant in DNA extraction

The liquid portion that remains after centrifugation, containing dissolved DNA.

Pellet in DNA extraction

The solid material that collects at the bottom of the tube after centrifugation, typically containing larger, insoluble components.