Lecture 8 - Genetic instability

Question 1

What is a problem with genetic stability and cells that lose that capacity to make a target protein

Answer 1

Cells that lose the capacity to make the target protein often grow more quickly and can displace the original recombinant strain

Question 2

Genetic instability may occur due to (3)

Answer 2

Segregational loss
Structural Instability
Host cell regulatory mutations

These may cause
Growth rate dominated instability

Question 3

What is segregational loss influenced by? (4)

When does it occur

Answer 3

DO Conc
Temperature
Medium composition
Dilution rate

occurs during cell devision where one of the daughter cells does not receive a plasmid

Question 4

What is the equation for the probability of plasmid containing cells losing their plasmids during growth

Answer 4

P = 2^(1-z)

Question 5

What is significant about low copy number cells

Answer 5

They have specific mechanisms to ensure equal distribution among daughter cells

Question 6

What is significant about high copy number cells

Answer 6

Plasmids distributed randomly according to binomial distribution

Question 7

What is structural stability

Answer 7

Where some cells alter the plasmid to reduce harmful effects on the cell.
cells will stop producing the target protein to reproduce faster

Question 8

What are host cell mutations

Answer 8

Sometimes the host cell will mutate and make cells less useful to the production system

Question 9

What is growth rate dominated instability

Answer 9

If the altered system has a distinct growth rate advantage it will eventually dominate the culture

Question 10

Write 2 modelling equations that can be used to find the accumilation of plasmid containing and plasmid free cells

Answer 10

dX-/dt = p(mu+)X+ + (mu-)X-
The rate of plasmid free cells is the rate of growth of the culture multiplied by the probability of loss of cells plus the growth rate of plasmid free cells

dX+/dt = (1-p)(mu+)X+
The growth rate of plasmid containing cells multiplied by the probability that they will not lose their plasmids

Question 11

What is the fraction of plasmid containing cells

Answer 11

f = X+/X++X-

Question 12

what is n

Answer 12

n = (mu+)t/ln2

The number of cell generations

Question 13

On slides 18 - 20 Discuss the changes in the graph

Answer 13

Slides 18 - 20

Question 14

What key considerations must be made for reactor design

Answer 14

• copy number of cells
• Promotor
• Inducer
• compatibility with host cell
• Selective pressure