Microbiology- Chapter 10 (Genetic Engineering)

Front 1

what is biotechnology?

Back 1

Biotechnology – application of biological systems (microorganisms) to obtain a product (food, antibiotics, vitamins)

Front 2

what is Recombinant DNA technology?

Back 2

Recombinant DNA technology – procedures by which a fragment of DNA of one organism is incorporated into the genom of a different organism

Front 3

What are the goals of genetic engineering?

Back 3

Goals of Genetic Engineering
-Eliminate undesirable phenotypic traits in humans, animals, plants, and microbes, (suppression of ripening in tomatoes)
-Create organisms that synthesize products humans need (insulin)

Front 4

What are the tools and techniques of Genetic engineering?

Back 4

Tools and Techniques of Genetic Engineering

-Restriction enzymes – major tool
-Analysis of DNA – gel electrophoresis
-Nucleic acid hybridization
-DNA sequencing
-Polymerase Chain Reaction: PCR

Front 5

What are restriction enzymes?

Back 5

Restriction Enzymes

-DNA cutting enzymes
-They recognize and cut specific fragments of DNA (sequences of nucleotides in DNA)
-They leave single stranded sticky ends of DNA
-DNA from different sources cut with the same restriction enzyme will produce the same type of sticky end

Front 6

How do restriction enzymes work?

Back 6

-DNA is cut on a specific Palindromic sequence -Palindromes are sequences that are identical when read in opposite directions on two strands

Front 7

Describe DNA Gel electrophoresis.

Back 7

DNA Gel Electrophoresis

-Separation of DNA fragments based on their size
-In agar gel, DNA fragments are subjected to an electrical current
-DNA molecule has a negative charge – moves toward the positive pole
-Smaller fragments move faster

Front 8

What is acid hybridization?

Back 8

Nucleic Acid Hybridization

-A fragment of a single-stranded nucleic acid (DNA, RNA) can hybridize (unite) with another
fragment that has a complementary sequence of nucleotides

*when discovered, used to measure questions of similarity between two organisms *

Front 9

describe Hybridization with a probe

Back 9

Hybridization with a probe

-The method used to detect specific nucleotide sequence in an unknown sample by using a gene
probe
-Gene probe is a short segment of DNA of a known sequence
-A probe carries a radioactive level

Front 10

What is the process in which exact sequence of nucleotides in a DNA segment is determined?

Back 10

DNA sequencing

Front 11

what is Polymerase chain reaction PCR?

Back 11

-Technique by which a small amount of specific DNA fragment can be amplified in vitro

Front 12

What is needed for Polymerase chain reaction PCR?

Back 12

-PCR machine – thermal cycler
-Target DNA that serves as a template
-Supply of 4 nucleotides
-DNA polymerase
-Primers
-Primers are short fragments of DNA that are complementary to the target DNA

Front 13

Describe one PCR cycle.

Back 13

One PCR cycle has 3 basic steps:

-Denaturation: 94 degrees Celsius – separation of DNA strands
-Priming: 50-65 C primer attached to complementary strand of DNA
-Extensions: 72 C DNA polymerase extends the molecule by adding nucleotides
-Typically we use 20-40 cycles – millions of copies of DNA

Front 14

How is the process of Recombinant DNA Technology (if you want to clone a gene)?

Back 14

Recombinant DNA Technology (if you want to clone a gene)

-A selected gene is removed from the genetic donor
-This gene is incorporated into a vector (plasmid or virus)
-The vector is inserted into the cloning host (bacteria yeast)

Front 15

What are Vectors?

Back 15

-Vectors are DNA molecules into which a segment of foreign DNA can be incorporated
(plasmids, transposons, and viruses)

Front 16

What are the characteristics of Vectors?

Back 16

-Vector’s characteristics:

-self-replicating
-circular shape
-proper size – to be able to accept foreign DNA
-must have a promoter
-must have a gene for antibacterial resistance

Front 17

How is foreign DNA inserted?

Back 17

Inserting foreign DNA into cells

Transformation

-Plasmid from the surrounding environment is taken up by a cell
-Cells have to be made competent – by soaking them in calcium chloride

Front 18

Describe Screening of bacteria that contain foreign DNA.

Back 18

-The vector (plasmid) contains the gene for ampicilin resistance
-Just those cells that have been transformed can grow on medium containing ampicilin

Front 19

What is synthetic DNA?

Back 19

-DNA synthesis machine
-Short fragments of DNA (120 molecules) can be synthesized
-We must know the sequence of the DNA fragment that we want to synthesize

Front 20

What are the applications of Recombinant DNA technology.

Back 20

-Pharmaceutical and Therapeutic Applications

-Protein synthesis
-Vaccines, DNA vaccines
-Genetic screening
-DNA fingerprinting
-Gene therapy

Front 21

What is Somatostatin?

Back 21

Somatostatin

-Human hormone used for treatment of giantism (an excessive secretion of growth hormone)
-Before 500,000 sheep brains were needed to produce 5mg of somatosin
-Today – 8 liters culture of generally engineered bacteria to obtain equivalent amount

Front 22

WHat are subunit vaccines?

Back 22

-A protein portion of the virus is cloned
-Hepatitis B vaccine (Saccharomyces cerevisaie carries the virus gene on a plasmid)
-Advantage – there is no chance of becoming infected during vaccination

Front 23

What are DNA vaccines?

Back 23

A single gene from that pathogen is artificially copied and multiplied

-That gene is then injected into a muscle. Muscle cells tend to take up this gene and use it as
one of their own genes, making the product
-The immune system will recognize that product as foreign, and will start producing antibodies

Front 24

What are the agricultural applications of Recombination DNA?

Back 24

Agriculture Applications:

-Transgenic organisms – recombinant plants and animals altered by addition of genes from

other organisms
-Improving Crops
-Herbicide resistance
-Salt tolerance
-Freeze resistance
-Pest resistance
-Improvements in nutritional value and yield

Front 25

How are transgenic plants created?

Back 25

-Agrobacterium tumefaciens – plant parasite that can incorporate its DNA into plant’s genom by
using Ti plasmid
-Ti plasmid can be engineered to contain a new gene

Front 26

What are transgenic animals created?

Back 26

Why to create a transgenic animal?

-Many human genes have better expression in animals than in bacteria
-The product (protein) can be collected in milk or semen
-Foreign genes are inserted into an embryo by using a virus or an injection

Front 27

What is Gene therapy?

Back 27

-Mosty preliminary work
-Missing or defective genes replaced with normal copies
-Possible treatment for: cystic fibrosis, sickle cell anemia, some types of hemophilia, some types of diabetes

Front 28

what is Antisense DNA and RNA?

Back 28

-Antisense strand of DNA recognizes and binds to the complementary mRNA fragment
-This results in blocking the expression (translation) of the harmful gene
-Antisense drugs are being researched to treat cancers and other diseases

Front 29

Describe genetic screening.

Back 29

-Many genetic diseases can be detected by genetic engineering techniques
-Technique: Southern blotting (Ed Southern 1975)
`-Inherited forms of breast cancer