Presentation on theme: "A Little More Advanced Biotechnology Tools"— Presentation transcript:
1 A Little More Advanced Biotechnology Tools Better Plasmids
2 Engineered plasmids Building custom plasmids restriction enzyme sites antibiotic resistance genes as a selectable markerEcoRIBamHIHindIIIrestriction sitesSelectable markerantibiotic resistance gene on plasmidampicillin resistanceselecting for successful transformationsuccessful uptake of recombinant plasmidHow do we know what’s the right combination of genes on a plasmid?Trail and error research work.selectable markershigh copy rateconvenient restriction sitesThere are companies that still develop plasmids, patent them & sell them.Biotech companies (ex. New England BioLabs)plasmidoriamp resistance
3 Selection for plasmid uptake Antibiotic becomes a selecting agentonly bacteria with the plasmid will grow on antibiotic (ampicillin) plateonly transformed bacteria growall bacteria growaaaaaaaaaaaaaaaaaLB plateLB/amp platecloning
4 Need to screen plasmids Need to make sure bacteria have recombinant plasmidrestriction sitesrecombinant plasmidampresistancebroken LacZ geneinserted gene of interestEcoRIall in LacZ geneBamHIHindIIILacZ genelactose blue colorlactose white colorXplasmidampresistanceorigin of replication
5 Screening for recombinant plasmid Bacteria take up plasmidFunctional LacZ geneBacteria make blue colorBacteria take up recombinant plasmidNon-functional LacZ geneBacteria stay white colorWhich colonies do we want?
7 Finding your gene of interest DNA hybridizationfind sequence of DNA using a labeled probeshort, single stranded DNA moleculecomplementary to part of gene of interestlabeled with radioactive P32 or fluorescent dyeheat treat DNA in gelunwinds (denatures) strandswash gel with probeprobe hybridizes with denatured DNAGATClabeled probegenomic DNACTAGTCATC
8 Southern blotting restriction digest gel electrophoresis blot DNA off of gel onto filter paperexpose filter paper to X-ray filmwash filter with labeled probe
9 Southern blot IDing one gene Southern blot illustration Edwin SouthernSouthern blottingNorthern blot: RNA on filter & DNA probe measures gene expression because grabbing mRNA from cell -- only expressed genesWestern blot: protein on filter labeled directlygel of genomic DNASouthern blot IDing one geneSouthern blot illustration
10 DNA libraries Cut up all of nuclear DNA from many cells of an organism restriction enzymeClone all fragments into many plasmids at same time“shotgun” cloningCreate a stored collection of DNA fragmentspetri dish has a collection of all DNA fragments from the organism
11 Making a DNA library21engineered plasmid with selectable marker & screening systemall DNA from many cells of an organism is cut with restriction enzymesgene of interest3all DNA fragments inserted into many plasmidshuman genome = 3 billion basesfragments are cut to ~5000 basestherefore ~ 600,000 fragments per cell.But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes.4clone plasmids into bacteria
12 But how do we find colony with our gene of interest in it? DNA libraryrecombinant plasmids inserted into bacteriagene of interesthuman genome = 3 billion basesfragments are cut to ~5000 basestherefore ~ 600,000 fragments per cell.But you have to use many cells to make sure you have a complete set in the library, so… you may have millions of cells that you extracted the DNA from, so… you would need millions of colonies, so the human genome cloned into bacteria would be a walk-in freezer full of petri dishes.?DNA Library plate of bacterial colonies storing & copying all genes from an organism (ex. human)
13 Find your gene in DNA library Locate Gene of Interestto find your gene you need some of gene’s sequenceif you know sequence of protein…can “guess” part of DNA sequence“back translate” protein to DNAif you have sequence of similar gene from another organism…use part of this sequenceComplementationif you have a mutant that lacks YFG, you can transform bacteria with plasmids from the library until one “cures” (complements) the mutation?Which bacterial colony has our gene?Like a needle in a haystack!
14 Colony Blots 4 1 3 2 Locate expose film Cloning locate colony on plate from film1Cloning- plate with bacterial colonies carrying recombinant plasmidsplateplate + filterfilm32Replicate platepress filter paper onto plate to take sample of cells from every colonyHybridization- heat filter paper to denature DNA- wash filter paper with radioactive probe which will only attach to gene of interestfilter
15 Problems… Human Genome library Clean up the junk! introns are there only genes in there?nope! a lot of junk!human genomic library has more “junk” than genes in itClean up the junk!if you want to clone a human gene into bacteria, you can’t have…introns
16 How do you clean up the junk? Don’t start with DNA…Use mRNAcopy of the gene without the junk!But in the end, you need DNA to clone into plasmid…How do you go from RNA DNA?reverse transcriptase from RNA virusesretrovirusesreverse transcriptase
17 cDNA (copy DNA) libraries Collection of only the coding sequences of expressed genesextract mRNA from cellsreverse transcriptaseRNA DNAfrom retrovirusesclone into plasmidApplicationsneed edited DNA for expression in bacteriahuman insulinCould you imagine how much that first insulin clone was worth to Genentech? One little piece of DNA in a plasmid worth billions! It put them on the map & built a multi-billion dollar biotech company.
18 reverse transcriptase Where do we go next….DNARNAproteintraitWhen a gene is turned on, it creates a traitwant to know what gene is being expressedextract mRNA from cellsmRNA = active genesHow do you match mRNA back to DNA in cells???reverse transcriptase
19 Microarrays slide with spots of DNA each spot = 1 gene Create a slide with a sample of each gene from the organismeach spot is one geneConvert mRNA labeled cDNAmRNA cDNAmRNA from cellsreverse transcriptase
20 Microarrays Labeled cDNA hybridizes with DNA on slide slide with spots of DNAeach spot = 1 geneMicroarraysLabeled cDNA hybridizes with DNA on slideeach yellow spot = gene matched to mRNAeach yellow spot = expressed genecDNA matched to genomic DNAmRNA cDNADeveloped by Pat Brown at Stanford in late 1980sRealized quickly he needed an automated system: robot spotterDesigned spotter & put plans on Internet for benefit of scientific community.
21 Application of Microarrays “DNA Chip” 2-color fluorescent taggingComparing treatments or conditions = Measuring change in gene expressionsick vs. healthy; cancer vs. normal cellsbefore vs. after treatment with drugdifferent stages in developmentColor coding: label each condition with different colorred = gene expression in one samplegreen = gene expression in other sampleyellow = gene expression in both samplesblack = no or low expression in bothIt’s all about comparisons!Powerful research tool.
22 I may be very selective… But still Ask Questions! EcoRIBamHIHindIIIrestriction sitesplasmidoriamp resistance