Biotechnology Terms Starting With L
Biotechnology Glossary: L
Jump to Biotechnology Term
Library Screening
/ LY-brer-ee SKREE-ning / · Latin libraria, collection; Old English screnian, to sift
Library screening is the process of searching a collection of cloned DNA fragments, either a genomic or cDNA library, to identify and isolate a specific gene or sequence of interest.
Libraries are constructed by fragmenting source DNA, inserting pieces into vectors such as plasmids or bacteriophage lambda, and introducing them into host cells to create millions of individual clones, each carrying a different insert. Screening is then performed by hybridization with a labeled nucleic acid probe, immunological detection of an expressed protein, or activity-based selection in which only clones producing a functional enzyme survive selective conditions. Colony hybridization, one of the earliest screening methods, was described by Grunstein and Hogness in 1975 and could survey tens of thousands of bacterial colonies on a single nylon membrane.
Library screening was the primary method for gene isolation before whole-genome sequencing made most library approaches obsolete for well-studied organisms, though cDNA library screening remains useful for identifying tissue-specific splice variants.
Before genomic databases existed, the human insulin gene was isolated in 1977 by screening a cDNA library prepared from pancreatic islet cells, a discovery that directly enabled the first recombinant human insulin produced in Escherichia coli.
A genomic library is a physical building with books. It is a collection of bacterial colonies or viral particles, each carrying a different fragment of cloned DNA, stored in freezers at minus 80 degrees Celsius.
Researchers screening a pituitary cDNA library with a radiolabeled probe complementary to a known hormone sequence identified positive colonies within 48 hours; each positive clone carried an insert averaging 1.5 kilobases encoding the target gene, which was then sequenced to confirm identity. The library contained roughly 200,000 independent clones derived from poly-A-selected mRNA, representing most transcripts expressed above one copy per cell in the source tissue. Filter lifts from each agar plate captured DNA from all colonies simultaneously, allowing a single hybridization experiment to screen the entire library at once.
Endocrine System Fun Facts →Lipid Nanoparticle
/ LIP-id NAN-oh-PAR-tih-kul / · From Greek lipos, fat, plus Latin particula, small part, with nano from Greek nanos, dwarf
Lipid nanoparticle is a spherical delivery vehicle composed of ionizable lipids, phospholipids, cholesterol, and PEGylated lipids, typically 50 to 200 nanometers in diameter, designed to encapsulate and deliver nucleic acids or drugs into cells.
Lipid nanoparticles protect fragile RNA molecules from nuclease degradation in the bloodstream and facilitate cellular uptake through endosomal membrane fusion. The Moderna and Pfizer-BioNTech COVID-19 vaccines both employ LNPs containing ionizable lipids that carry a positive charge at acidic endosomal pH, promoting RNA release, but become neutral at physiological pH to reduce toxicity. Manufacturing involves mixing lipids dissolved in ethanol with aqueous mRNA solutions using microfluidic devices, producing particles with over 90 percent encapsulation efficiency.
Each Moderna vaccine dose contains approximately 100 micrograms of mRNA packaged in trillions of individual nanoparticles, while the Pfizer-BioNTech dose contains 30 micrograms. Because LNPs accumulate preferentially in the liver after intravenous injection, several approved and investigational therapies target hepatic diseases, including patisiran for hereditary transthyretin amyloidosis.
The ionizable lipid SM-102 used in Moderna's COVID-19 vaccine was synthesized in quantities measured in metric tons within months of the vaccine's authorization, representing one of the fastest pharmaceutical ingredient scale-ups ever documented. Liposomal drug delivery predates mRNA vaccines by decades; Doxil, a liposomal doxorubicin formulation, received FDA approval in 1995 for Kaposi's sarcoma.
Lipid nanoparticles are a new invention created for COVID-19 vaccines. Liposomal drug delivery systems received FDA approval as early as 1995, and the ionizable lipid formulations used for RNA delivery were refined through more than a decade of research before the pandemic.
Alnylam Pharmaceuticals developed patisiran, an LNP-formulated siRNA drug approved in 2018 for hereditary transthyretin amyloidosis; the nanoparticles deliver therapeutic RNA specifically to liver hepatocytes, silencing the mutant TTR gene, and clinical trials showed a 56 percent reduction in serum transthyretin levels after 18 months of dosing at 0.3 milligrams per kilogram every three weeks. Each LNP particle averages approximately 80 nanometers in diameter and carries a cargo of roughly 10 siRNA duplexes per particle, with the ionizable lipid component adopting a positive charge at endosomal pH to disrupt the membrane and release siRNA into the cytoplasm. Patisiran's approval marked the first RNA interference drug cleared by any regulatory agency.
Luciferase Reporter
/ loo-SIF-er-ays ree-POR-ter / · From Latin lucifer, light-bearing, plus -ase enzyme suffix, and reporter
Luciferase reporter is a genetic construct in which the luciferase enzyme gene is placed under the control of a promoter of interest, so that bioluminescent light emission provides a quantitative readout of gene expression.
Luciferase reporters produce quantifiable light when the enzyme oxidizes its substrate luciferin in an ATP-dependent reaction, enabling real-time, non-destructive monitoring of gene activity. Firefly luciferase from the firefly Photinus pyralis generates 550-nanometer yellow-green light with quantum efficiency approaching 90 percent, making it detectable at femtomolar enzyme concentrations. Researchers clone promoters or regulatory sequences upstream of the luciferase gene to study transcriptional responses to hormones, drugs, or environmental signals.
Detection spans six orders of magnitude of enzyme concentration, with sensitivity 100 to 1,000 times greater than fluorescent reporters, making the system well suited to high-throughput drug screening. Bioluminescence from luciferase-expressing tumor cells penetrates several centimeters of mouse tissue, allowing researchers to track tumor growth and metastasis in living animals without surgery.
Scientists have engineered luciferases from the sea pansy Renilla reniformis and from click beetles to emit colors ranging from blue to red, allowing simultaneous monitoring of two or more independent promoters in the same living cell without spectral overlap.
Luciferase reporters make cells glow continuously. Cells only emit light when exogenous luciferin substrate and ATP are supplied, typically producing a detectable signal for 30 to 60 minutes before the reaction substrate is exhausted.
Researchers at Vanderbilt University used firefly luciferase reporters driven by the Per2 promoter to monitor circadian clock gene activity in isolated mouse liver cells, recording bioluminescence continuously for 14 days and demonstrating that individual hepatocytes maintain autonomous 24-hour rhythms independent of the suprachiasmatic nucleus. The photon output from each well containing approximately 50,000 cells peaked at roughly 10,000 counts per second, detected by a photomultiplier tube cooled to reduce background noise. Temperature compensation within the cells held the oscillation period to within 15 minutes of 24 hours across a temperature range from 30 to 37 degrees Celsius.