Flashcards in 2: Micobiology: Biotechnology and Recombinant DNA Technology Deck (36):
What is biotechnology?
is the use of microorganisms or mammalian cells to carry out defined chemical processes fir industrial purposes.
Over the years microbes have been used in the chemical production of what?
What does Genetic Engineering encompasses what?
the many procedures used to join together DNA fragments to produce genes of interest and the procedures to introduce such recombinant DNA molecules into expression systems for large-scale production of proteins (or DNA) of interest.
What are the steps in Gene cloning?
the gene of interest (eg, insulin gene) is inserted into the vector DNA (eg, plasmid) in vitro.
the recombinant vector DNA is introduced into a host organism (eg, E. coli) where it can multiply.
Detection and purification of the desired clone.
the cell containing the recombinant vector is the grown in culture to form a clone of several genetically identical cells, each of which carries a coy of the vector.
the final step depends on whether the gene itself or the product of the gene is of interest.
What can the gene of interest be from?
1. gene libraries containing either natural copies of genes or cDNA synthesized from the mRNA template reverse transcriptase.
2. synthetic DNA
What happens to E. coli if it is treated with high concentrations of calcium ions and then stored in the cold?
becomes transformable at low efficiency.
What are Electroporation?
small pores are produced in the membranes of cells that are exposed to pulsed electrical fields. DNA molecules present outside the cells during the electrical pulse can then enter the cells through these pores.
What can large quantities of the gene of interest be harvested and used for?
a variety of purposes
(eg, diagnostic DNA probes)
What can be done if the gene of interest is expressed (ie, transcribed and translated) on the cell clone?
its protein product can be harvested
What are restrictions endonucleases?
certain bacteriophages failed to replicate in particular species of bacteria due to the rapid hydrolysis of the bacteriophage DNA by speific bacterial endonucleases = restriction endonucleases.
the first restriction enzymes were purified in 1970.
What is restriction endonuclease?
an enzyme that recognizes short specific palindromic sequences in double-stranded DNA and cleave the duplex.
What is a cloning vector?
is a plasmid or viral DNA into which foreign DNA may be inserted to be propagated in a host cell.
Cloning vectors are designed to allow what?
integration of foreign DNA at a restriction site that cuts the vector in a way that does not affect its replication.
What i the most important property of a vector?
once in a cell, a vector must be capable or replicating.
What is important about small size vectors?
small vectors are more easily manipulated than larger DNA molecules, which tend to be more fragile.
What is important about preservation when relating to cloning vectors?
the circular form of DNA molecules is more important in protecting the DNA of the vector from destruction by the recipient of the vector.
Certain regions of the DNA are not essential for what?
are not essential for replication which can be removed and placed with the foreign DNA fragment.
What are marker genes?
plasmids frequently have antibiotic resistance genes or genes for an enzyme that carries out an easily identified reaction, making detection and selection of the plasmid-containing clones easier.
What can viral DNA accept more than plasmids can?
can accept larger pieces of foreign DNA.
What happens to DNA after it is inserted into the viral vector?
it can be cloned in the host cell the virus replicates in.
What type of viruses are being used in gene therapy and how?
used in gene therapy to insert corrective genes into the human cells that have defective genes.
What are the most useful hosts for cloning?
are microorganisms which grow well and for which a lot of genetic information is available.
What are the ideal characteristics of a microbial host for cloned genes?
rapid growth; capable of growth in cheap culture medium.
not harmful or pathogenic.
transformable by DNA.
stable in culture.
examples: E. coli, yeasts; transgenic animal systems; and mammalian cell culture systems.
pBR322 possesses what?
possesses two antibiotic resistance markers, ampicillin and tetracycline, which permit ready selection of hosts containing the plasmid.
What happens if a foreign DNA is inserted into one of the antibiotic resistance sites?
the antibiotic resistance conferred by this site is lost = phenomenon known as INSERTIONAL INACTIVATION
What happens to the clone if pBR322 is digested with BamHI, linked with foreign DNA, and the transformed bacterial clones are isolated?
those clones which are both ampicillin resistant and tetracycline resistant lack the foreign DNA.
whereas those cells still resistant to ampicillin but sensitive ti tetracycline contain the plasmid with inserted foreign DNA.
Ampicillin resistance and tetracycline resistance can be determined how?
can be determined independently on agar plates using replica plating.
How is replica plating performed?
a sterile velvet pad is pressed lightly down on a plate containing microcolonies of the organism.
the velvet pad is then pressed lightly on a sterile agar plate containing ampicillin or tetracycline.
by noting the position of the colonies that grew, it is possible to go back to the original plate and subculture the colony of interest.
What are some products that have been obtained from genetically engineered E. coli?
epidermal growth factor (heals wounds, burns, and ulcers)
human growth hormone
taxol (plant product used for treatment of ovarian cancer)
relaxin (used to ease childbirth)
Products obtained from mammalian cell culture?
Factor VIII (treatment of hemophilia)
bone morophogenic proteins (induces new bone formation)
Products obtained from yeasts?
Subunit vaccines (consists of a protein portion of a pathogen)
Describe Environment Biotechnology
genes for the biodegradation of many toxic wastes and wastewater pollutants have been show to exist in natural isolates of bacteria.
Genetic engineering is trapping these resources for the purpose of environmental cleanup.
The use of pest repellents
potato resistant to the Colorado potato beetle.
Bacillus thuringiensis, a soil-dwelling bacterium produces a protein crystal that is toxic to the potato beetle but is harmless to humans.
The gene for the toxin is cleaved from the bacterium and spliced into potato plant cells. thus, the new plant is able to produce its own insecticide.
What is Polymerase chain reaction (PCR)?
this is a technique that uses DNA polymerase to make copies of a DNA template in vitro.
The DNA can be used to detect the presence of infectious agents in specimens.
Restriction enzymes can produce what?
hundreds of restriction enzymes are known and each producing DNA fragments with characteristic ends.