Biotechnology Flashcards
what is biotechnology?
use of biological systems to generate useful products
examples of areas where biotechnology used / can be used?
- transgenic animals
- gene editing
- multigene cassettes
- tissue regeneration
- protein production + protein engineering
- gene therapy
what is exogenous gene expression?
expression of non native genes in organism
expression via plasmids/integration into genome
give examples of expression vectors
- F1 phage origin: viral packaging
- AMP resistance: antibiotic resistance
- ori : origin/replicaion
when cloning any gene, what do you need?
restriction sites at ends of gene
cloning process
- cut both dna mol with same RE
- gel purify 2 fragments - gel electrophoresis
- join fragments using dna ligase
- verify cloning via gel electrophoresis
why do you usually use different RE?
to stop re-ligation + to preserve orientation of insert
gel electrophoresis
used to verify sizes and separate/purify fragments
purified product used for transforming cell
product can = digested with REs to verify that ligation has worked
what do eukaryotes use for strong expression?
viral promoters (CMV/SV40)
what are more specific promoters used for?
lower expression levels
for most biotech processes, what is used?
e.coli
mammalian cell expression system
- correctly folded + fully post-transcriptionally modified
- transported to culture media
available for purification
bacterial/yeast cell expression host
- intracellular expression
- soluble/insoluble
- cell breakage needed
available for purification and modification if needed
give an example of a recombinant vector?
lac operon
what is the lac operon used for?
control elements for heterologous protein expression
what can be improved with the promoter-expression-cassette
needs = more tightly controlled
as is prone to leakage
leaky expression leads to plasmid instability - plasmid loss
T7 RNA polymerase
taken from bacteriophage T7
state the cassette location, promoter, operator, and whether its induced/un induced for the LacI cassette?
- e coli chromosome
- rna polymerase
- operator unknown
- un-induced constitutive ON
- induced constitutive ON
state the cassette location, promoter, operator, and whether its induced/un induced for the T7 polymerase?
- e coli chromosome
- rna polyermase
- operator -> lacI controlled
- uninduced constitutive OFF
- induced constitutive ON
state the cassette location, promoter, operator, and whether its induced/un induced for the gene of interest?
- plasmid vector
- t7 polymerase
- lacI controlled
- uninduced constitutive OFF
- induced constitutive ON
what do CHO cells need in their medium for growth?
proline
CMV promoter
- presence of SV40 replication —> high dna replication levels in SV40 replication permissive cells
what is CMV?
herpes virus that infects most cell types and establishes latency in leukocytes
the enhancer sequence is…
primary control point
