Bioprocessing Mid Term Cards

Front 1

What are the Recombinant and Expression Systems?

Back 1

Microorganisms (Such as E. coli, yeast)
Cell cultures from higher organisms (such as: animal cells, insect cells and plant cells)
Whole higher organisms (such as, transgenic plants, animals)

Front 2

What are the benefits of recombinant DNA technology?

Back 2

1. Overcomes source availability
2. Overcome problems of product safety
3. Alternative to inappropriate and dangerous source material
4. We can engineer in desired properties

Front 3

What are Post-translational modifications (PTM) and expression systems?

Back 3

Bacterial systems-E. coli cannot attach sugars to proteins so
proteins requiring such sugars
have to be made in the cells of eukaryotes such as yeast cells and mammalian cells
Eukaryotic systems-Yeast

Front 4

What are the Advantages of Recombinant Protein Expression in E.coli?

Back 4

•Grows quickly (8hrs to produce protein)
•High Yields (50-500mg/L)
•Low cost of media (simple media constituents)
•Low fermentor costs

Front 5

What are the Disadvantages of Recombinant Protein Expression in E.coli?

Back 5

•Difficulty expressing large proteins (>50kD)
•No glycosylation or signal peptide removal
•Eukaryotic proteins are sometimes toxic
•Can’t handle S-S rich proteins

Front 6

Why Do We Use the K-12 of the E.coli Strain vs The Wild Type Strains?

Back 6

Cultivated strains (e.g. E. coli K12) are well-adapted to the laboratory environment
Modified strains are used to optimize expression of recombinant proteins
They are non-pathogenic and have lost their ability to thrive in the intestine

Front 7

What is on the pGLO Plasmid-What expresses GFP?

Back 7

Beta Lactamase

Green Fluorescent Protein

araC regulator protein

Front 8

What does each factor Do on the pGLO Plasmid?

Back 8

Beta Lactamase is for ampicillin resistance
Green Fluorescent Protein is the aequorea victoria jellyfish gene
araC regulator protein regulates GFP transcription

Front 9

What two factors must be added to the bacteria’s environment for you to see the green color?

Back 9

Arabinose and Ampicillin

Front 10

What are each of the two added factors you listed doing to cause the genetically transformed bacteria to turn green?

Back 10

Arabinose-Induces GFP procduction (added in log phase)
Ampicillin-B-lactamase ampicillin resistance for selection

Front 11

Why do we need inducible promoters?

Back 11

E. coli growth is very sensitive to expressed foreign proteins

Bacteria expressing foreign proteins can grow slowly, and may lose the ability to express the foreign protein.

The most efficient way to express foreign proteins is to use a promoter that is “off” then add inducer to induce foreign protein expression

Front 12

What is the Purpose Double Induction by IPTG T7 RNA polymerase (98 kDa)
Target Gene??

Back 12

Tight control of protein
expression to avoid toxicity

Front 13

In Recombinant Gene Expression Systems Why is IPTG Used Instead of Lactose?

Back 13

its not metabolized

Front 14

What is IPTG?

Back 14

Lactose analog, isopropyl-beta-D-thiogalactopyranoside

Front 15

Two Conditions Must be Satisfied for the Lactose Genes to be Transcribed?

Back 15

Low glucose: high levels of cAMP accumulate. cAMP binds to catabolite activator protein (CAP) and together they will bind to a promoter sequence on the lac operon.
High lactose: must be present inside the cell to remove the lactose repressor from the operator sequence
When these two conditions are satisfied, it means for the bacteria that glucose is absent and lactose is available.

Front 16

What is Involved in Upstream Processing

Back 16

Cell culture steps-Steps that produce the drug. Bioreactors are our drug factories

Use strategy of increasign culture volume

Thaw vial or cell bank
laboratory scale production
procution scale production
Production scale bioractor

Front 17

What is Involved in Downstream Processing

Back 17

Purification steps-steps that remove cell debris

use of microfiltration,centrifugation or depth filtration

Front 18

Draw and Lable the E.coli Curve

Back 18

curve

Front 19

Describe the different E. coli Growth Phases

Back 19

Lag phase - growth and reproduction are just beginning
Log phase - reproduction is occurring at an exponential rate
Stationary phase - environmental surroundings and food supply cannot support any more exponential growth
Death phase - when all of the nutrients have been exhausted, the population dies off

Front 20

What measures Cell Concentration?

Back 20

Turbidity by Absorption @ 600nm

Dry cell mass


Wet cell mass

Front 21

What Affects the Growth of E.coli?

Back 21

•Temperature
•Nutrient supply
•Changes in pH
•Aeration/dissolved oxygen
•Toxic products
•Heat produced during growth

Front 22

What is the Optimal Growth Temperature for E.coli

Back 22

37 degrees celcius

Front 23

Why is Dissolved Oxygen Important?

Back 23

Its a substrate in aerobic fermentation but can be a limiting substrate, since oxygen is sparingly soluble in water

Front 24

What are the Different types of Fermentation?

Back 24

•Batch

•Fed-batch

•Continuous

Front 25

What Goes on in The Batch Reactor? and Draw the graph.

Back 25

Stirred tank
Permanent change in conditions, nutrient concentration decreases, cell and waste product concentration increases
Oxygen and CO2: Continuous supply and removal, but no steady state
Fully unregulated system

Front 26

What Goes on in The Fed-Batch Reactor? and Draw the graph.

Back 26

Continuous or bolus supply of nutrients
Goal: steady concentration of nutrients
Looking for steady cell growth with constant growth rate
Limitation: accumulation of toxic metabolites
Less concentrated feed solution can help to dilute toxins to some extent

Front 27

What Goes on in Continuous? and Draw the graph.

Back 27

Steady state of everything nutrient introduction and waste removal

Front 28

Draw the Basic Fermentor Design

Back 28

Aeration by gas sparging
Impeller – even distribution of nutrients and cells
Baffles prevent vortex formation
Ports for probes to measure pH, temp, metabolite conc.
Ports to add acid/base or nutrients
Circulating water in outer jacket to dissipate heat

Front 29

What is the Clark Electrode?

Back 29

it regulates glucose

Front 30

What is Ideal for Growing E. coli in Fermenters?

Back 30

High gas flow rates, use oxygen supplementation to air
High impeller speed

Front 31

How can we Maximize Production of Recombinant Proteins in E.coli?

Back 31

Increase size of fermentation facility
Improvements in protein expression
-Expression/induction systems
Improving protein folding and stability
E.coli strains with reduced protease activity
E.coli strains with increased expression of chaperones and or redox enzymes
Improvements in fermentation processes
Metabolic engineering strategies

Front 32

In E.coli: Aerobic respiration and mixed-acid fermentation, After glycolysis what does E.coli do?

Back 32

shuttle the sugar
equivalents into the citric acid cycle for generation
of energy or into mixed-acid production

Front 33

What can E. coli cells produce as an extracellular co-product of aerobic fermentation?

Back 33

acetate

Front 34

How does the formentation of Acetate by E. coli as an Extracellular Product Happen?

Back 34

When glucose is the limiting nutrient (sole carbon source)
When cells grow above a threshold growth rate
Not related to availability of oxygen, but to the rate of oxygen consumption
E.coli can only consume oxygen up to a maximum rate

Front 35

What are th eProblems with Acetate?

Back 35

Retards growth and inhibits protein formation
Acetate production represents a diversion of carbon that might otherwise have generated biomass or the protein product
Reduces rate of RNA, DNA, protein and lipid synthesis
Acetate is more inhibitory to protein-producing cells than wild type cells

Front 36

If a Protein is Expressed Intracellulary it is Typically from What type of Cell? and What is required?

Back 36

bacterial

Cell fraction required

Front 37

If a Protein is Expressed Extracellulary it is Typically from What type of Cell? And What is Required?

Back 37

Mammalian

Culture Medium is Required

Front 38

For microbial expression: How do we get the protein out? (3 ways)

Back 38

Mechanical – high pressure, agitation with beads
Non-mechanical - sonication
Enzymatic – lysozyme to digest cell wall (research scale)

Front 39

What are Inclusion Bodies?

Back 39

Insoluble aggregates of misfolded protein.
contain mostly the recombinant protein



Insoluble aggregates of misfolded protein.
contain mostly the recombinant proteinInsoluble aggregates of misfolded protein.

.

Front 40

How Do We Deal With Inclusion Bodies?

Back 40

After isolating the inclusion bodies, they have to be dissolved using chatropic agents
---8 M urea
---6 M guanidine HCl
The conditions that dissolve the inclusion bodies also completely denature the recombinant protein
The recombinant protein has the be “refolded” carefully to restore its functionality – optimize redox conditions
further purified