Biotechnology

This book is meant for students and professionals who are looking for reference on different areas in this field, to bring a new student or new hire up to speed.

A scientific revolution less than 20 years old that has already changing the foods we eat and react to the environment.

To bring out the best in nature.

What is Biotech?

Farmers and bakers were the pioneers of the biotech. Remember Grandma's freshly baked bread? How Grandpa kept the seeds of those really big pepper or tomatoes? Your grandparents were practicing biotechnology. Maybe you still do the same, that is the basis of biotechnology.

Defining "Biotechnology"

The application of the principles of engineering and the use of technology in the field of life sciences-bioengineering.

1. The use of living things to make products.
2. The study, application and control of a biological processes.
3. The application of any of the above or derivatives thereof, to make or modify products or processes for specifically defined uses.

The use of microorganisms (such as bacteria or yeasts) or biological substances (such as enzymes) to perform specific industrial or manufacturing processes. Applications include the production of certain drugs, synthetic hormones, and bulk foodstuffs, as well as the bioconversion of organic waste and the cleanup of oil spills.

Cloning, genetic manipulation, cell fusion, and mutation.

Modifying the genetic material of organisms directly and with increasing precision, has enabled the transfer of genes between extremely diverse organisms, in combinations unlikely to occur by non-technological means, allowing speedier and more specific results.

Essentially, doing "more and faster" building on what we have known and done for centuries and going beyond.

Life- Defined as:

Movement

(Though some living organisms many organisms remain non-motile or some may show negligible movement from their position throughout their lives even when they are alive)

Growth

(increase in size in terms of mass, volume, and/or number of cells in case of multicellular organisms till peak growth is attained.)

Eating

(though autotrophic organisms can produce their own food e.g. most plants (except carnivorous plants) are able to make their own food via photosynthesis in chloroplasts in presence of light)

Reproduction

Response to stimuli

Presence of metabolism

(ability to carry out metabolic biochemical reactions essential for survival e.g., glycolysis)

Presence of genetic material

(e.g. DNA in eukaryotes)

Interaction with abiotic and biotic components of environment

Homeostasis

Products

Products

Traditional

1. Beer / Wine (See Fermentation)
2. Yeast
3. Ethanol
4. Cheese
5. Idli

Protein Therapeutics

1. Herceptin
2. Neupogen
3. Insulin - 1982 U.S. pharmaceutical manufacturer Eli Lilly the first to market genetically-engineered human insulin
4. Somatostatin
5. Growth hormone

Agriculture

1. Bacillus thuringiensis (Bt) Crops Bt crops
2. Herbicide tolerance
3. Disease resistance
4. Pest resistance

Industrial / Environmental

1. Hazardous Waste
2. Wastewater Treatment
3. Bioremediation

Other

1. Plastics
2. Biopulping
3. GMO (Genetically modified organism)

21 CFR 58

Good laboratory practice for nonclinical laboratory studies:

http://www.access.gpo.gov/nara/cfr/waisidx_02/21cfr58_02.html

21 CFR 11

Title 21 Code of Federal Regulations (21 CFR Part 11) Electronic Records; Electronic Signatures

http://www.fda.gov/ora/compliance_ref/part11/

21 Code of Federal Regulations Parts 210 and 211

Part 210 - current good manufacturing practice in manufacturing, processing, packing, or holding of drugs; general

Part 211 - current good manufacturing practice for finished pharmaceuticals

http://www.fda.gov/cder/dmpq/cgmpregs.htm

SOPs

SOP's (Standard Operating Procedures)

1. Approved by management
2. You don't put data in a SOP
3. Written at 6th grade level
4. Clear, current, complete
5. Simple, Concise, Accurate
6. Archived Where?

Notebooks

Notebook

1. If it is not written, it is not done.
2. pages signed
3. Can you read it?
4. Black or blue ink
5. pages numbered
6. Leave some pages in the front for TOC
7. Required by GLP / GMP regs
8. Signed by witness
9. Traceability

Documentation for Integrity and traceability

1. What you did
2. Why you did it
3. Results
4. Reference earlier work?
5. Also for Quality

Keys to Successful Biotech products

Keys to Successful Biotech products

• Identify unmet clinical needs
• Explore and estimate business value
• Evaluate the feasibility and efforts
  

1. Availability of labor force
2. Skilled labor force
3. Power
4. Good Universities nearby:
   1. For retraining
   2. For new ideas

1. National and international labor pool
2. Raw materials
3. Marketing
   1. Clear trend and need

1. FDA approval

Record Keeping

1. Inventory control logs
2. Process Steps
3. SOP
4. et cetera
5. If it's not written it's not done
   1. Love it
   2. Live it
6. Clinical trials
   1. GMP apply to:
      1. Manufacture
      2. Processing
      3. Packing
      4. Storage
   2. Validation of each process
   3. PAT (Process Analytical Technology)
7. Quality should be built in and by design

1. Environmental Concerns
   1. Use of energy
   2. Use of water
   3. Environmental Laws
   4. Waste streams

1. Do we have the technology?

Requirements

1. Safety
2. Identity and Strength
3. Quality and purity
4. The plant:
5. Can we Measure
   1. pH
   2. Aeration
   3. Baffiling
   4. impellers
   5. medium
   6. Seed train
6. Are we Better than our competition?
   1. why?

Processes

Development / Upstream / Downstream processes

Development

1. Need / market analysis
2. Select a solution
3. Process Development
4. Understand the growth requirements
5. Calculate yields of biomass
6. Run small scale test

• Fermentation

Chemical

Yeast

1. High biomass yields
2. Will secrete the protein

• Picha Pastoris
• Fungi
• Mammalian Cells

Upstream

1. Media prep

Fermentation?

Find best conditions

Expensive Labor intensive Open Ended Time Consuming

Constraints

Raw Materials Batch to Batch variations Transportation costs Storage

1. Seed Prep

Find cell line

Composition Growth kinetics Yield Seed Bank

Master Seed Bank MSB

Original Stored Cells

Working Seed Bank WSB

Used in actual fermentation

Downstream

1. Slurry Handling
2. Excreted product
3. Contamination
4. Product purification
5. Separation / purification / sterilization

How?

1. Plate and frame
2. Bulk filtration
3. Spiral wound

       Plugs up easily
       Usually not used for bulk filtration

1. Storage

Technician: Skills Needed

The Biotech Technician must be a person possessing skills with ability to solve problems and meet the customer in such a way that the translations of what is possible can be made clear. They have to maintain a notebook, one that can be read by someone else. Present results in a clear manner, and work with others to meet objectives.

Laboratory Skills

A technician must use the tools of the trade not unlike any other trade, we are farmers but our herd is tiny tiny wildlife. To take care of our herd we must measure certain aspects of their environment.

Solute,solvent, and solution

Solute = Dry Material

Solvent = What you mix with

Solution = Solute + Solvent

pH

Measurement

1. Probe and meter

most accurate more expensive piece of equipment Store in buffer Check for clogging

1. Litmus paper

very coarse measurement of pH

1. Field kit

The letters pH stand for "power of hydrogen"

The most abundant element in the universe is hydrogen, which makes up about 3/4 of all matter!

Stronger acids give up more protons, H+ (hydrogen ions); stronger bases give up more OH- (hydroxide ions). Neutral substances have an even balance of H+ and OH-, E.g. Pure (distilled) water.

>7 base -- 7 Neutral -- <7 Acid

Depending on your definition, an acid is a hydrogen ion or proton donator and a base is a hydrogen ion acceptor, hydroxide ion donator, or electron acceptor.

Acids produce H+ ions in aqueous solutions, whereas bases produce OH- ions in aqueous solutions

pH electrode compared to a battery

Store in buffer not H2O

Mercury tube Good for metals and biologicals and up to 80 degrees C

The common Silver-Silver Chloride reference electrode used with most combination pH electrodes has a Potassium Chloride salt-bridge which is saturated with Silver Chloride.

Works well in most samples, but not in biological samples containing proteins or related materials

Calibration

Span error Difference b/w perfect and actual pH Electrode at 25C produces 59.12 mV/pH unit

Offset error

Difference from Perfect reading from actual reading is the offset error

signal @ pH 7.0 @ 25 C is 0 mV

Three point calibration

pH's 4, 7 and 10

Calibrate W/I range you going to use

Buffer and reagent Prep

Chemist use buffers to moderate the pH of a reaction. Buffers stabilize a solution at a specific pH value. Resist pH change when small amounts of acid or alkali are added.

KPO4

KPO4 buffer is highly recommended for most P450 assays (microsomal or recombinant enzymes) with the exception of CYP 2C9 and 2A6 where a Tris buffer system is more appropriate.

TRIS buffer

TRIS buffers are used by biochemists to control pH in the physiological range (about 7 to 8 pH) because phosphates cause undesirable side reactions with the biological substances in their test samples.

"Good" buffers

These buffers were well received by the research community because "Good" buffers are nontoxic, easy to purify and their pKa is typically between 6.0 and 8.0, the range at which most biological reactions occur.

The "Good" buffers also feature minimal penetration of membranes, minimal absorbance in the 240-700 nm range and minimal effects due to salt, concentration or temperature.

pKa = dissociation constant

In chemistry and biochemistry, a dissociation constant or an ionization constant is a specific type of equilibrium constant used for dissociation (ionization) reactions. Dissociation in chemistry and biochemistry is a general process in which complexes, molecules, or salts separate or split into smaller molecules, ions, or radicals, usually in a reversible manner. Dissociation is the opposite of association and recombination.

Problems

1. Ionic Strength
2. Temperature
3. Counter ion

colony agar plating

What is Agar?

A gelatinous material derived from certain marine algae.

1. Growth Media
2. Carbon Sources
3. Carbohydrates
4. Proteins
5. Nitrogen Sources

Two types:

Simple

Complex

Components required for preparing a minimal agar

1. minerals
2. H2O
3. vitamins
4. Precursors
5. Compounds that may lead to your product - inducers
6. Compounds added to induce gene expression

LB (Luria-Bertani) Media

Agar Plates

Blood

contains blood cells from an animal (e.g. a sheep). Most bacteria will grow on this medium

Chocolate

This contains lysed blood cells, and is used for growing fastidious (fussy) respiratory bacteria.

Mannitol Salt

Purpose Mannitol salt agar is both a selective and differential growth medium.

Brilliant Green

Inhibits Gram+ MacConkey

This type of agar is used since it is one of the most forgiving media available - it is hard to contaminate, and E. coli usually grow up as red colonies.

(Almost all spore forming bacteria are Gram-positive, but these cannot grow on MacConkey agar because of the detergent in it (bile salts), and very few Gram-negative bacteria can tolerate either the initial dryness of the plates, or the boiling temperatures needed to make the MacConkey agar. Also, while fungal spores can tolerate the dryness, they cannot tolerate the boiling.)

This is an agar upon which only Gram-negative bacteria can grow

Starch

An agar plate is a sterile Petri dish that contains agar plus nutrients, and is used to culture bacteria or fungi.

Neomycin agar

contains the antibiotic neomycin.

Sabouraud agar

Used for fungi. It contains gentamicin and has a low pH that will kill most bacteria.

LB

+ Complex + pH 7.2

UV/VIS Spectroscopy

Common UV/ VIS spectrophotometers Following is a list of commonly used spectrophotometers: GeneSys 20 HP8452A Diode Array Spectronic 20

Ultraviolet-Visible spectroscopy or Ultraviolet-Visible spectrophotometry (UV/ VIS) involves the spectroscopy of photons (spectrophotometry). It uses light in the visible and adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this region of energy space molecules undergo electronic transitions.

A=elc

1. A=Absorbance
2. e=Molar Absorbtivity
3. l=Path length (cm)
4. c=Concentration (M)

Laboratory calculations

1. + - Prep of Sterile solid and Liquid media

Sterilization Methods

There are different types of Sterilization techniques. Some of them are 1. Physical sterilization 2. Chemical sterilization

Under Physical sterilization a) Heat b) Filtration c) Ionising Radiation etc., In Heat sterilization i. Temperature above 100 C ii. Temperature at 100 C iii. Temperature below 100 C.

i. Temperature above 100 C There are two methods involved in it a. Moisture heat sterilization b. Dry heat sterilization

Pipetting

1. Growing concerns regarding repetitive strain injuries (RSIs)
2. Adopting good pipetting techniques is critical for the accuracy of your analysis and your health.
3. Contamination Prevention
4. Care of

Measuring / Mixing

Using a balance Calibration / documentation

UV/VIS spectroscopy

   * http://www.scienceofspectroscopy.info/
   * Use correct Reagent
   * Use correct Wavelength

Gel Electrophoresis

Gel electrophoresis is a method that separates macromolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties. Researchers can typically control the charge at the top and bottom of the gel. DNA is negatively charged so to run it through the gel, the top would have to be set to - and the bottom to +.

materials

Two basic types

agarose and polyacrylamide

agarose

Agarose is a natural colloid extracted from sea weed It is very fragile and easily destroyed by handling Agarose gels have very large "pore" size and are used primarily to separate very large molecules with a molecular mass greater than 200 kDaltons Agarose gels can be processed faster than polyacrylamide gels, but their resolution is inferior.

Agarose is a linear polysaccharide (average molecular mas about 12,000) made up of the basic repeat unit agarobiose, which comprises alternating units of galactose and 3,6-anhydrogalactose. Agarose is usually used at concentrations between 1% and 3%. Agarose is a chain of sugar molecules, and is extracted from seaweed.

Perhaps you have seen the terms TBE or TAE.

These are names of two commonly used buffers in electrophoresis.

The "T" stands for Tris, a chemical which helps maintain a consistent pH of the solution.

The "E" stands for EDTA, which itself is another anacronym. EDTA chelates (gobbles up) divalent cations like magnesium. This is important because most nucleases require divalent cations for activity, and you certainly wouldn't want any stray nucleases degrading your sample while it's running through the gel, would you?

Finally, the "B" or "A" stand for Boric acid or Acetic acid, which provide the proper ion concentration for the buffer.

polyacrylamide

The polyacrylamide gel electrophoresis (PAGE) technique was introduced by Raymond and Weintraub (1959).

Polyacrylamide is the same material that is used for skin electrodes and in soft contact lenses.

provide a wide variety of electrophoretic conditions:

By controlling the percentage (from 3% to 30%), precise pore sizes can be obtained, usually from 5 to 2,000 kdal. Polyacrylamide gels can be cast in a single percentage or with varying gradients Polyacrylamide gels offer greater flexibility and more sharply defined banding than agarose gels.

Spectrophotometer

Absorbance

Transmission

Colormetric

400 to 700 nm

Pertaining to measurement of concentration of a solution based on its absorption or transmission of light, or on the intensity of color in a liquid.

Centrifugation

• Speed
• Distance from Center of rotation
• RCF= relative Centrifugal Force

factors

         o Higher RCF the faster the sedimentation
         o viscosity
         o size of particle
         o difference b/w particle and medium

Application

         o Separation
         o + - Safety
               + Never exceed Max speed of rotor
               + Never use a cracked tube or bottle
         o + - Maintenance
               + Clean with mild detergent
               + air dry rotor
               + Check rotor O-rings

Types

+ - low speed

               + rpm<10k
               + g<8000

+ - High Speed

               + rpm<30k
               + g<100k
               + refrigeration used

+ - ultracentrifuge

               + rpm<120k
               + g<700k
               + refrigeration used
               + vacuum

+ - microfuge

               + tabletop
               + rpm<15k
               + g<21k
               + 1-2ml volumes

parts

               + + - rotors
                     # horizontal
                     # + - fixed angle
                           * b/w 15 & 40 degrees
                     # vertical

tubes / bottles

                     # glass
                     # stainless steel
                     # polycarbonate
                     # + - Teflon
                           * expensive
                     # + - polypropylene
                           * What most people use

Aseptic techniques

Aseptic techniques is defined as a method that keeps undesirable microbes from contaminating a pure culture.

Only a single species of an organism is what one should work with in a microorganismal laboratory all materials that will be used for transfer, growth, and experimentation of the microbe must be sterilized media and glassware most often is sterilized by using an autoclave.

Lab bench needs to be clean with a disinfectant before and after working.

Hands should be washed upon entering and leaving the laboratory.

Inoculation tools and the tops of tubes need to be sterilized over an flame

Inoculating Loop - held like a pencil

Employee traits

Computer Skills

1. Popular OS'es and file management
2. Excel
3. PowerPoint
4. Word
5. Linux

People Skills

Energy and interest

WHAT CAN I DO FOR YOU?

How you are qualified

Desire to continue learning

Brainpower:

In today's world, it's "be sharp or die."

Because we are status quo creatures. We like things just the way they are, thank you. Change unsettles us. If it works (or appears to work, actually), don't fix it.

Basic Microbiology

Basic Microbiology

Cell Identification

Enterotube

cell growth

Log Phase

cell maintenance & storage

Cryopreservation

http://nalgenelab.nalgenunc.com/techdata/technical/manual.asp

The protection of biological molecules during freezing and freeze-drying( lyophilization) is a subject of considerable practical importance, particularly in the pharmaceutical industry.

   wide variety of compounds as cryoprotectants

Saccharides are often used in this capacity

They have been found to protect proteins during freezing and drying stresses.

They have also been shown to prevent damage to cells during freezing and drying.

Basic Molecular Biology

Basic Molecular Biology

Genetic engineering

"Geneticist and science writer Steve Jones argues that humanity does not, and will never have the technology that proponents of transhumanism seek. He once joked that the letters of the genetic code, A, C, G and T should be replaced with the letters H, Y, P and E. Jones claims that technologies like genetic engineering will never be as powerful as is popularly believed."

-http://en.wikipedia.org/wiki/Transhumanism

E. Coli The workhorse of molecular biology

Theodor Escherich isolates a microbe from the colon that is later given the name Escherichia coli in his honor.

1. Ecoli
   1. HB101
      1. Fairly safe
      2. Needs
      3. 37 C
      4. Food
      5. H2O
      6. Vitamins

Escherichia coli, a subgroup of fecal coliform bacteria that is present in the intestinal tracts and feces of warm-blooded animals.

It is used as an indicator of the potential presence of pathogens. There are many different strains of E. coli that are classified into more than 170 serogroups.

Although most strains of E. coli are harmless and live in the intestines of healthy humans and animals, the E. coli O157:H7 strain produces a powerful toxin and can cause severe illness.

Its presence in groundwater is a common indicator of fecal contamination.

("Enteric" is the adjective that describes organisms that live in the intestines. "Fecal" is the adjective for organisms that live in feces, so it is often a synonym for "enteric.") The name comes from its discoverer, Theodor Escherich.

Basic Protein Separation

Basic Protein Separation

1. Protein Separation
2. Protein Analysis

Basic Tissue Culture

Basic Tissue Culture

1. Cultures
2. Cell line
3. Culture methods
4. Animal handling

Basic Chromatography

Basic Chromatography

1. HPLC Systems

High Performance Liquid Chromatography (HPLC):

HPLC is a popular method of analysis because it is easy to learn and use and is not limited by the volatility or stability of the sample compound

Modern HPLC has many applications including separation, identification, purification, and quantification of various compounds

Textbook on High Performance Liquid Chromatography (HPLC) http://hplc.chem.shu.edu/NEW/HPLC_Book/

GC-MS

Biotech HOT SPOTS in the US

1. RTP - Research Triangle Park, created in 1959, located between Duke University in Durham, North Carolina, the State University in Raleigh, and the University of North Carolina at Chapel Hill.
2. Boston
3. Albany
4. South San Francisco

Acronyms / definitions

Acronyms / definitions

GLP

GLP Good Laboratory Practices

GMP

Good Manufacturing Practice http://www.fda.gov/cder/dmpq/cgmpregs.htm

Biocidol

   Kills cells
   Disinfectant

Biostat

   Chemical that stops cell growth - does not kill.

Supernatants

Liquid removed from a tank once the solids have settled. Usually a clear liquid left after material (like cells) has been precipitated or centrifuged.

The material remaining above the pellet after centrifugation of a suspension.

Authors of this Wikibook

• Tom Maioli tmaioli