Microbial Biotechnology

Microbial Biotechnology

Application of genetic engineering to microorganisms to produce useful products such as proteins, antibiotics, biofuels, and vaccines.

Genetic Engineering (GE)

Modification of an organism's genetic material by adding, removing, or altering DNA.

DNA Cloning

Production of many identical copies of a specific DNA sequence using a cloning vector and host cell.

Plasmid

Small circular DNA molecule that replicates independently of the bacterial chromosome and is commonly used as a cloning vector.

Cloning Vector

Engineered plasmid that carries foreign DNA into a host cell for replication and/or expression.

Origin of Replication (OriR)

DNA sequence where plasmid replication begins; allows independent replication and determines plasmid copy number.

Restriction Site

Specific DNA sequence recognized and cut by a restriction endonuclease; often located in the multiple cloning site (MCS).

Multiple Cloning Site (MCS)

Region containing several unique restriction sites that allows insertion of foreign DNA into a vector.

Selectable Marker

Gene, usually antibiotic resistance, that allows identification of bacteria carrying the plasmid.

Reporter Gene

Gene used to indicate successful DNA insertion or gene expression, commonly lacZ for blue-white screening.

Blue-White Screening

Method using the lacZ reporter gene and X-gal to distinguish recombinant (white) from non-recombinant (blue) bacterial colonies.

Restriction Endonuclease

Bacterial enzyme that recognizes specific palindromic DNA sequences and cleaves DNA at or near those sites as a defense against bacteriophages.

DNA Methylation

Addition of methyl groups to bacterial DNA that protects host DNA from cleavage by its own restriction enzymes.

Palindromic Sequence

DNA sequence that reads the same in the 5'→3' direction on both complementary strands and is commonly recognized by restriction enzymes.

EcoRI

Restriction endonuclease that recognizes the sequence GAATTC and produces sticky (cohesive) ends.

Sticky Ends

Single-stranded DNA overhangs produced by staggered restriction enzyme cuts that readily base-pair with complementary sequences to facilitate ligation.

Blunt Ends

DNA ends lacking overhangs that can be ligated to any other blunt-ended DNA fragment.

Recombinant DNA

DNA molecule formed by joining DNA fragments originating from different organisms.

DNA Ligase

Enzyme that joins DNA fragments by forming phosphodiester bonds between adjacent nucleotides.

Transformation

Introduction of recombinant plasmid DNA into competent bacterial cells.

Chemical Transformation

Method of bacterial transformation using chemical treatment followed by heat shock to increase membrane permeability for DNA uptake.

Electroporation

Transformation method that uses a brief electrical pulse to create temporary membrane pores through which DNA enters the cell.

Transduction

Delivery of recombinant DNA into bacteria using bacteriophages as vectors.

Polymerase Chain Reaction (PCR)

Technique that exponentially amplifies a specific DNA sequence through repeated cycles of denaturation, annealing, and extension.

Template DNA

DNA containing the target sequence that serves as the template for PCR amplification.

Primer

Short single-stranded DNA that anneals to sequences flanking the target DNA and determines PCR specificity.

dNTPs

Deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, dTTP) that serve as substrates for DNA synthesis during PCR.

Taq DNA Polymerase

Heat-stable DNA polymerase from Thermus aquaticus that synthesizes DNA during PCR and remains active after repeated heating.

PCR Denaturation

PCR step at ~95°C in which double-stranded DNA separates into single strands.

PCR Annealing

PCR step at ~55–65°C in which primers bind to complementary target sequences.

PCR Extension (Elongation)

PCR step at ~72°C in which Taq DNA polymerase synthesizes new DNA strands from the primers.

PCR Amplification

Process in which the amount of target DNA approximately doubles every PCR cycle (2ⁿ).

Primer Design

Selection of primers complementary to known flanking sequences to determine which DNA fragment is amplified.

Complementary DNA (cDNA)

DNA synthesized from mature mRNA by reverse transcriptase that contains only coding sequences and lacks introns.

Reverse Transcriptase

Enzyme that synthesizes complementary DNA (cDNA) from an RNA template.

Bacterial Expression Vector

Vector containing a bacterial promoter, ribosome-binding site (RBS), and gene of interest to enable protein expression in bacteria.

Recombinant DNA Technology

Use of genetically engineered microorganisms to produce useful biological products for medicine, agriculture, food, and industry.

Chymosin

Recombinant milk-clotting enzyme used in cheese production as a replacement for animal-derived rennin.

Bioluminescence

Production of light by living organisms; used as biological indicators and reporter systems in research.

Recombinant Protein

Protein produced by genetically engineered organisms expressing a foreign gene.

DNase

Recombinant enzyme that digests extracellular DNA to reduce mucus viscosity in cystic fibrosis patients.

Erythropoietin (EPO)

Hormone that stimulates red blood cell production and is used to treat anemia.

Factor VIII

Blood-clotting protein produced recombinantly for treatment of hemophilia A.

Granulocyte Colony-Stimulating Factor (G-CSF)

Protein that stimulates neutrophil production and is used to treat neutropenia and aid bone marrow recovery.

Growth Hormone

Hormone that stimulates bone and muscle growth and is used to treat growth hormone deficiency.

Insulin

Hormone that promotes glucose uptake into cells and is produced recombinantly for diabetes treatment.

Interferons

Immune signaling proteins used in the treatment of certain cancers and viral diseases.

Interleukins

Immune signaling proteins used in immunotherapy for cancers and blood disorders.

Superoxide Dismutase (SOD)

Antioxidant enzyme that converts superoxide radicals into less harmful molecules, reducing oxidative tissue damage.

Tissue Plasminogen Activator (tPA)

Recombinant enzyme that dissolves blood clots during heart attack and ischemic stroke treatment.

Recombinant Vaccine

Vaccine produced using recombinant DNA technology to stimulate protective immunity.

Recombinant Bovine Somatotropin (rBST)

Recombinant growth hormone administered to dairy cattle to increase milk production.

Transgenic Plant (GMO)

Plant genetically modified by introducing genes from another organism to confer desirable traits.

Binary Vector System

Modified Ti plasmid system used by Agrobacterium to transfer desired genes into plants.

Herbicide Resistance Gene

Transgene that enables plants to survive herbicides such as glyphosate.

Bt Toxin

Insecticidal protein encoded by the Bacillus thuringiensis gene and expressed in transgenic crops to kill insect pests.

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats that form the adaptive immune system of bacteria and archaea by storing viral DNA spacers.

Spacer

Short viral DNA sequence stored within the CRISPR array that enables recognition of previously encountered viruses.

Cas9

RNA-guided endonuclease that cleaves DNA at a sequence specified by a guide RNA.

Guide RNA (gRNA)

RNA molecule complementary to the target DNA that directs Cas9 to the correct editing site.

CRISPR-Cas9

Genome-editing system in which a guide RNA directs Cas9 to create a double-stranded break at a specific DNA sequence for gene knockout, insertion, or replacement.

Jennifer Doudna

Co-developer of CRISPR-Cas9 gene editing and recipient of the 2020 Nobel Prize in Chemistry.

Emmanuelle Charpentier

Co-developer of CRISPR-Cas9 gene editing and recipient of the 2020 Nobel Prize in Chemistry.

He Jiankui

Scientist who controversially used CRISPR to edit human embryos, raising major ethical concerns about human germline editing.