1. The Production of a Recombinant Biotechnology Product Chapter 8

2. 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.

3. 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.

4. 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.

5. Overview of Genetic Engineering

6. How do you find your gene of interest?  1. membrane removal  A. Bacterial chromosomal  B. Bacterial plasmids

7. 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

8. Cell Contents  Removing other molecules from the cell lysate.  Detergents  Dissolves membrane lipids and precipitates proteins.  Sarkosyl  SDS  Proteases  Remove proteins  RNase  Removes RNA

9. 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.

10. 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.

11. 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

12. 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.

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

14. 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.

15. Producing Large Numbers of Transformed Cells

16.  During each scale-up the following variables are measured:  Cell growth rate  Product concentration  Product activity  Possible contamination

17. 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.

18. 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.

19. 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.

20. 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

21. 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.

22. 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 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.

23. 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.

24. 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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