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RESTRICTION ENDONUCLEASES CUT AT SPECIFIC SITES & LEAVE STICKY ENDS
EcoR1 animation
Leave “sticky ends” that can be used to join DNA from different organisms
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PLASMIDS
• Small circular self replicating DNA molecule in bacteria separate from bacterial chromosome
• 2-30 genes
• Often carry genes for antibiotic resistance or genetic recombination
• Can be exchanged between bacteria• Bacterial “sex” = conjugation (facilitated by F plasmids)
• Role in rapid evolution
• Method for spreading “antibiotic resistance”•R PLASMIDS
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PLASMIDS
• PLASMID MOVIE Part 2• LAB 6:• Cells can be made “competent” by using
calcium chloride and “heat shock” to change their cell walls - makes them better able to pick up plasmids;
• rapidly growing cells are made competent more easily
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Can be cut with restriction enzymes and used to incorporate foreign DNA into bacteria
Bacteria reproduce, copying the inserted gene along with plasmid
PLASMIDS & RECOMBINANT DNA
• Ti plasmid movie
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Green Fluorescent Protein(GFP)
• Genetic tool
• Originally from jellyfish
• Way to tell if gene has been incorporated
http://www.vet.upenn.edu/schoolresources/communications/publications/bellwether/61/stem_cells.html
http://mabryonline.org/blogs/larkin/GFP%5CGFP_aequorea_victoria-1.jpeg
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REVERSE TRANSCRIPTASE
• Found in RETROVIRUSES (EX: HIV)• Uses RNA message to make a DNA copy• Info flows in reverse RNA → DNA
• Can take eukaryotic RNA message after introns have been removed and change it into a DNA sequence to be read by bacteria (no RNA processing in prokaryotes)
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REVERSE TRANSCRIPTASE
http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/AAAreverse.jpg
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GENE CLONING
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http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/pcranimatie.gif
PCR movie
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Genetics of Viruses & Bacteria Chapter 18
http://www.awesomebackgrounds.com/s-energy-and-power.htm
Control of gene expression in Eukaryotes
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Bacteria• Bacteria review
– one-celled organisms– prokaryotes– reproduce by mitosis
• binary fission
– rapid growth• generation every ~20 minutes• 108 (100 million) colony overnight!
– dominant form of life on Earth– incredibly diverse
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Bacterial genome
• Single circular chromosome– haploid– naked DNA
• no histone proteins
– ~4 million base pairs• ~4300 genes• 1/1000 DNA in eukaryote
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No nucleus!• No nuclear membrane
– chromosome in cytoplasm– transcription & translation are coupled
together• no processing of mRNA
– no introns– but Central Dogma
still applies• use same
genetic code
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Binary fission• Replication of bacterial
chromosome• Asexual reproduction
– offspring genetically identical to parent
– where does variation come from?
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Variation in bacteria
• Sources of variation– spontaneous mutation– transformation
• plasmids• DNA fragments
– transduction– conjugation– transposons
bacteria shedding DNA
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Spontaneous mutation• Spontaneous mutation is a
significant source of variation in rapidly reproducing species
• Example: E. coli– human colon (large intestines)– 2 x 1010 (billion) new E. coli each day!– spontaneous mutations
• for 1 gene, only ~1 mutation in 10 million replications• each day, ~2,000 bacteria develop mutation in that gene• but consider all 4300 genes, then:
4300 x 2000 = 9 million mutations per day per human host!
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TRANSFORMATION in bacteria• Bacteria are opportunists
– pick up naked foreign DNA wherever it may be hanging out
• have surface transport proteins that are specialized for the uptake of naked DNA
– import bits of chromosomes from other bacteria
– incorporate the DNA bits into their own chromosome
• express new gene• form of recombination
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Swapping DNA
• Genetic recombination by trading DNA1 3 2
arg+trp-
arg-trp+
minimalmedia
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Plasmids • Plasmids
– small supplemental circles of DNA• 5000 - 20,000 base pairs• self-replicating
– carry extra genes• 2-30 genes
– can be exchanged between bacteria• bacterial sex!!• rapid evolution • antibiotic resistance
– can be imported from environment
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Plasmids This will beimportant!
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Plasmids & antibiotic resistance• Resistance is futile?
– 1st recognized in 1950s in Japan
– bacterial dysentery not responding to antibiotics
– worldwide problem now• resistant genes are
on plasmids that are swapped between bacteria
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Biotechnology
• Used to insert new genes into bacteria– example: pUC18
• engineered plasmid used in biotech
antibiotic resistance gene on plasmid is used as a selective agent
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TRANSDUCTION with viruses
Phage viruses carry bacterial genes from one host to another
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CONJUGATION - Bacteria “sex”
movie Conjugation • Direct transfer of DNA between 2 bacterial cells
that are temporarily joined– results from presence of F plasmid with F factor
• F for “fertility” DNA
– E. coli “male” extends sex pilli, attaches to female bacterium
– cytoplasmic bridge allows transfer of DNA
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• TRANSFORMATION in bacteria
• TRANSDUCTION with viruses
• CONJUGATION - Bacteria “sex”
movie Conjugation