XII Chap 11 Biotechnology Flashcards
Define biotechnology.
Examples?
using live organisms or enzymes to produce useful products / processes
e.g. GMO or IVF
Synthesizing a gene, correcting a gene or developing DNA vaccine are all examples of biotechnology. T or F?
True
Making curd, bread and wine can also be thought of as biotechnology. T or F?
True, but not using genetically modified organisms / large scale
What is the definition of biotechnology as per the European Federation of Biotech?
integration of natural science and organisms, cells, parts thereof, and molecular analogues for products & services
Two core techniques that led to modern biotechnology?
- Genetic engineering
2. Bioprocess engineering
Define genetic engineering
Techniques to alter the chemistry of genetic material (DNA/RNA)
=> introduce into host
=> change the phenotype of host
What is bioprocess engineering?
Maintaining sterile ambience in chemical eng. processes to enable growth of only the desired microbes/eukaryotic cells in large quantities
________ reproduction preserves genetic information, while ________ reproduction permits variation
Asexual;
Sexual
Traditional hybridisation procedures allow us to choose isolate and transfer only desired set of genes without introducing undesirable genes. T or F?
False; only genetic engineering allows us to do that e.g. recombinant DNA, gene cloning and gene transfer
What is origin of replication?
specific DNA sequence in chromosome responsible for initiating replication
What is cloning?
Alien DNA is linked with chromosome(s) that contain the origin of replication => alien piece can replicate and multiply inside the host organism
First instance of construction of artificial recombinant DNA molecule was ?
gene encoding antibiotic resistance
native plasmid
salmonella typhimurium
Stanley Cohen, Herbert Boyer - 1972
What is a plasmid?
Autonomously replicating circular extra-chromosomal DNA;
acts as vector to transfer alien DNA to into host organism
Restriction enzymes are also called ___________
molecular scissors
What do restriction enzymes allow us to do?
cut DNA at specific locations
What are vectors in recombinant DNA?
plasmid DNA;
molecules that are able to covalently bond to and carry foreign DNA into cells
What is DNA ligase?
enzyme that joins cut ends of DNA molecules
What is recombinant DNA ?
a new combination of circular autonomously replicating DNA created in vitro
When DNA is transferred into E. coil instead of Salmonela it ______
replicates using the new host DNA’s polymerase enzyme and make multiple copies i.e. cloning
What are the 3 basic steps in genetically modifying an organism?
- identification of DNA with desirable genes
- introducing identified DNA into host
- maintenance of introduced DNA in the host & transfer of DNA to progeny
What are the key tools in recombinant DNA technology?
- restriction enzymes
- ligases
- vectors
- host organism
- polymerase enzymes
What is a restriction endonuclease?
same as restriction enzyme
What is recognition sequence?
Specific base sequence where an enzyme cuts DNA
How many base pairs in the recognition sequence for Hind II?
6
Since discovering Hind II, how many restriction enzymes have been isolated?
900+ from 230+ strains of bacteria
What is the convention for naming enzymes?
First letter from genus
Second 2 letters from species
One letter(?) for name of strain
Roman Numbers showing order in which isolated
e.g. EcoRI
What are exonucleases?
Enzymes that remove nucleotides from the ends of DNA
Endonuclease vs exonuclease
Endo - make cut in DNA
Exo - remove nucleotides from ends of DNA
What are the steps in endonuclease’s functioning?
Inspect the length of DNA
Finds recognition sequence/palindromic nucleotide sequence
Binds to DNA
Cut both DNA strands at the same site (little away from center but between same two bases on both ends => sticky ends
(Does this for vector and foreign DNA)
DNA ligase joins foreign DNA and vector DNA
Vector and source DNA can be cut with different restriction enzymes. T or F?
False, has to be same (recognition sequence)
EcoRI is an ________ enzyme isolated from ____________ organism and has the recognition sequence __________
endonuclease;
Escherichia coli R Y 13;
GAATTC
What is gel electrophoresis?
Technique to separate DNA fragements
DNA fragments are positively or negatively charged?
Negatively charged
How does gel electrophoresis work?
Negatively charged DNA fragments are forced to move towards the anode under an electric field through a medium/matrix => DNA resolve/separate according to their size through sieving effect
smaller => moves farther
What is the most commonly used matrix in gel electrophoresis? Where does it come from?
Agarose gel - natural polymer extracted from sea weeds
How do we make DNA fragments visible in gel electrophoresis?
Staining often with ethidium bromide + exposure to UV radiation => bright orange colored bands
What is elution?
Separated bands of DNA are cut out from the agarose gel and extracted => purified to use in recombinant DNA
_____ and _____ have the ability to replicate within bacterial cells independent of control of chromosomal DNA
Plasmids
Bacteriophages
What are the copy numbers of bacteriophages and plasmids?
Bacteriophages - HIGH copy numbers of genome
Plasmids - one or two copies per cell OR 15-100 copies per cell
Two factors that determine using something as a cloning vector?
- easy linking of foreign DNA
2. easy selection of recombinants from non-recombinants
Features required for cloning into vectors:
- Origin of replication (ori) - controls copy number of linked DNA
- Selectable marker - identify and eliminate non-transformant, permit growth of transformants
- Cloning sites - very few or single recognition site for restriction enzyme
- Vectors for cloning genes into plants and animals
In E. Coli, the ligation of alien DNA is carried out at a restriction site present in one of the two ____________ genes
antibiotic resistance genes
Alternative selectable markers (so not antibiotics) have been developed. What are these and how do they work?
marker - colour in presence of chromogenic substrate
DNA inserted within coding sequence of β-galactosidase; insertional inactivation (i.e. no color) of gene; present of insert would have given blue color colonies
What is insertional inactivation?
when inserting a recombinant DNA results into inactivation of the gene for synthesis of a particular enzyme
______________ a pathogen is able to deliver piece of DNA known as _____ to transform normal plant cells into tumor cells
Agrobacterium tumifaciens
T-DNA
Retroviruses have what effect in animals?
Normal cells => cancerous cells
What are the two pathogens that have been converted into vectors for cloning?
Agrobacterium tumifaciens (Ti plasmid) Retroviruses
What is transformation?
Procedure through which piece of DNA is introduced in host bacterium
What is done to make bacterial cells ‘competent’ to host DNA?
Treatment with specific concentration of a divalent cation e.g. calcium => increased efficiency for DNA entry through cell wall pores;
incubation on ice with recombinant DNA => heat shock (42°C) => again on ice
What is micro-injection?
Method to make host cell competent for DNA entry
recombinant DNA injected directly into nucleus of animal cell
What is gene gun? Another name for it?
for plant cells; bombarded with high velocity micro-particles of gold or tungsten coated with DNA
Another name: biolistics
What are the methods to make host competent?
- Divalent cation + ice-heat-ice
- Micro-injection (animal cells)
- Biolistics / gene gun (plant cells)
- using ‘disarmed pathogen’ vectors that ‘infect’ cells and transfer the recombinant DNA
Steps in Recombinant DNA technology?
- Isolation of DNA
- Fragmentation of DNA
- Isolation of desired DNA fragment
- ligation of DNA fragment + vector
- transfer of recombinant DNA into host
- culturing the host cells at large scale
- extraction of desired product
Nucleic acid is the genetic material of all organisms without exception. T or F?
True
Most animals have RNA. T or F?
False, DNA
DNA needs to be in ___ form free from _____ in order to be cut
pure form;
free from other macro-molecules
Why do we have to break the cell open in recombinant DNA technology?
Since DNA is enclosed in membranes, to release other macromolecules (RNA, proteins, etc)
What are the macro-molecules present in a cell along with DNA?
RNA, proteins, polysaccharides, lipids
How is DNA isolated from other macromolecules in cell?
By treating the cell / tissue with enzymes e.g. RNA removed with ribonuclease, proteins removed by protease
What do each of these enzymes act on?
lyosozyme?
cellulase?
chitinase?
What is their effect?
lyosozyme - bacteria
cellulase - plant cells
chitinase - fungus
Break cell open to release macromolecules
The last step in isolating DNA involves precipitating it out after the addition of ________
chilled ethanol
What is PCR?
Polymerase Chain Reaction
- multiple copies of the gene of interest
- in vitro
- using 2 sets of primers + DNA polymerase
What is a primer?
small chemically synthesised oligonucleotides
complementary to the regions of DNA
What are the 3 steps in a cycle of PCR?
- Denaturation
- Primer annealing
- Extension of primers
How is repeated amplification in PCR achieved?
use of a thermostable DNA polymerase (from bacterium Thermus aquaticus)
What is a recombinant protein?
Protein encoding gene expressed in a heterologous host
What type of culturing method produces larger biomass and higher yields of desired proteins?
Continuous culture system => used medium is drained out from one side while fresh added from other => cells maintained in their physiologically most active/exponential phase
What is technology is required to produce cultures in large quantities?
Bioreactors (100-1000 litres) with optimum growth conditions (oxygen, temp, pH, substrate, salts, vitamins); cylindrical
+ agitator system
+ foam control system
+ sampling ports
Most commonly used bioreactors are of type _______
stirring type
What are the two types of stirred tanks?
Simple and sparged (sterile air bubbles)
What is downstream processing?
Separation and purification of product in recombinant DNA technology;
formulation with suitable preservatives;
clinical trials (if drugs)
QC testing
Why does a host cell need to be made competent to take up a foreign DNA?
Because DNA is a hydrophilic molecule
Why is taq polymerase used in PCR?
Because it is thermostable
_________ was the first type II restriction endonuclease that could cut dsDNA specific site
Hind II
Restriction enzymes are synthesized by which kind of cells?
bacteria, yeast, eukaryotic cells
bacteria only
PCR steps occur at what temperatures?
95-98° C
55° C
72° C
What is the tag polymerase enzyme obtained from?
A thermophilic bacterium - Thermus aquaticus
__________ is the first artificial cloning vector constructed
By whom?
In which year?
pBR322
Boliver and Rodriguez
1977
1960s, 2 enzymes were discovered in bacteria that provided immunity against bacteriophages. What are they?
Restriction endonuclease
Methylase
What are the names associated with the construction of the first Recombinant DNA?
Annie Chang, Boyer, Berg and Cohen
Variations as a result of sexual reproduction are not always good for the individual but are always good for the population. T or F?
False, not always good for either
Which of the following is NOT associated with the first recombinant DNA molecule?
plasmid of salmonella typhimurium - antibiotic resistance gene - 1975 - Stanley Cohen
1975
EcoRI cuts between
G and A in GAATTC
EcoRI cuts both strands at the same time a little away from center. T or F?
True
Primers are not:
small - biologically synthesized - oligonucleotides - complimentary to the regions of DNA
biologically synthesized
After 30 cycles of PCR, how many times will DNA be amplified?
1 billion
Use of primers in PCR occurs in which step?
Annealing
Which of the following processes is not associated with the protein produced by recombinant DNA?
Restriction Digestion - Optimization - Purification - Expression
Restriction Digestion
Increased surface area for oxygen transfer is a feature found specifically in _________________
sparged stirred-tank bioreactor
Quality control testing a part of downstream processing. T or F?
False, happens after
A molecular technique in which DNA sequences between 2 oligonucleotide primers can be amplified is known as _________
PCR
Which of the following is not a feature of the plasmids?
circular structure - transferable - single-stranded - independent replication
single-stranded
In recombinant DNA, antibiotics are used to:
- keep medium bacteria-free
- to detect alien DNA
- to impart disease-resistance to the host plant
- as selectable markers
- to detect alien DNA
