Lecture 8 Flashcards
Scaling up and downstream processing
what parameters influence design of processing system at all scales
temp pH rate and type of mixing oxygen demand colour jk sterility and containment SA:V
With what increase in size do optimal conditions normally change
10 fold increase
Describe the general scheme for large scale fermentation process
formulate growth medium
sterilise equipment
grow a stock culture up to 1l
inoculate seed fermenter then finally production fermenter
seperate cells from culture medium once finished - centrifugation or filtration
When separating cells form culture medium what must considered
if protein is secreted or not - if yes can destroy cells if not must somehow get out of cells
What is the most common design for a fermentor and it’s main principle
Stirred tank - growing microbes under defined conditions
What are the features of a stirred tank reactor
parameters are controllable
cooling jacket for temp
stirrers for aeration
sensors to measure pH
Name the 2 growing modes fermentation can accommodate, describing each
Batch
Closed system, fixed volume of medium inoculated, culture grows, medium changed as result of microbe growth
Fed batch
media added to vessel as process proceed
fermentation stopped when vessel is full
means you can extend exponential phase by adding more medium, diluting culture as you go along so you;re also diluting TOXINS
Give 2 examples where fed batch was better and what expression was controlled by
IFNgamma
initially produced from ecoli in batch - expression controlled by pl and cI (you get temp sensitive cI mutants, so denture this therefore get more expression cos less control)
Now in fed batch system, grown till late exponential then growth medium added at high temp.
20xgreater yield
Monoclonal AB fragments - fab
greatly imporved in fed
use lacI so cheap lactose as inducer. Cheap yay.
if you have multicopy plasmids, is radnom diffusion sufficient to stabilise them and pass on desired genes?
Yup.
If you are not a high copy plasmid microbe (give an example) how can the problem of plasmids not being passed on to some duaghters be solved
Plasmid based toxin-anti-toxin systems
What’s the example of the toxin anti toxin system and which plasmid is it on in ecoli?
Hok/soc system, on R1 plasmid
What encodes a toxin in the h/s system?
hok
What’s occuring if hok/sok plasmid is present
mok/hok transcript is made
also an anti-sense sok transcript is made
sok transcript INHIBITS TRANSLATION of mok/hok.
hok protein not made, cells ok.
What’s occuring if no hok/soc plasmid made it to daughter cell
Sok (which is encoded by something else, so is present anyway) is an sRNA so degrades faster than the Hok mRNA.
Therefore the hok protein is translated and cell dies.
Ensures you;re only left with cells whihc have a specific plasmid present
How are mammalian cells normally cultured? What growth levels achieved over 3-4 days
stiirred tank reactor, batch culture
10^6cells/ml
