Biotechnology
From Wikibooks, the open-content textbooks collection
To create a textbook for use in 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's already changing the foods we eat and react to the environment.
To bring out the best in nature.
[edit] What is Biotech?
"Defining "biotechnology""
- The use of living things to make products.
- The application of a biological process to produce a useful product.
- Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific 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.
The application of the principles of engineering and technology to the life sciences--bioengineering.
Cloning, genetic manipulation, cell fusion, and mutation.
Essentially, doing "more and faster" what we have known and done for centuries. It is almost as old as agriculture iself.
Farmers and bakers are the biotechs of days gone by. Remember Grandma's freshly baked bread? How Grandpa kept the seeds of those really big pepper or tomatoes? Your grandparents were practicing biotechnology.
Modifying the genetic material of organisms directly and precisely. It enables the transfer of genes between diverse organisms, allowing combinations unlikely to occur by conventional means. Allowing speedier and more specific results.
Life- Define:
- Moving
- Growing
- Eating
- Breeding
- Response to stimuli
[edit] Products
Products
[edit] Traditional
- Beer / Wine (See Fermentation)
- Yeast
- Ethanol
- Cheese
[edit] Protein Therapeutics
- Herceptin
- Neupogen
- Insulin - 1982 U.S. pharmaceutical manufacturer Eli Lilly the first to market genetically-engineered human insulin
[edit] Agriculture
- Bacillus thuringiensis (Bt) Crops Bt crops
- Herbicide tolerance
- Disease resistance
- Pest resistance
[edit] Industrial / Environmental
- Hazardous Waste
- Wastewater Treatment
- Bioremediation
[edit] Other
- Plastics
- Biopulping
- GMO (Genetically modified organism)
[edit] 21 CFR 58
Good laboratory practice for nonclinical laboratory studies:
http://www.access.gpo.gov/nara/cfr/waisidx_02/21cfr58_02.html
[edit] 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/
[edit] 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
[edit] SOPs
SOP's (Standard Operating Procedures)
- Approved by management
- You don't put data in a SOP
- Written at 6th grade level
- Clear, current, complete
- Simple, Concise, Accurate
- Archived Where?
[edit] Notebooks
Notebook
- If it is not written, it is not done.
- pages signed
- Can you read it?
- Black or blue ink
- pages numbered
- Leave some pages in the front for TOC
- Required by GLP / GMP regs
- Signed by witness
- Tracability
Documentation for Integrity and traceablity
- What you did
- Why you did it
- Results
- Reference earlier work?
- Also for Quality
[edit] Keys to Successful Biotech products
Keys to Successful Biotech products
- Availability of labor force
- Skilled labor force
- Power
- Good Universities nearby:
* For retraining * For new ideas
- National and international labor pool
- Raw materials
- Marketing
* Clear trend and need
- FDA approval
Record Keeping
Inventory control logs
Process Steps
SOP
ect
If it's not written it's not done
Love it
Live it
* Clinical trails
* GMP apply to:
+ Manufacture
+ Processing
+ Packing
+ Storage
* Validation of each process
* PAT (Process Anylitical Technology)
Quality should be built in and by desighn
- Evironmental Concerns
* Use of energy * Use of water * Evironmental Laws * Wastestreams
- Do we have the technology?
requirements
Safety Identity and Strength Quality and purity
The plant:
Can we Measure
+ pH
+ Aeration
+ Baffiling
+ impellers
+ medium
+ Seed train
- Are we Better than our competition?
why?
[edit] processes
Development / Upstream / Downstream processes
[edit] Development
- Need / market analysis
- Select a solution
- Process Development
- Understand the growth requirements
- Calculate yields of biomass
- Run small scale test
Chemical
Yeast
High biomass yields
Will secrete the protein
Picha Pastoris
Fungi
Mammalian Cells
[edit] Upstream
- Media prep
Fermentation?
[edit] Find best conditions
Expensive Labor intensive Open Ended Time Consuming
[edit] Constraints
Raw Materials Batch to Batch variations Transportation costs Storage
- Seed Prep
[edit] Find cell line
Composition Growth kenetics Yield Seed Bank
- Master Seed Bank MSB
Original Stored Cells
- Working Seed Bank WSB
Used in actual fermentation
[edit] Downstream
- Slurry Handling
- Excreted product
- Contamination
- Pruduct purification
- Seperation / purification / sterilization
-
- How?
-
-
- Plate and frame
- Bulk filtration
- Spiral wound
-
Plugs up easily
Usually not used for bulk filtration
- Storage
[edit] Technician: Skills Needed
The Biotech Technician must be a person posessing 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 maintian a notebook, one that can be read by someone else. Present results in a clear manner, and work with others to meet objectives.
[edit] 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.
[edit] Solute,solvent, and solution
- Solute = Dry Material
- Solvent = What you mix with
- Solution = Solute + Solvent
[edit] pH
[edit] Measurement
- Probe and meter
most accurate more expensive piece of equipment Store in buffer Check for clogging
- Litmus paper
very coarse measurement of pH
- Field kit
The letters pH stand for "power of hydrogen"
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-, Eg. 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
[edit] 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
[edit] 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 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 (ionizationation) 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
- Ionic Strength
- Temperature
- Counter ion
[edit] colony agar plating
[edit] What is Agar?
A gelatinous material derived from certian marine algae.
- Growth Media
- Carbon Sources
- Carbohydrates
- Proteins
- Nitrogen Sources
Two types:
- Simple
- Complex
Compounents required for preparing a minimal agar
- minerals
- H2O
- vitamins
- Precursors
- Componds that may lead to your product - inducers
- Componds added to induce gene expression
LB (Luria-Bertani) Media
[edit] Agar Plates
[edit] Blood
contains blood cells from an animal (e.g. a sheep). Most bacteria will grow on this medium
[edit] Chocolate
This contains lysed blood cells, and is used for growing fastidious (fussy) respiratory bacteria.
[edit] Mannitol Salt
Purpose Mannitol salt agar is both a selective and differential growth medium.
[edit] Brillant 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.
[edit] Neomycin agar
contains the antibiotic neomycin.
[edit] Sabouraud agar
Used for fungi. It contains gentamicin and has a low pH that will kill most bacteria.
[edit] LB
+ Complex + pH 7.2
[edit] 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
- A=Absorbance
- e=Molar Absorbtivity
- l=Path length (cm)
- c=Concentration (M)
[edit] Laboratory calculations
- + - Prep of Sterile solid and Liquid media
[edit] Sterilization Methods
There are different types of Sterilization techniques. Some of them are 1. Physical sterilzation 2. Chemical sterilzation
Under Physical sterilzation a) Heat b) Filteration c) Ionisation 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
[edit] Pipetting
- Growing concerns regarding repetitive strain injuries (RSIs)
- Adopting good pipetting techniques is critical for the accuracy of your analysis and your health.
- Contamination Prevention
- Care of
[edit] Measuring / Mixing
Using a balance Calibration / documentation
[edit] UV/VIS spectroscopy
* http://www.scienceofspectroscopy.info/ * Use correct Reagent * Use correct Wavelength
[edit] 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.
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 wiht a molecular mass greater than 200 kdal 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.
[edit] 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.
[edit] Centrifugation
- Speed
- Distance from Center of rotation
- RCF= relative Centrifugal Force
[edit] factors
o Higher RCF the faster the sedimentation
o viscosity
o size of particle
o difference b/w particle and medium
[edit] Application
o Seperation
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
[edit] 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
[edit] parts
+ + - rotors
# horizontal
# + - fixed angle
* b/w 15 & 40 degrees
# vertical
[edit] tubes / bottles
# glass
# stainless steel
# polycarbonate
# + - Teflon
* expensive
# + - polypropylene
* What most people use
[edit] 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
[edit] fermentor design & application
[edit] Employee traits
[edit] Computer Skills
- Popular OS'es and file management
- Excel
- PowerPoint
- Word
- Linux
[edit] People Skills
Energy and interest
WHAT CAN I DO FOR YOU?
How you are qualified
[edit] Desire to continue learning
[edit] Brainpower:
In today's world, it's "be sharp or die."
Most people use only 5% of their potential cognitive brainpower.
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.
[edit] Ongoing education / cross pollination of other areas:
[edit] Basic Microbiology
Basic Microbiology
[edit] Cell Identification
Enterotube
[edit] cell growth
Log Phase
[edit] cell maintenance & storage
[edit] 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.
[edit] Basic Molecular Biology
Basic Molecular Biology
[edit] Organism Design
[edit] Genetic Analysis
[edit] Strain Validation Applications
[edit] 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
[edit] 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.
- Ecoli
- HB101
- Fairly safe
- Needs
- 37 C
- Food
- H2O
- Vitamins
- HB101
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.
[edit] Basic Protein Separation
Basic Protein Separation
- Protein Separation
- Protein Analysis
[edit] Basic Tissue Culture
Basic Tissue Culture
- Cultures
- Cell line
- Culture methods
- Animal handling
[edit] Basic Chromatography
Basic Chromatography
- 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
[edit] Biotech HOT SPOTS in the US
- 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.
- Boston
- Albany
- South San Francisco
[edit] Acronyms / definitions
Acronyms / definitions
[edit] GLP
GLP Good Laboratory Practices
[edit] GMP
Good Manufacturing Practice http://www.fda.gov/cder/dmpq/cgmpregs.htm
[edit] Biocidol
Kills cells Disinfectant
[edit] Biostat
Chemical that stops cell growth - doesnt kill
[edit] 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.
[edit] Authors of this Wikibook
This Wikibook was written by Tom Maioli tmaioli
