Biotechnology: Principles and Processes

Flashcards covering the fundamental concepts, tools, and processes of biotechnology, including genetic engineering principles, restriction enzymes, cloning vectors, and large-scale product recovery.

Biotechnology (EFB definition)

The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services.

Genetic engineering

Techniques to alter the chemistry of genetic material ($DNA$ and $RNA$), to introduce these into host organisms and thus change the phenotype of the host organism.

Bioprocess engineering

The maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of products like antibiotics, vaccines, or enzymes.

Plasmids

Small, autonomously replicating circular extra-chromosomal $DNA$ found in certain bacterial cells that float freely in the cytoplasm and replicate independently from the chromosomal $DNA$.

Restriction enzymes

Commonly known as 'molecular scissors', these enzymes have the capability of cutting $DNA$ strands at specific locations.

Sticky ends

Single stranded overhanging portions at the ends of $DNA$ strands left after restriction enzyme cuts, which form hydrogen bonds with their complementary counterparts and facilitate the action of $DNA$ ligase.

DNA ligase

An enzyme that acts on cut $DNA$ molecules and joins their ends together, enabling the linking of an antibiotic resistance gene with a plasmid vector.

Recombinant DNA

A new combination of circular autonomously replicating $DNA$ created in vitro by linking a foreign piece of $DNA$ with a vector $DNA$.

Cloning

The process of making multiple identical copies of any template $DNA$.

Origin of replication (ori)

A specific $DNA$ sequence responsible for initiating replication and controlling the copy number of the linked $DNA$.

Hind II

The first restriction endonuclease isolated and characterized, which recognizes a specific sequence of six base pairs to cut $DNA$ molecules.

Exonucleases

A class of nucleases that remove nucleotides from the ends of the $DNA$.

Endonucleases

A class of nucleases that make cuts at specific positions within the $DNA$.

Palindromic nucleotide sequences

A sequence of base pairs that reads the same on two strands when the orientation of reading is kept the same, such as $5' \rightarrow 3'$ on both strands.

Gel electrophoresis

A technique used to separate $DNA$ fragments according to size through the sieving effect of an agarose gel matrix under an electric field.

Agarose

A natural polymer extracted from sea weeds used as a matrix in gel electrophoresis.

Ethidium bromide

A compound used to stain $DNA$ fragments so they can be visualized as bright orange coloured bands under $UV$ radiation.

Elution

The step in which separated bands of $DNA$ are cut out from the agarose gel and extracted from the gel piece.

Selectable marker

A feature of a vector that helps in identifying and eliminating non-transformants and selectively permitting the growth of the transformants.

Transformation

A procedure through which a piece of $DNA$ is introduced into a host bacterium.

Insertional inactivation

The inactivation of a gene (such as $\beta$-galactosidase) due to the insertion of recombinant $DNA$ within its coding sequence, used to differentiate recombinant from non-recombinant colonies.

Ti plasmid

The tumor inducing plasmid of $\text{Agrobacterium tumifaciens}$ which has been modified into a cloning vector to deliver genes of interest into plants.

Competent host

A bacterial cell treated with a divalent cation such as calcium and subjected to heat shock to increase the efficiency with which $DNA$ enters the cell through wall pores.

Micro-injection

A method where recombinant $DNA$ is directly injected into the nucleus of an animal cell.

Biolistics (Gene gun)

A method for plant cells where they are bombarded with high velocity micro-particles of gold or tungsten coated with $DNA$.

Lysozyme

An enzyme used to break open bacterial cells to release $DNA$.

Chitinase

An enzyme used to break open the cell walls of fungi during $DNA$ isolation.

Polymerase Chain Reaction (PCR)

An in vitro reaction used to synthesize multiple copies ($1 \text{ billion}$ or more) of a gene of interest using primers and $DNA$ polymerase.

Taq polymerase

A thermostable $DNA$ polymerase isolated from the bacterium $\text{Thermus aquaticus}$ that remains active during the high temperature denaturation step of $PCR$.

Recombinant protein

A protein produced when a protein-encoding gene is expressed in a heterologous host.

Bioreactor

Large volume vessels ($100-1000 \text{ litres}$) that provide optimal growth conditions (temperature, $pH$, substrate, salts, vitamins, oxygen) for biologically converting raw materials into specific products.

Downstream processing

The series of processes including separation and purification that a product undergoes after the biosynthetic stage before it is ready for marketing.