21 Flashcards
Two key features of a plasmid vector that you could use to generate corn plants that express GFP only in corn kettles
- promoter ( drive expression in corn Kernals)
- transgene (clone into plasmid )
- ori
- antibiotic resistance
Why can’t proteins with complex post-translational modifications be produced in bacteria? What is al alternative protein production system that will allow successful expression of glycosyalted proteins?
- they dont have a golgi or ER
- they doing have enzymes required to glycosylation
Mammalian cells
Transgenic animals
Transgenic plants
Worst and best choices of protein production
In protein production ____ is a problem for bacteria
Folding
Is insulin PTM and where is it best produced in?
No
Bacteria
Is EPO PTM and where is it best produced in?
- yes, gycoslated
- mammalian cells (CHO)
Is antithrombin PTM and where is it best produced in?
Gamma - carboxylated
(Not modified in mammalian cell) there for its best produced in transgenic animals (goats)
GFP is usually found in
Jelly fish
Insulin found in the
Pancreas
Antithrombin is found in the
Kidney
Purpose of protein evolution
- speeding up evolution process to try and alter or try and improve a protein for a new or better use
What were the different colours of GFP made from
Protein evolution
How protein evolution of GFP worked to make diff colours
- amplified the cDNA using degenerate PCR
- degenerate PCR allows for mutations as degerate doesnt have proof reading mechanisms, making it prone to errors
- take mutated PCR products and clone into plasmid (using restriction sites) then transform into bacteria.
- select out the variants from the plate
- can then sequence to discover what to base pair change was
Recombinant DNA technologies in the clinic =
Recombinant proteins that require repeated administration of drugs for the life of the patient / illness
?????
How can we provide a permanent treatment ?
Use patients as host for protein production
Plasmids can be carries into proteins ALSO by using ____ _____
Viral vectors
Viral and bacterial plasmids look the same
Yes
In gene therapy we introduce a plasmid into a… which is then…….
Why?
…Viral vector (virus is diablesd) which is then inserted into a patient and gets taken up into cells and gene of interest is expressed under control of an appropriate promoter
- good at getting genetic info into cells
Invivo vs exvico
Exvivo - take cells out of body to insert gene
Invivo - insert directly into cells
Conventional biotechnology vs gene therapy
What kind of promoter do we use for gene therapy insulin production in humans
Mammalian glucose responsive promoter
Virus is used as a ____ to ___ ______ ____ patient _____
Virus is used as a vector to carry gene into patient cell
In insulin gene therapy what cells of what organ are target and why
- liver rather then pancreas where cells are dying
Which system is virus injected into in the permanent gene therapy hypothesis and what do they target
Venous
Liver - so they stay whole life time - only one treatment
Why can’t u target pancrease B cells for gene therapy with insulin?
B cells are destroyed by autoantibodies so we use liver to make insulin (using promote that responds to glucose in the blood)
Insulin - what kind of cDNA
- prepreprtein
Fulll one
Different protein factories are required depending on
Complexity of the protein u want to produce
DNA technology can be used to..
Create new proteins
Using humans as a protein factory (gene therapy) can be used to…
Permanently treat disease
