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


22 Οκτ 2013 (πριν από 4 χρόνια και 8 μήνες)

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The Production of a
Recombinant Biotechnology

Chapter 8


Give a basic overview of genetic

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

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:

DNA or protein is identified or isolated.

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

Recombinant cells are produced.

Vector produced is inserted into a host cells.

Recombinant cells are grown in culture

Recombinant protein product is isolated and

Product is tested and sent to market.

Overview of Genetic Engineering

How do you find your gene of

1. membrane removal

A. Bacterial chromosomal

B. Bacterial plasmids

How do you find your gene of

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

Cell Contents

Removing other molecules from the cell


Dissolves membrane lipids and precipitates




Remove proteins


Removes RNA

Cell Contents


Spin sample

pellet (precipitate) forms at the bottom of the tube

Bacterial DNA remains in solution

Supernatant is poured off leaving debris pellet

Cold ethanol extraction performed.

Layer cold ethanol applied followed by high
centrifuge spin.

Precipitated pellet that forms is DNA.

How do you find your gene of

B. Plasmids:

Similar process to getting bacterial chromosomal

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

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)


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

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

Transformed cells are grown in ever
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 “

defined as either:

Alcoholic fermentation (glucose into CO


acid fermentation (glucose into lactic acid)

In both cases cells utilize glucose under anaerobic

In biotechnology fermentation is defined as
growth of cells

under optimum conditions
for maximum cell division and product

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.


Up to 20
µg/500 µL


800 µg/mL


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

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

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

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

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

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.


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

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

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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