2: Micobiology: Biotechnology and Recombinant DNA Technology

Question 1

Over the years microbes have been used in the chemical production of what?

Answer 1

vaccines
industrial enzymes
foods
vitamins
antibiotics
hormones
etc.

Question 2

What does Genetic Engineering encompasses what?

Answer 2

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.

Question 3

What are the steps in Gene cloning?

Answer 3

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.

Question 4

What can the gene of interest be from?

Answer 4

1. gene libraries containing either natural copies of genes or cDNA synthesized from the mRNA template reverse transcriptase.
2. synthetic DNA

Question 5

What happens to E. coli if it is treated with high concentrations of calcium ions and then stored in the cold?

Answer 5

becomes transformable at low efficiency.

Question 6

What are Electroporation?

Answer 6

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.

Question 7

What can large quantities of the gene of interest be harvested and used for?

Answer 7

a variety of purposes

eg, diagnostic DNA probes

Question 8

What can be done if the gene of interest is expressed (ie, transcribed and translated) on the cell clone?

Answer 8

its protein product can be harvested

Question 9

What are restrictions endonucleases?

Answer 9

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.

Question 10

What is restriction endonuclease?

Answer 10

an enzyme that recognizes short specific palindromic sequences in double-stranded DNA and cleave the duplex.

Question 11

What is biotechnology?

Answer 11

is the use of microorganisms or mammalian cells to carry out defined chemical processes fir industrial purposes.

Question 13

What is a cloning vector?

Answer 13

is a plasmid or viral DNA into which foreign DNA may be inserted to be propagated in a host cell.

Question 14

Cloning vectors are designed to allow what?

Answer 14

integration of foreign DNA at a restriction site that cuts the vector in a way that does not affect its replication.

Question 15

What i the most important property of a vector?

Answer 15

self-replication.

once in a cell, a vector must be capable or replicating.

Question 16

What is important about small size vectors?

Answer 16

small vectors are more easily manipulated than larger DNA molecules, which tend to be more fragile.

Question 17

What is important about preservation when relating to cloning vectors?

Answer 17

the circular form of DNA molecules is more important in protecting the DNA of the vector from destruction by the recipient of the vector.

Question 18

Certain regions of the DNA are not essential for what?

Answer 18

are not essential for replication which can be removed and placed with the foreign DNA fragment.

Question 19

What are marker genes?

Answer 19

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.

Question 20

What can viral DNA accept more than plasmids can?

Answer 20

can accept larger pieces of foreign DNA.

Question 21

What happens to DNA after it is inserted into the viral vector?

Answer 21

it can be cloned in the host cell the virus replicates in.

Question 22

What type of viruses are being used in gene therapy and how?

Answer 22

Retroviruses
Adenoviruses
Herpesviruses
used in gene therapy to insert corrective genes into the human cells that have defective genes.

Question 23

What are the most useful hosts for cloning?

Answer 23

are microorganisms which grow well and for which a lot of genetic information is available.

Question 24

What are the ideal characteristics of a microbial host for cloned genes?

Answer 24

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.

Question 25

pBR322 possesses what?

Answer 25

possesses two antibiotic resistance markers, ampicillin and tetracycline, which permit ready selection of hosts containing the plasmid.

Question 26

What happens if a foreign DNA is inserted into one of the antibiotic resistance sites?

Answer 26

the antibiotic resistance conferred by this site is lost = phenomenon known as INSERTIONAL INACTIVATION

Question 27

What happens to the clone if pBR322 is digested with BamHI, linked with foreign DNA, and the transformed bacterial clones are isolated?

Answer 27

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.

Question 28

Ampicillin resistance and tetracycline resistance can be determined how?

Answer 28

can be determined independently on agar plates using replica plating.

Question 29

How is replica plating performed?

Answer 29

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.

Question 30

What are some products that have been obtained from genetically engineered E. coli?

Answer 30

Human insulin
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)
cytokines
etc.

Question 31

Products obtained from mammalian cell culture?

Answer 31

Erythropoietin
Factor VIII (treatment of hemophilia)
bone morophogenic proteins (induces new bone formation)
monoclonal antibodies 
etc.

Question 32

Products obtained from yeasts?

Answer 32

Subunit vaccines (consists of a protein portion of a pathogen)
Cytokines
etc.

Question 33

Describe Environment Biotechnology

Answer 33

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.

Question 34

The use of pest repellents

Answer 34

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.

Question 35

What is Polymerase chain reaction (PCR)?

Answer 35

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.

Question 36

Restriction enzymes can produce what?

Answer 36

hundreds of restriction enzymes are known and each producing DNA fragments with characteristic ends.

Question 37

A bacteria cell can do what when using modification enzymes (eg, methylases)?

Answer 37

a bacteria cell modifies its own DNA by attaching small methyl groups to some of the cytosines or adenines in its DNA.
the modification has no effect on reading the genetic code but labels the DNA molecule as belonging to the host cell.