The Production of a Recombinant Biotechnology ... - Workforce 3 One

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22 Οκτ 2013 (πριν από 3 χρόνια και 10 μήνες)

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The Production of a
Recombinant Biotechnology
Product

Chapter 8

Objectives


Give a basic overview of genetic
engineering.


Describe the processes involved in
isolating a piece DNA of interest


Mass producing DNA or it’s protein product,
and recovering product.


Describe how DNA concentrations and
purity can be calculated.


Define cGMP.

8.1 Overview of Genetic
Engineering


Genetic engineering involves the
manipulation of the genetic information of
an organism.


Genetic engineering can result in the
production of organisms with new and
improved characteristics.


Can bring about new and improved products.

Overview of Genetic Engineering


Steps in genetic engineering:

1.
DNA or protein is identified or isolated.


In case of DNA a specific sequence is cut out
and placed into a vector (produces recombinant
DNA, rDNA).

2.
Recombinant cells are produced.


Vector produced is inserted into a host cells.

3.
Recombinant cells are grown in culture
(cloning).

4.
Recombinant protein product is isolated and
purified.


Product is tested and sent to market.

Overview of Genetic Engineering

How do you find your gene of
interest?


1. membrane removal


A. Bacterial chromosomal


B. Bacterial plasmids


How do you find your gene of
interest?


A. Chromosomal:


Good amount of cells grown on agar or in
broth culture


Cultures transferred to buffer solution contain
enzyme lysozyme


Lysozyme degrades bacterial cell wall by breaking down
structural carbohydrates


When cell walls gone….osmotic pressure
causes cell to rupture dumping their cell
contents


Cell Contents


Removing other molecules from the cell
lysate.


Detergents


Dissolves membrane lipids and precipitates
proteins.


Sarkosyl


SDS


Proteases


Remove proteins


RNase


Removes RNA

Cell Contents


Centrifugation


Spin sample


pellet (precipitate) forms at the bottom of the tube


Bacterial DNA remains in solution


Supernatant is poured off leaving debris pellet
behind.


Cold ethanol extraction performed.


Layer cold ethanol applied followed by high
-
speed
centrifuge spin.


Precipitated pellet that forms is DNA.

How do you find your gene of
interest?


B. Plasmids:


Similar process to getting bacterial chromosomal
DNA.


Exception: Cell lysis also contain a base such as,
NaOH, in addition to using SDS


Allows for degradation of bacterial chromosomal DNA, In
addition to cell wall and plasma membrane.


Requires a series of isopropanol and ethanol
precipitations follow.


Isolating animal, plant, or fungal DNA is similar
to bacterial chromosomal DNA with some minor
differences.


8.2 Getting the DNA into a cell


Prior to getting DNA
recombinant DNA (rDNA)
needs to be produced.


Recombinant DNA can be
created from putting your
DNA of interest into a vector,
or into the DNA of a virus.


Making recombinant DNA
results from cutting your DNA
and either the plasmid or
viral DNA with restriction
enzymes (molecular scissors)


Endonucleases


Isolated from bacteria


Named based on origin




Getting the DNA into a cell


The DNA cut and the vector
it is inserted into have
complementary base
pairing ends.


These “sticky ends” are
“glued” together using DNA
ligase


Once successfully inserted
the rDNA can be used.




Getting the DNA into a cell


Once you have your rDNA the next step is to get
it into a cell or virus.




8.3 Producing Large Numbers of
Transformed Cells


To get a protein or piece of DNA of interest you
must have large volumes of transformed cells.


The process by which this is done is called
scaling
-
up.


Transformed cells are grown in ever
-
increasing
amounts, in larger and larger containers.


50 mL broth solution


1 or 2 L spinner flasks


10 L fermenters


100 L fermenters


1000 L fermenters


10,000 L or more fermenters


Bacteria exhibit exponential growth so under maximal
conditions it generally does not take long for large
volumes to be obtained from a seed colony.





Producing Large Numbers of
Transformed Cells





Producing Large Numbers of
Transformed Cells


During each scale
-
up the following
variables are measured:


Cell growth rate


Product concentration


Product activity


Possible contamination





8.4 Producing Large Numbers of
Transformed Cells


Clarifying fermentation.


Fermentation is “
generally”

defined as either:


Alcoholic fermentation (glucose into CO
2

and
ethanol)


Lactic
-
acid fermentation (glucose into lactic acid)


In both cases cells utilize glucose under anaerobic
conditions.


In biotechnology fermentation is defined as
the
growth of cells

under optimum conditions
for maximum cell division and product
production.





8.5 Plasmid Retrieval from Cells


Plasmid retrieval is performed:


to make sure that the correct plasmid has been
inserted into the cell.


to collect plasmids for future transformations.


Preparation (prep)


Extraction of plasmids from transformed cells.


Miniprep


Up to 20
µg/500 µL


Midipreps


800 µg/mL


Maxipreps


1 mg/mL or higher


Each process follows the same overall process but as larger
amounts of plasmid are required larger volumes and
equipment are needed.





Plasmid Retrieval from Cells


Outline of the miniprep procedure:


Transformed cells separated from the broth.


Resuspended cells are treated with high pH SDS
-
NaOH followed up with potassium acetate.


destroys cell wall and membrane


chromosomal DNA and proteins precipitate out


Mixture spun again and supernatant mixed with
isopropanol.


Nucleic acids precipitate out.


Mixture spun again and ethanol washes preformed to
remove everything except the DNA.


Your plasmid is in the pellet formed after the spin.


RNase may be added to limit chance of RNA
contamination.


Plasmid pellet is resuspended in TE buffer.






Plasmid Retrieval from Cells


Determining the amount and the purity of
your plasmid DNA.


Quick visual exam


Cut with restriction enzymes and run on gel
electrophoresis.


Stain with ethidium bromide





Plasmid Retrieval from Cells


Determining the amount and the purity of
your plasmid DNA.


UV spectroscopy


Measure at 260 nm


Usually want a minimum of 0.005 µg/µL


Restriction digest require a minimum concentration of 0.1
µg/µL.


How to calculate concentration?


Known that 50 µg/mL of pure double stranded DNA
absorbs 1 au of light at 260 nm.


50 µg/mL

=
X µg/mL

1 au at 260 nm the absorbance of sample at 260 nm


Sample must have an absorbance of 0.02 to 2.0 au to be
used.






Plasmid Retrieval from Cells


Determining the amount and the purity of
your plasmid DNA.


UV spectroscopy


Calculating DNA purity


Must know the DNA & protein concentration


Use a ratio of DNA to protein to calculate


Absorbance (au) at 260 nm


Absorbance (au) at 280 nm


Ratio value between 1.8 to 2.0 is desired


Greater than 2.0 RNA contamination.


Lesser than 1.8 protein contamination.


Purity values 1.0 or less indicate plasmids recovered
not worth using for future transformations or
restriction digest.






cGMP


Products that are produced and under FDA
(Food and Drug Administration) must comply
with current good manufacturing practices
(cGMP).


cGMP is outlined in Title 21, Parts 210 and 211, of
the Code of Federal Regulations.


Outlines quality management & organization, device design,
buildings, equipment, purchase, and handling of
components, production and process controls, packaging
and labeling controls, device evaluation, distribution,
installation, complaint handling, servicing, and record
keeping.


Regular site audits are carried out by the FDA.





Homework 8


Section 8.1


Questions 2, 3, 4


Sec 8.2


Questions 2, 3


Think like biotech


Questions 1,3,4, 5, 7, 8




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