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 CO2 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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