Biotecnologie
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Si tratta di un settore disciplinare nel quale la tecnologia è impiegata per la produzione o la modificazione di molecole, per la manipolazione di organismi viventi, in modo da realizzare prodotti e sviluppare processi utili per l'umanità. Le biotecnologie comprendono un'ampia e diversificata gamma di settori di ricerca, che va dalle scienze biologiche, alla chimica, informatica, medicina, medicina veterinaria, scienze agrarie, farmacologiche, ambientali, bioingegneria...
Le Biotecnologie si sono sviluppate a partire dalla scoperta del DNA (acido desossiribonucleico), il materiale genetico di base che regola la composizione delle cellule e lo sviluppo degli organismi viventi.
[modifica] Prodotti
Prodotti
[modifica] Tradizionale
- Birra / Vino (Vedi Fermentazione)
- Lievito
- Etanolo
- Formaggio
[modifica] Terapie Proteiche
- Herceptin
- Neupogen
- Insulina - 1982 U. S. Eli Lilly - Multinazionale farmaceutica, il primo a mettere in commercio l'insulina umana creata con Ingegneria genetica
[modifica] Agricoltura
- - Bacillus thuringiensis (Bt) Crops Bt crops
- Tolleranza erbicida
- Resistenza alle malattie
[modifica] Industriale / Ambientale
- Rifiuti Speciali/Pericolosi
- Trattamento delle acque di scarico
- Biorimediazione
[modifica] Altro
[modifica] 21 CFR Code of Federal Regulations (Codice delle regolazioni federali) 58
GLP - GOOD LABORATORY PRACTICE PER STUI DI LABORATORIO NON CLINICI:
http://www.access.gpo.gov/nara/cfr/waisidx_02/21cfr58_02.html
[modifica] 21 CFR Code of Federal Regulations (Codice delle regolazioni federali) 11
Titolo 21 Codice delle regolazioni federali (21 CFR Part 11) Registrazioni elettroniche; Firme elettroniche
http://www.fda.gov/ora/compliance_ref/part11/
[modifica] 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
[modifica] SOPs
SOP's (Standard Operating Procedures)| in Italiano POS (Le procedure operative standard)
- Approvazione dal management
- Non mettere i dati in una procedura operativa standard
- Scritto a livello di grado 6
- Cancella, corrente, completa
- Semplice, concisa, accurata
- Archiviare, Dove?
[modifica] Blocco Note
Caratteristiche del Blocco Note
- Se non è scritto, e' inutile!
- Pagine firmate
- Riuscite a leggere? fate in modo che sia capibile
- Inchiostro nero o blu?
- Pagine numerate
- Lasciate alcune pagine all'inizio per la Tabella dei contenuti (indice)
- Richiesto dalle regole del GLP / GMP
- Firmato con testimonianza
- Tracciabilità
Documentazione per l'Integrità e tracciabilità
- Che cosa hai fatto
- Perché l'avete fatto
- Risultati
- Riferimento lavori precedenti?
- Anche per la Qualità
[modifica] Chiavi di successo di prodotti biotecnologici
Chiavi di successo di prodotti biotecnologici
- La disponibilità di forza lavoro
- Forza lavoro specializzata
- Potenza, abilità
- Buona Università nelle vicinanze:
* Per la riqualificazione
* Per le nuove idee
- Bacino di lavoro Nazionale e internazionale
- Materie prime
- Marketing
* Avere chiare le tendenze e le necessità
- Approvazione U.S. Food and Drug Administration (FDA)
Tenuta dei registri
Inventario di controllo log
Fasi processo
Processo operativo standard
ECT (Environmental Compliance Technician) in Italiano - Rispetto delle norme tecnicho/ambientali
Se non è scritto non è fatto
Amalo
Vivilo
- Percorsi clinici
* BPF applica:
+ Fabbricazione
+ TRasformazione
+ Imballaggio
+ immagazzinaggio
* Convalida di ogni processo
* PAT (Process Technology Anylitical)
La qualità dovrebbe essere resa dal e con il design
- Preoccupazioni Ambientali
* L'utilizzo di energia
* L'uso di acqua
* Le leggi Ambientali
* Flussi di rifiuti
- Abbiamo la tecnologia?
Requisiti
Sicurezza Identità e Forza Qualità e Purezza
La Pianta:
Possiamo misurare:
+ pH
+ Areazione
+ Baffiling
+ giranti
+ medio
+ Semi terreni
- Siamo meglio della nostra concorrenza?
perche?
[modifica] Processi
Sviluppo / a Monte / a Valle dei processi
[modifica] Lo sviluppo
- Neccessità / analisi di mercato
- Selezionare una soluzione
- Processo di sviluppo
- Comprendere la crescita o cambiamento dei requisiti
- Calcolare le rese di biomassa
- Esegue test su piccola scala
Chimico
Lievito
Rendimenti elevati della biomassa
Si secernono la proteina
Picha pastoris
Funghi
Su cellule di mammifero
[modifica] a Monte
- Preparato Medio
Fermentazione?
[modifica] Trovare le migliori condizioni
- Costoso
- Lavoro intensivo
- A tempo indeterminato
- In termini di tempo
[modifica] Costrizioni
Materie Prime Variazione lotti I costi di trasporto Immagazzinaggio
- Preparazione Semi
[modifica] Trovare la linea cellulare
- Composizione
- Growth kenetics
- Lievito
- Banca del seme
- Master Seed Bank MSB
- Cellule Originali memorizzate
- Working Seed Bank WSB
- Utilizzati nella fermentazione attuale
[modifica] a Valle
- Slurry Handling
- Excreted product
- Contaminazione
- Prodotto di depurazione
- Separazione / purificazione / sterilizzazione
-
- Come?
-
-
- Plate and frame
- Bulk filtration
- Spiral wound
-
Plugs up easily
Usually not used for bulk filtration
- Immagazzinaggio
[modifica] Tecnico: competenze necessarie
Il Bio tecnico deve essere una persona in possesso di competenze con la capacità di risolvere problemi e soddisfare il cliente in modo tale che il lavoro svolto sia chiaro e leggibile da tutti. Il Tecnico deve mantenere un blocco note, per mantenere il lavoro e in modo che possa essere passato a qualcun altro per continuare il lavoro svolto. Presentare i risultati in maniera chiara, e lavorare con gli altri per soddisfare gli obiettivi.
[modifica] Capacità di laboratorio
Un tecnico deve utilizzare gli strumenti del mestiere, non diversamente da ogni altro lavoro, siamo agricoltori, ma la nostra piccola mandira è come piccola fauna selvatica. Per prendersi cura del nostro allevamento, bisogna tener conto di alcuni aspetti del loro ambiente.
[modifica] Soluto,solvente, e soluzione
- Soluto = Materiale asciutto
- Solvente = Quello con cui mescolate
- Soluzione = Soluto + Solvente
[modifica] pH
[modifica] Misura
- Sonda e metro
più preciso apparecchio più costoso Conservare in tampone Verificare la presenza di intasamento
- Carta Litmus
misura del pH molto grossolana
- Kit da campo
Le lettere pH significano "potenza di idrogeno"
L'idrogeno
l'elemento più abbondante nell'universo è l'idrogeno, che costituisce circa 3 / 4 di ogni cosa!
Acidi molto forti rilasciano più protoni, H + (ioni di idrogeno); basi più forti rilasciano piu OH-(ioni idrossido). Sostanze neutre hanno un equilibrio di H + e OH-, ad esempio. Acqua Pura (distillata).
>7 basico -- 7 Neutro -- <7 Acido
A seconda della definizione, un acido è una ione d'idrogeno o donatore di protoni. Una base è un accettatore di ioni d'idrogeno, un donatore di idrossido di ioni o un accettatore di elettroni.
Gli Acidi producono ioni H + in soluzioni acquose, come per la produzione di basi con ioni OH-
elettrodo pH rispetto ad una batteria
Conservare in tampone non in 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
[modifica] 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
[modifica] 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
[modifica] colony agar plating
[modifica] What is Agar?
A gelatinous material derived from certian marine algae.
- Growth Media
- Carbon Sources
- Carbohydrates
- Proteins
- Nitrogen Sources
Two types:
- Simple
- Complex
minerals H2O vitamins Precursors
- Componds that may lead to your product - inducers
- Componds added to induce gene expression
LB (Luria-Bertani) Media
[modifica] Agar Plates
[modifica] Blood
contains blood cells from an animal (e.g. a sheep). Most bacteria will grow on this medium
[modifica] Chocolate
This contains lysed blood cells, and is used for growing fastidious (fussy) respiratory bacteria.
[modifica] Mannitol Salt
File:Mannitolsalt.jpg Purpose Mannitol salt agar is both a selective and differential growth medium.
[modifica] Brillant Green
Inhibits Gram+ Maconkey
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.
[modifica] Neomycin agar
contains the antibiotic neomycin.
[modifica] Sabouraud agar
Used for fungi. It contains gentamicin and has a low pH that will kill most bacteria.
[modifica] LB
+ Complex + pH 7.2
[modifica] 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 legth (cm)
- c=concentration (M)
[modifica] Laboratory calculations
- + - Prep of Sterile solid and Liquid media
[modifica] Sterilization Methods
- heat
- Autoclave
- 200C
- 2-4hrs
Radiation
- UV 250-270nm
- Gamma
Chemicals
- Chlorine
- H2O2
- Alcohol
Gas
- Formaldehyde
Filtration
.1 or 0.22 micron filters
- Of Reagents
- Batch
- Time consuming
- Lots of Energy
- Continuous
- More equipment
- Heat Exchanger
- Filtration
- For Air
- For Heat sensitive nutrients
[modifica] 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
[modifica] Measuring / Mixing
Using a balance Calibration / documentation
[modifica] UV/VIS spectroscopy
* http://www.scienceofspectroscopy.info/ * Use correct Reagent * Use correct Wavelength
[modifica] 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.
[modifica] 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.
[modifica] Centrifugation
- Speed
- Distance from Center of rotation
- RCF= relative Centrifugal Force
[modifica] factors
o Higher RCF the faster the sedimentation
o viscosity
o size of particle
o difference b/w particle and medium
[modifica] 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
[modifica] 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
[modifica] parts
+ + - rotors
# horizontal
# + - fixed angle
* b/w 15 & 40 degrees
# vertical
[modifica] tubes / bottles
# glass
# stainless steel
# polycarbonate
# + - Teflon
* expensive
# + - polypropylene
* What most people use
[modifica] 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
[modifica] fermentor design & application
[modifica] Computer Skills
- Popular OS'es and file management
- Excell
- PowerPoint
- Word
- Linux
[modifica] People Skills
Energy and interest
WHAT CAN I DO FOR YOU?
How your qualified
[modifica] Desire to continue learning
[modifica] 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.
[modifica] Ongoing education / cross pollination of other areas:
[modifica] Basic Microbiology
Basic Microbiology
[modifica] Cell Identification
[modifica] cell growth
Log Phase
[modifica] cell maintenance & storage
[modifica] 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.
[modifica] Basic Molecular Biology
Basic Molecular Biology
[modifica] Organism Design
[modifica] Genetic Analysis
[modifica] Strain Validation Applications
[modifica] 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
[modifica] 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.
[modifica] Basic Protein seperation
Basic Protein seperation
- Protein Seperation
- Protein Analysis
[modifica] Basic Tissue Culture
Basic Tissue Culture
- Cultures
- Cell line
- culture methods
- Animal Handling
[modifica] 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
[modifica] Biotech HOT SPOTS in the US
- RTP - Research Triangle Park, created in 1959 located between Duke University in Durham, N.C. State University in Raleigh, and the University of North Carolina at Chapel Hill.
- Boston
- Albany
[modifica] Acronimi / definizioni
Acronimi / definizioni
[modifica] GLP
GLP Good Laboratory Practices
[modifica] GMP
Good Manufacturing Practice http://www.fda.gov/cder/dmpq/cgmpregs.htm
[modifica] Biocidol
Uccide le cellule Disinfettante
[modifica] Biostat
Agente chimico che ferma la crescita della cellula - non uccide
[modifica] Supernatants
Liquid removed from a tank once the solids have settled a usually clear liquid left after material (like cells) has been precipitated or centrifuged.
The material remaining above the pellet after centrifugation of a suspension
