A Little More Advnced Biotechnology Tools Better Plsmids Engineered plsmids Building custom plsmids restriction enzyme sites ntibiotic resistnce genes s selectble mrker EcoRI BmHI HindIII restriction sites Selectble mrker ntibiotic resistnce gene on plsmid mpicillin resistnce selecting for successful trnsformtion successful uptke of recombinnt plsmid plsmid ori mp resistnce Selection for plsmid uptke Antibiotic becomes selecting gent only bcteri with the plsmid will grow on ntibiotic (mpicillin) plte ll bcteri grow only trnsformed bcteri grow LB plte LB/mp plte cloning
Need to screen plsmids Need to mke sure bcteri hve recombinnt plsmid EcoRI BmHI restriction sites inserted gene of interest ll in LcZ gene HindIII LcZ gene broken LcZ gene lctose blue color X white color lctose plsmid recombinnt plsmid mp resistnce mp resistnce origin of AP Biology repliction Screening for recombinnt plsmid Bcteri tke up plsmid Functionl LcZ gene Bcteri mke blue color Bcteri tke up recombinnt plsmid Non-functionl LcZ gene Bcteri sty white color Which colonies do we wnt? Finding your Gene of Interest
Finding your gene of interest DNA hybridiztion find sequence of DNA using lbeled probe short, single strnded DNA molecule complementry to prt of gene of interest lbeled with rdioctive P32 or fluorescent dye het tret DNA in gel wsh gel with probe unwinds (dentures) strnds probe hybridizes with dentured DNA lbeled probe genomic DNA G A T C A G T A G C T A G T C A T C Southern blotting restriction digest gel electrophoresis blot DNA off of gel onto filter pper expose filter pper to X-ry film wsh filter with lbeled probe Edwin Southern Southern blotting gel of genomic DNA Southern blot IDing one gene Southern blot illustrtion
DNA librries Cut up ll of nucler DNA from mny cells of n orgnism restriction enzyme Clone ll frgments into mny plsmids t sme time shotgun cloning Crete stored collection of DNA frgments 1 petri dish hs collection of ll DNA frgments from the orgnism Mking DNA librry 2 engineered plsmid with selectble mrker & screening system ll DNA from mny cells of n orgnism is cut with restriction enzymes gene of interest 3 ll DNA frgments inserted into mny plsmids 4 clone plsmids into bcteri But how do we find colony with our gene of interest in it? DNA librry recombinnt plsmids inserted into bcteri gene of interest DNA Librry plte of bcteril colonies storing & copying ll genes from n orgnism (ex. humn)?
Find your gene in DNA librry Locte Gene of Interest to find your gene you need some of gene s sequence if you know sequence of protein cn guess prt of DNA sequence bck trnslte protein to DNA if you hve sequence of similr gene from nother orgnism? use prt of this sequence Which bcteril colony hs our gene? Like needle in hystck! Colony Blots 4 Locte - expose film - locte colony on plte from film 1 Cloning - plte with bcteril colonies crrying recombinnt plsmids plte plte + filter film 3 2 Replicte plte - press filter pper onto plte to tke smple of cells from every colony filter Hybridiztion - het filter pper to denture DNA - wsh filter pper with rdioctive probe which will only ttch to gene of interest Problems Humn Genome librry re there only genes in there? nope! lot of junk! humn genomic librry hs more junk thn genes in it Clen up the junk! if you wnt to clone humn gene into bcteri, you cn t hve introns
How do you clen up the junk? Don t strt with DNA Use mrna copy of the gene without the junk! But in the end, you need DNA to clone into plsmid How do you go from RNA DNA? reverse trnscriptse from RNA viruses retroviruses reverse trnscriptse cdna (copy DNA) librries Collection of only the coding sequences of expressed genes extrct mrna from cells reverse trnscriptse RNA DNA from retroviruses clone into plsmid Applictions need edited DNA for expression in bcteri humn insulin Where do we go next. DNA RNA protein trit When gene is turned on, it cretes trit wnt to know wht gene is being expressed extrct mrna from cells mrna = ctive genes How do you mtch mrna bck to DNA in cells??? reverse trnscriptse
slide with spots of DNA ech spot = 1 gene Microrrys Crete slide with smple of ech gene from the orgnism ech spot is one gene Convert mrna lbeled cdna mrna cdna mrna from cells reverse trnscriptse slide with spots of DNA ech spot = 1 gene Microrrys Lbeled cdna hybridizes with DNA on slide ech yellow spot = gene mtched to mrna ech yellow spot = expressed gene mrna cdna cdna mtched to genomic DNA Appliction of Microrrys DNA Chip 2-color fluorescent tgging Compring tretments or conditions = Mesuring chnge in gene expression sick vs. helthy; cncer vs. norml cells before vs. fter tretment with drug different stges in development Color coding: lbel ech condition with different color red = gene expression in one smple green = gene expression in other smple yellow = gene expression in both smples blck = no or low expression in both
I my be very selective But still Ask Questions! EcoRI BmHI HindIII restriction sites plsmid ori mp resistnce