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Biotechnology
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Amgen Lab Objectives Before the lab: become proficient in basic biotechnology lab skills (using a micropipette and loading a gel) 1) Use restriction enzymes to digest a plasmid (pARA-R) containing rfp gene 2) Verify that plasmid contained rfp gene through gel electrophoresis 3) Transform bacteria with recombinant plasmid
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Biotechnology today Genetic Engineering Our tool kit…
manipulation of DNA if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with Our tool kit…
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Bacteria Review: single-celled 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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How have these little guys gotten to be so diverse??
Bacterial genome Single circular chromosome haploid naked DNA no histone proteins ~4 million base pairs ~4300 genes 1/1000 DNA in eukaryote Genome = all the DNA of an organism Eukaryotes • 1000 times more DNA • only 10 times more genes - introns, spacers, inefficiency How have these little guys gotten to be so diverse??
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Transformation 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 genes transformation form of recombination mix heat-killed pathogenic & non-pathogenic bacteria While E. coli lacks this specialized mechanism, it can be induced to take up small pieces of DNA if cultured in a medium with a relatively high concentration of calcium ions. In biotechnology, this technique has been used to introduce foreign DNA into E. coli. mice die
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Plasmids Small supplemental circles of DNA carry extra genes
,000 base pairs self-replicating carry extra genes 2-30 genes genes for antibiotic resistance can be exchanged between bacteria conjugation (bacterial sex) rapid evolution can be imported from environment Mini-chromosomes
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How can plasmids help us?
A way to get genes into bacteria easily insert new gene into plasmid insert plasmid into bacteria = vector bacteria now expresses new gene bacteria make new protein transformed bacteria gene from other organism recombinant plasmid vector plasmid cut DNA + glue DNA
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insert “gene we want” into plasmid... “glue” together
Biotechnology Plasmids used to insert new genes into bacteria cut DNA gene we want like what? …insulin …HGH …lactase cut plasmid DNA ligase insert “gene we want” into plasmid... “glue” together recombinant plasmid
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How do we cut DNA? Restriction enzymes restriction endonucleases
discovered in 1960s evolved in bacteria to cut up foreign DNA “restrict” the action of the attacking organism protection against viruses & other bacteria bacteria protect their own DNA by methylation (blocks access of restriction enzymes)
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What do you notice about these phrases?
radar racecar Madam I’m Adam Able was I ere I saw Elba a man, a plan, a canal, Panama Was it a bar or a bat I saw? go hang a salami I’m a lasagna hog palindromes
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Restriction enzymes Action of enzyme Many different enzymes
cut DNA at specific sequences restriction site symmetrical “palindrome” produces protruding ends sticky ends will form H bonds to complementary DNA Many different enzymes named after organism they are found in EcoRI, HindIII, BamHI, SmaI CTGAATTCCG GACTTAAGGC CTG|AATTCCG GACTTAA|GGC
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restriction enzyme cut site restriction enzyme cut site
Restriction enzymes Cut DNA at specific sites leave “sticky ends” restriction enzyme cut site GTAACGAATTCACGCTT CATTGCTTAAGTGCGAA restriction enzyme cut site GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA
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chromosome want to add gene to
Sticky ends Cut other DNA with same enzymes leave “sticky ends” on both can glue DNA together at “sticky ends” GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA gene you want GGACCTG AATTCCGGATA CCTGGACTTAA GGCCTAT chromosome want to add gene to GGACCTG AATTCACGCTT CCTGGACTTAA GTGCGAA combined DNA
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Sticky ends help glue genes together
cut sites gene you want cut sites TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTT AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA isolated gene sticky ends AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTT TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA chromosome want to add gene to cut sites Recombinant DNA molecule DNA ligase joins the strands chromosome with new gene added TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC sticky ends stick together
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How can bacteria read human DNA? human insulin gene in bacteria
Why mix genes together? Gene produces protein in different organism or different individual human insulin gene in bacteria TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC aa “new” protein from organism ex: human insulin from bacteria bacteria human insulin
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The code is universal Since all living organisms… use the same DNA
use the same code book read their genes the same way Strong evidence for a single origin in evolutionary theory.
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Copy (& Read) DNA Transformation
insert recombinant plasmid into bacteria grow recombinant bacteria in agar cultures bacteria make lots of copies of plasmid “cloning” the plasmid production of many copies of inserted gene production of “new” protein transformed phenotype DNA RNA protein trait
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Grow bacteria…make more
transformed bacteria gene from other organism + recombinant plasmid vector plasmid grow bacteria harvest (purify) protein
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Uses of genetic engineering
Genetically modified organisms (GMO) enabling plants to produce new proteins Protect crops from insects: BT corn corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn) Extend growing season: fishberries strawberries with an anti-freezing gene from flounder Improve quality of food: golden rice rice producing vitamin A improves nutritional value For example, a transgenic rice plant has been developed that produces yellow grains containing beta-carotene. Humans use beta-carotene to make vitamin A. Currently, 70% of children under the age of 5 in Southeast Asia are deficient in vitamin A, leading to vision impairment and increased disease rates.
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Cut, Paste, Copy, Find… Word processing metaphor… cut paste copy find
restriction enzymes paste ligase copy plasmids bacterial transformation is there an easier way?? find ????
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Comparing cut up DNA How do we compare DNA fragments?
separate fragments by size How do we separate DNA fragments? run it through a gelatin agarose made from algae gel electrophoresis
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“swimming through Jello”
Gel electrophoresis A method of separating DNA in a gelatin-like material using an electrical field DNA is negatively charged so when it’s in an electrical field it moves toward the positive side Size of DNA fragment affects how far it travels Small pieces travel farther Large pieces travel slower & lag behind DNA – + “swimming through Jello”
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DNA & restriction enzyme
Gel Electrophoresis DNA & restriction enzyme - wells longer fragments power source gel shorter fragments + completed gel
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fragments of DNA separate out based on size
Gel Electrophoresis fragments of DNA separate out based on size (DNA is cut with restriction enzymes) 1 2 3 4 Stain DNA ethidium bromide binds to DNA fluoresces under UV light
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DNA fingerprints Comparing blood samples on defendant’s clothing to determine if it belongs to victim DNA fingerprinting comparing DNA banding pattern between different individuals ~unique patterns
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DNA patterns for DNA fingerprints
Allele 1 GCTTGTAACGGCCTCATCATCATTCGCCGGCCTACGCTT CGAACATTGCCGGAGTAGTAGTAAGCGGCCGGATGCGAA repeats cut sites Cut the DNA GCTTGTAACG GCCTCATCATCATCGCCG GCCTACGCTT CGAACATTGCCG GAGTAGTAGTAGCGGCCG GATGCGAA 1 2 3 – DNA + allele 1
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Engineered plasmids Building custom plasmids restriction enzyme sites
antibiotic resistance genes as a selectable marker EcoRI BamHI HindIII restriction sites Selectable marker antibiotic resistance gene on plasmid ampicillin resistance selecting for successful transformation successful uptake of recombinant plasmid How do we know what’s the right combination of genes on a plasmid? Trail and error research work. selectable markers high copy rate convenient restriction sites There are companies that still develop plasmids, patent them & sell them. Biotech companies (ex. New England BioLabs) plasmid ori amp resistance
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Selection for plasmid uptake
Antibiotic becomes a selecting agent only bacteria with the plasmid will grow on antibiotic (ampicillin) plate only transformed bacteria grow all bacteria grow a a a a a a a a a a a a a a a a a LB plate LB/amp plate cloning
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Need to screen plasmids
Need to make sure bacteria have recombinant plasmid restriction sites recombinant plasmid amp resistance broken LacZ gene inserted gene of interest EcoRI all in LacZ gene BamHI HindIII LacZ gene lactose blue color lactose white color X plasmid amp resistance origin of replication
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Screening for recombinant plasmid
Bacteria take up plasmid Functional LacZ gene Bacteria make blue color Bacteria take up recombinant plasmid Non-functional LacZ gene Bacteria stay white color Which colonies do we want?
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PCR process It’s copying DNA in a test tube! What do you need?
template strand DNA polymerase enzyme nucleotides ATP, GTP, CTP, TTP primer Thermocycler
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PCR primers The primers are critical!
need to know a bit of sequence to make proper primers primers can bracket target sequence start with long piece of DNA & copy a specified shorter segment primers define section of DNA to be cloned PCR is an incredibly versatile technique: An important use of PCR now is to “pull out” a piece of DNA sequence, like a gene, from a larger collection of DNA, like the whole cellular genome. You don’t have to go through the process of restriction digest anymore to cut the gene out of the cellular DNA. You can just define the gene with “flanking” primers and get a lot of copies in 40 minutes through PCR. Note: You can also add in a restriction site to the copies of the gene (if one doesn’t exist) by adding them at the end of the original primers. 20-30 cycles 3 steps/cycle 30 sec/step
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What does 90°C do to our DNA polymerase?
PCR process What do you need to do? in tube: DNA, DNA polymerase enzyme, primer, nucleotides denature DNA: heat (90°C) DNA to separate strands anneal DNA: cool to hybridize with primers & build DNA (extension) What does 90°C do to our DNA polymerase?
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