Biotechnology Flashcards
What is pharmaceutical biotechnology?
a field that uses micro and macro-organisms and genetically engineered cells to create pharmaceuticals
What is the goal of pharmaceutical biotechnology?
create safer and more cost effective than conventionally produced pharmaceuticals
What are some applications of biotechnology in pharmacy?
antibiotic production
antibody production
gene therapy and gene silencing
vaccines
personalized medicine
transgenic animals
What is the pharmacists role in biotechnology?
product evaluation and selection
patient education and counselling
provision of drug information
assistance in patient monitoring
drug control and preparation
FACILITATING BETTER CARE
Differentiate between eukaryotes and prokaryotes.
eukaryotes:
-nucleus and other organelles enclosed by a plasma membrane
-large (10-100um)
-most are multicellular, some are unicellular
-linear DNA
prokaryotes:
-unicellular
-lack membrane bound structures, even the nucleus
-nucleoid contains only one circular chromosome
-small (0.1-5um)
-extra chromosomal material: plasmids
How many nucleotides are in a strand of DNA?
four
What is the central dogma of molecular biology?
DNA–>RNA–>protein
transcription then translation
What is a gene?
a portion of the DNA that codes for a mRNA that then translates into protein
include the coding sequence and adjacent sequence required for regulation of expression
Differentiate gene organization of eukaryotes and prokaryotes.
prokaryotes:
-most of the DNA
-very little inter-genic DNA
-no introns
-1 gene=1 protein
eukaryotes:
-75% of the DNA is non-coding (regulatory)
-most of the DNA is inter-genic DNA in the genome
-genes are split: exons (1.5%) and introns (23.5%)
-1 gene=multiple proteins
What are promoters?
DNA sequence that promote gene expression
required for DNA transcription
typically located upstream of the genes
RNA polymerase binding site
direct the exact place to initiate DNA transcription
determine when an how a gene is transcribe
True or false: gene expression is highly regulated
true
What is epigenetics?
the shape of chromatin influences gene expression
What happens to gene expression in the following scenarios:
open DNA
compact DNA
highly compacted DNA
open DNA: easy to read, gene expression
compact DNA: difficult to read, some gene expression
highly compacted DNA: very hard to read, no gene expression
What are housekeeping genes?
expressed in all cells all the time (almost)
responsible for routine metabolic functions common to all cells
How are some other genes asides from housekeeping genes expressed?
some are expressed as a cell enters a particular pathway of differentiation
some are expressed all the time in only those cells that have differentiated in a particular way
some are expressed only as conditions around and in the cell change
What does separation of transcription and translation in space and time allow eukaryotes to do?
greater control in regulating gene expression
What are the four types of nucleotides in mRNA?
A, U, G, C
not thymine
pairs: A-U and G-C
multiple codons code for 1 amino acid
What is recombinant DNA?
form of artificial DNA that is created by combining two sequences from different sources
allows proteins to be produced via artificial means
-engineer gene for more protein productivity
-produce desired proteins in vitro for therapeutic use
What are some uses of rDNA?
insulin
growth hormone deficiency
What are advantages of engineering prokaryotes?
cultivation of prokaryotes is easy (grow and maintain)
gene manipulation is easy as plasmid DNA is easy to isolate and manipulate
Which cells are used to mass produce proteins?
bacterial
yeast
mammalian
How are clones typically produced for prokaryotes?
placing a DNA fragment of interest into a vector DNA molecule, which can replicate in a host cell
What are the steps in DNA cloning?
- isolation of gene of interest
- isolation of plasmid DNA (cloning vector)
- manipulation of DNA sequence
a. cutting-restriction enzymes
b. joining-DNA ligase - transformation of bacteria
- selection of “correct” bacteria
- replication of the cells carrying rDNA molecules to get a genetically identical cells or clone
What are cloning vectors?
specifically modified DNA that are used to propagate foreign DNA in bacteria
What are the three essential features for a cloning vector?
origin of replication
dominant selectable marker
restriction sites
What are restriction endonucleases?
primarily bacterial enzymes that cut foreign DNA into fragments by recognizing specific nucleotide base pairs
What is DNA ligase?
unique ability to link or paste together DNA fragments that have been produced by exposure to restriciton endonucleases
What are methods of transformation?
heat shock
CaCl2 transformation
Lipofectin and similar molecules
electroporation
microinjection
What is the size, structure, modification, manufacturing, characterisation, stability, and immunogenicity of small molecule drugs.
size: small (single molecule), low molecular weight
structure: simple, well defined, independent of manufacturing
modification: well defined
manufacturing: chemical synthesis, predictable chemical process, identical copy can be made
characterisation: easy to characterise completely
immunogenicity: mostly non-immunogenic
What is the size, structure, modification, manufacturing, characterisation, stability, and immunogenicity of biological drugs.
size: large (mixture of related molecules), high molecular weight
structure: complex (heterogenous), defined by the exact manufacturing
modification: many options
manufacturing: produced in living cell culture, difficult to control from starting material to final API, impossible to ensure identical copy
characterisation: cannot be characterised completely the molecular composition and heterogenicity
stability: unstable, sensitive to external conditions
immunogenicity: immunogenic
What is a big issue with biologics?
cost, especially for chronic conditions such as rheumatoid arthritis
small molecule drugs cost on average US $1 per day, a biological costs on average US $22 per day
What is an example of an early use of biotechnology in the pharmaceutical industry?
insulin was made using E.coli cells in 1978
How did we used to make insulin? What was the issue?
previously isolated from the pancreas of cows, pigs, and other farm animals
could elicit an immune response
True or false: animal growth hormones have therapeutic value in humans
false
How was growth hormone extracted before the use of recombinant DNA? What was the issue?
extracted from the pituitary of cadavers
resulted in significant shortage in availability and transfer of diseases such as Creutzfeldt-Jacob disease
Differentiate between lag phase, log phase, stationary phase, and death phase.
lag phase: number of bacteria does not change with time
log phase: number of bacteria increases exponentially over time
stationary phase: there is no net change in number of bacteria with time, bacteria divide but also die at same rate
death phase: number of bacteria decreases with time
True or false: most important biological products such as antibiotics are secondary metabolites
true
At what phase do primary and secondary metabolites peak?
primary: log phase
secondary: late log phase and early stationary phase
Describe rifamycin B.
medically useful antibacterials
produced by Stretopmyces mediterranei
inhibit transcription by binding prokaryotic but not eukaryotic RNA polymerases
inhibit chain elongation, leaving inactivated RNA polymerase bound to the promoter
sterically blocks the path of RNA elongation when the transcript two or three nucleotides long
What are limitations with bacterial expression systems?
bacteria are not capable of producing glycoproteins because they lack the capacity to glycosylate
proteolytic cleavage may lead to degraded product
What is cell & tissue culture?
tissue culture is the general name for the removal of cells, tissues or organs from an animal or plant and their subsequent placement into artificial environment conductive to growth
the environment usually consists of a support medium that supplies nutrients
when the cells are removed from the organ fragments, disrupting their normal relationship with neighboring cells, it is called cell culture
What are the two classes of cultures of animal cells?
primary cells
cell lines
Describe primary culture.
cells are surgically removed from an organism and placed into a suitable culture environment they will attach, divide, and grow
primary culture cells have a lifespan of 5-10 divisions in vitro
due to lifespan, cannot do long term experiments
considered to be physiologically similar to in vivo cells
What is Hayflicks Phenomenon?
cells will continue to grow and divide normally for a limited number of passages
when they get to a certain point even if they are given appropriate nutrients, they simply stop dividing and die
correlation between maximal number of passages and aging
number of passages decreases when cells are harvested from older individuals
Describe cell lines.
if the cells in a cell strain undergo a transformation process that makes them immortal they are called a cell line
it is unknown how a diploid cell strain becomes a cell line, although it may be mimicked with oncogenic viruses or chemical carcinogens
often have abnormal chromosome # and maybe tumorigenic
What are other techniques to transform cells?
chemical or gamma ray treated cells
viral infection with SV40 T antigen
Result is a cell with altered function, morphological, and growth characteristics
What is a suspension cell culture?
derived from cells which can divide and survive without being attached to a substrate
maintained in culture vessels that are not tissue-culture treated
requires agitation for gas exchange
easier to passage
What is an adherent cell culture?
must adhere to a surface to survive
form monolayers
growth is limited by surface area
dissociated enzymatically or mechanically from surface
Describe cell culture medium.
cells have complex nutritional requirements that must be met to permit propagation in vitro
different types of cells have different growth requirements and a number of chemically-defined formulations
some serum-free media are available and some cell lines have been adapted to growing in such a medium, most cell lines require the addition of 5-10% serum for multiplication
Describe culture vessels.
provide a contamination barrier to protect the cultures from external environment while maintaining the internal environment
for anchorage-dependent cells, surface for cell attachment
easy access to the cultures and optically clear viewing surfaces
List examples of culture vessels.
cell culture dishes:
-cheapest and provide access to the growth surface
-anchorage dependent cells
multiwell plates:
-savings in space, media, and reagents
flasks:
-wide range of growing areas, shapes, several neck designs
-anchorage dependent cells
surface coatings:
-vessel surface is treated to render hydrophilic
-coating surface with serum, collagen, laminin, gelatin, poly-L-lysine
