Microbial DNA Technologies Flashcards

This set of vocabulary flashcards covers key concepts, tools, and methodologies in microbial DNA technologies, including genetic engineering, PCR, cloning vectors, and directed evolution as outlined in the lecture notes.

Genetic engineering

The modification of the genetic code of a living organism, often used for crop improvement and breeding animals.

Recombinant DNA

DNA molecules composed of segments originating from different organisms.

DNA Cloning

The use of enzymes and bacterial cells to modify and amplify DNA.

Biotechnology

The use of organisms to produce useful products.

Restriction endonucleases

Enzymes that recognize and cleave specific DNA sequences; they are named for the bacterium from which they originated.

Sticky ends

Overhangs produced in a DNA target following cleavage by certain restriction enzymes.

Blunt ends

DNA fragments produced by restriction enzymes that do not have overhangs.

Jackson, Symons, and Berg

The researchers who generated the first recombinant DNA molecules.

Reverse transcriptase

An enzyme that synthesizes double-stranded DNA from an RNA template (mRNA).

Complementary DNA (cDNA)

DNA constructed from an mRNA template that can be cloned without the need for RNA processing.

Gel Electrophoresis

A technique using agarose or acrylamide gels to separate DNA fragments by size as they migrate from the negative to the positive end.

Cloning Vectors

DNA elements, such as plasmids, bacteriophages, viruses, cosmids, and artificial chromosomes, that replicate and carry additional genetic cargo.

Origin of replication (ori)

A feature of a cloning vector that allows independent replication and dictates the copy number.

Selectable marker

A gene on a plasmid that helps the cell survive under certain conditions and is used to differentiate transformants from non-transformed cells.

Multicloning site (MCS)

A cluster of restriction sites within a cloning vector.

Shuttle vector

A vector containing two different origins of replication (ori) that are recognized by different organisms, allowing it to move between species.

Phage vectors

Phage genomes genetically modified to include multiple cloning sites for DNA insertion, which are then packaged into viral capsids.

Cosmids

Engineered vectors containing features from both phages (cos site) and plasmids (selectable marker, ori, and MCS) that can accommodate large DNA fragments.

Artificial chromosomes

Vectors used for cloning large DNA fragments, such as entire biosynthetic pathways, with a capacity up to $300\,kb$.

Electroporation

A mechanism for introducing recombinant DNA into host cells by using a brief pulse of high-voltage electricity to temporarily permeate the plasma membrane.

Polymerase Chain Reaction (PCR)

A technique for the rapid synthesis of billions of copies of a specific DNA fragment from a complex mixture of DNA molecules.

Oligonucleotides primers

Single-stranded DNA molecules, generally between $15$ and $30$ nucleotides long, used to initiate DNA synthesis in PCR.

Taq polymerase

A thermostable DNA polymerase used in the PCR reaction mix.

Denaturation

The first step of a PCR cycle where double-stranded DNA (dsDNA) is heated briefly to separate it into single-stranded DNA (ssDNA).

Annealing

The second step of a PCR cycle where the reaction is cooled to allow primers to hydrogen bond with the ends of the target sequence.

Extension

The third step of a PCR cycle where DNA polymerase adds nucleotides to the $3'$ end of each primer.

Real-time PCR (qPCR)

A type of PCR that can quantify the amount of DNA in a sample by monitoring the process as it occurs.

Genomic library

A collection containing all the DNA derived from a single organism.

Metagenomic library

A collection containing all the DNA derived from a single environment.

Heterologous gene expression

The process where genes from one organism are cloned into another, followed by transcription and translation.

Expression vectors

Vectors containing inducible promoters designed to results in high-level transcription of recombinant genes.

Polyhistidine tagging (His-tagging)

A protein purification method where a tag allows for the isolation of a protein using metal ion resin.

Green fluorescent protein (GFP)

A protein used in fluorescent labeling to study in vivo expression and regulation of recombinant genes.

Cas9

An endonuclease ribonucleoprotein, consisting of a polypeptide and guide RNA, used in CRISPR technology for in vivo genome editing.

Directed evolution

A process of targeting specific genes for mutagenesis to create higher yields of products with desirable features.

Site Directed Mutagenesis

A technique where the nucleotide sequence of a specific gene is altered before being moved into an organism of interest.

DNA shuffling

A directed evolution technology where similar genes from different species are randomly fragmented and pooled to generate new DNA fragments via in vitro homologous recombination.

Synthetic Biology

The forward engineering of biological systems to perform completely novel functions, such as the design of an ideal genome from scratch.