Genetic Engineering Technology Midterm 1

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organelles

Back 1

membrane bound structures in eukaryotic cells that conduct specific functions
•Composed of different types of macromolecules

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lipids

Back 2

o define a broad group of hydrophobic o rganic compounds
oPlay a vital role in forming membranes
•Polar head, hydrophobic tail
•Spontaneously form bilayers

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Polysaccharides

Back 3

o Long chains of monomers called monosaccharides

o Generally function as structural components or for energy storage

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Proteins

Back 4

o Large organic compounds that are the major determinants of an organism’s characteristics
o Machines in the cellular factory
• Cells may have 100s-1000s of different proteins
o Monomers:
• 20 different AAs connected by peptide bonds (condensation)

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Levels of protein structure

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• Primary:
Sequence of AAs (most important bc it determines all other levels of structure)
• Secondary:
Beta pleated sheet
Alpha helix
Random coil
• Tertiary:
Folded polypeptide chain
• Quaternary:
2 or more polypeptide chains

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nucleic acids

Back 6

o Involved in the storage and transmission of genetic information within the cell
o Long polymers composed of nucleotides

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DNA

Back 7

• Primary function is storage of genetic information
• Made of 4 different deoxyribonucleotides,
each with a phosphate group and sugar backbone (phosphodiester backbone)
and one of 4 bases (Adenine, Thymine Guanine Cytocine)

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what kind of bases are each of the 4 nucleic acids?

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• A and G are purines

T and C are pyridines

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bonding between base pairs

Back 9

o A=T (2 H bonds)
o C=G (3 H bonds)

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RNA

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• Different from DNA in structure and function
 One less O in DNA (deoxy…)
 Use Uracil instead of Thymine
 Generally single stranded

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Steps for cloning DNA

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• 1) Isolation of DNA
RE digestion and separating our DNA from other fragments
• 2) Ligating the DNA into a vector
• 3) Transforming the host cell with the recombinant DNA
• 4) Selection of only the host cells harboring the recombinant DNA vector
• 5) Screening cells for those that have the recombinant DNA or producing the appropriate protein product

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germplasm

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The genetic material within an organism, usually meaning plant geneitc material

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Gene

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a discrete stretch of bases on a DNA strand that serves as a unit of information. can be protein or RNA; includes coding sequences, introns, and noncoding regulatory regions.

each 3 bases is one codon

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Robert Hooke

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1665: looked at cork; called compartments cellulae

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Zacharias Janssen

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1590: first compound microscope, capable of 30x magnification

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Anton Van Leeuwenhoek

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1676: Examined pondwater and fermenting beer; called protozoa and fungi animalcules
submitted descriptions and drawings of yeast to the Royal Society of London

1683: first to see bacteria

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Matthias Schleiden

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1838: all living plant tissue is composed of cells, and that each plant arose from a single cell

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Theodor Schwann

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1839: all living animal tissue is composed of cells, and that each animal arose from a single cell

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Rudolf Virchow

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1858: refined cell theory: "all cells arise from cells, and cells are the basic unit of life."

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Vitalism

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prevailing theory before cell theory: only the complete organism, not its individual parts, possessed life.

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Fridrich Wohler

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1828: determined that organic molecules can come from inorganic sources by synthesizing urea from ammonium cyanate in the lab.

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Louis Pasteur

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1) established a link
between yeast and other microbes
and fermentation. Discovered that Yeast cells present in wine contributed to spoilage, turing old wine sour.

2) Wine is preserved if heated during the interval after the alcohol was made, and before lactic acid was produced (pasteurization).

3) Spontaneous generation of microorganisms does not occur. He sterilized beef broth in a swan-neck flask.

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Eduard Buchner

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1896: converted sugar to ethyl alcohol using yeast extracts, which were found to be enzymes. he called them "ferments"

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Ernst Ruska

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built first electron microscope in 1932

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Gregor Mendel

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father of genetics, published 1865 but not understood until 1900

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Johann Friedrich Miescher

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1869: isolated "nuclein" from white blood cell nuclei. Turned out to be nucleic acids

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Walter Flemming

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1882: described threadlike bodies visible during cell division which turned out to be chromosomes

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Walter Sutton

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1903: rediscovered mendel's experments and determined that chromosomes were the carriers of mendel's units of heredity.

Described meiosis: determined that meiosis was the mechnaism by which heredity units are distributed

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Charles Yanofsky

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along with others demonstrated a colinearity between the order of mutant sites within a gene, and the linear sequence of AAs in a protein.

experimented with trpA in E.coli.

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Fred Giffith

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1928: used 2 strains of Streptococcus pneumoniae: virulant smooth (S) and less viroulant rough (R).

Mice injected with:
1) live R: mice live
2) live S: mice die
3) heat-killed S: mice live
4) live R + heat-killed S: mice die, live S found in blood.

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Avery, MacLeod, and McCarthy

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extention of Griffith experiment: 1945

mixed live R strain with DNA from S strain: live S bacteria grew.

mixed live R strain with DNA from S strain and DNase: no S colonies grew

mixed live R strain with DNA from S strain and Protease: live S bacteria grew.

Thus: DNA is the transforming principle. not accepted unitl 1952

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Hershy and Chase

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experiments with T2 bacteriophagebacteriophage(virus that infects bacteria)
• Used 35S labeled viral proteins and 32P labeled DNA to follow viral infection of E. coli
• Analysis of the host and culture medium showed that the 35S labeled viral proteins was left behind
in the medium
• The 32P labeled DNA was inside the bacterial cells and used to used tosynthesize new viral progeny

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Discovering the structure of DNA

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1953 –Using Rosalind Franklin and Maurice Wilkins’ X-ray crystallography and diffraction data, and Erwin Chargaff’s predetermined base ratio, James D. Watson and Fracis H.C. Crick elucidated the structure of DNA

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The first recombinant DNA experiments

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1971 –Student in Stanley Cohen’s lab discovered the process of transformation: CaCl2 shocking
bacterial cells so that they take up genes (ampr)
• 1972 –Boyer purifies EcoRI; uses
gel electrophoresis and
EtBr for DNA work
• 1972 –Mertz and Davis dis-
covered a way to direct recombination and the orientation of the insert
• All of these scientists were from
the Bay Area (Stanford & UCSF),
but they joined forces in Hawaii!
• 1973 –First cloning experiment pub’d

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modern Biotech companies are born

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1980 –Patent awarded for the basic methods of cloning and transformation to Hebert Boyer and
Stanley Cohen
• Hebert Boyer became the founder of the biotechnology company,Genentech

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Bioremediation

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uses microorganisms to clean up toxic waste from industrial accidents/oil spills in 2 ways:
• Encourage growth and enhance the activity of naturally occurring bacteria in the soil or water
• The addition of new bacteria to the site (naturally ocurringor genetically engineered bacteria)
• Naturally occurring bacteria is usually used due to public perception.

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The discovery of Yeast

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Egyptians used a pure strain of the yeast Saccharomyces winlocki as early as ~1500 BC
• We generally use Saccharomyces cerevisiae now

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War and Biotechnology

Back 38

Fermentation of organic solvents began during WWI; by the 1940s (WWII), Germany had improved
techniques
• Fermented large quantities of glycerol for explosives
• Aseptic techniques improved and industrial fermentation; made it possible to produce rare & valuable
chemicals
• World War II also ushered in the age of the modern fermenter, or bioreactor, and antibiotics

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Origin of replication

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• Point at which replication is initiated
• Eukaryotic DNA may have multiple origins of replication, but circular prokaryotic DNA only has one origin of replication

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lagging strand synthesis

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o The lagging strand is synthesized in short segments as a series of Okazaki fragments, also in the 5’-3’ direction, but away from the replication fork
• Okazaki fragments are joined by the enzyme DNA ligase

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primers

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• Short oligonucleotides are 4-15 nt long
 Try to start primer with Gs and Cs
 RNA
 Required to start synthesis of both daughter strands
 Primases place primers about 50 nt apart in the lagging strand synthesis

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Transcription steps

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o 1) RNA polymersase binds DNA at the gene’s promoter; DNA helix unwinds
o 2) Initiation of transcription (RNA synthesis begins)
o 3) Elongation
o 4) Termination
o In eukaryotic cells, there is still one more step after this: RNA processing
• In prokaryotic cells, transcription and translation occur together

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mRNA processing

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o 5’ cap and 3’ tail for added stability
o noncoding sequences (introns) intervene between coding sequences (exons); the introns are removed via splicing

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Translation steps

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o 1) Initiation: requires mRNA transcript, ribosome, AAs, tRNAs, and energy
• tRNA has a site of AA attachment and an anticodon for the mRNA transcript
o 2) eleongation
• ribosome has a P site and an A site to cradle 2 tRNAs simultaneously, and the developing peptide (in p site)
o 3) Translocation
• kick out first tRNA, ratchet over, accept another tRNA
o 4) Termination
• Elongation ceases once a STOP codon is reached (UAA, UAG, UGA), then the complex falls off

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operons

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used to control multiple genes with the same switch in prokaryotes

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eukaryotic gene regulation

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o control transcription using transcription factors
o mRNA processing, nuclear export, and stability
o translational control (controlling protein synthesis)
o posttranslational control (regulating protein activity)

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DNA ligase

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• Catalyze the formation of covalent bonds in the sugar-phosphate backbone
• Does not discriminate between DNA of different origin; completes process of creating a recombinant DNA molecule

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ligation controls

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 Control 1: cut vector (no ligase)
• LB + amp plate does not grow colonies
 Control 2: cut vector + ligase
• LB + amp plate grows few colonies (only improperly cut)
 Control 3: cut vector + insert + ligase
• LB + amp plate grows many colonies

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Steps in gene cloning

Back 49

• 1) Isolation of DNA
 RE digestion and separating our DNA from other fragments
• 2) Ligating the DNA into a vector
• 3) Transforming the host cell with the recombinant DNA
• 4) Selection of only the host cells harboring the recombinant DNA vector
• 5) Screening cells for those that have the recombinant DNA or producing the appropriate protein product

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cloning vectors must have

Back 50

• ori: origin of replication so it can be replicated
• be small enough to be isolated and manipulated in the lab without degrading
• have several unique restriction sites for inserting DNA (MCS)
• selectable markers for determining whether the vector has been transferred into cells, and whether the insert is present
 alpha-complimentation; SEE selection

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bacterial vectors

Back 51

 Plasmids: extra-chromosomal circular dsDNA
• Can take a 10kb insert
 Bacteriophage: bacterial viruses
• Can take a 20kb insert
 Cosmids: hybrids of plasmid and bacteriophage
• Can take 35-45 kb insert

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non-bacterial vectors

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 Yeast artificial chromosomes (YAKs)
• Can take 200-1500kb inserts
• Study multiple gene interactions
 Bacterial artificial chromosomes (YAKs)
• Can take 100-300kb inserts
 Plant cloning vectors
 Mammalian cell vectors, retroviruses and more