Introduction to Biotechnology Transformation and more! Do you know how a car engine works? 4-stage cylinder? http://xorl.files.wordpress.com/2011/03/img11.gif Introduction to Biotechnology Transformation and more! Chapter 20 Section 20.1 Pgs. 396-404 Objective: I can describe several different types of biotechnology, such as plasmid cloning and transformation, in order to best determine how to cut a gene of interest out and insert it into a plasmid.

Prokaryotic DNA Packaging (Review) 1 circular bacterial chromosome NOT a plasmid… Plasmid can refer to small(er) circular molecules of DNA besides “main” chromosome Self-Replicating, so will pass on Confusing, because plasmid can be incorporated into “main” or stay isolated Prokaryotic Cell Division is called… Binary Fission, which is a type of… Asexual Reproduction

Prokaryotic Sexual “Reproduction” Conjugation Uses pili (singular pilus) Transfers DNA Not reproduction - recombination Can pass plasmid(s) or part of “main” Can either remain as plasmid in receiver Can be incorporated into main bacterial chromosome

Conjugation: A Natural Transformation Bacteria naturally transfer genetic info (in certain conditions)  helps survival (adaptation + evolution) Also viruses can transform genetic makeup of host… Similar to whose experiment…? Griffith’s Experiment

Can we artificially transfer genes? If bacteria can naturally take in foreign DNA (undergo transformation), can we artificially make them take up DNA we want them to? Yes: Biotechnology (DNA Technology) Genetic Engineering  Recombinant DNA What’s the point? Allow for gene cloning (just gene) Allows for GMO (Genetically Modified Organisms) How? (specifically)?

Bacteria of Choice E.coli Escherichia coli Use bacteria as “factory” Can clone a gene (DNA) by letting E.coli reproduce (divide)

A cell with DNA Plasmid containing the gene of interest E. coli bacterium 2 The cell’s DNA is isolated. Bacterial chromosome 1 A plasmid is isolated. Gene of interest DNA Figure 12.1B_s1 An overview of gene cloning (part 1, step 1) 8

A cell with DNA Plasmid containing the gene of interest E. coli Figure 12.1B_s2 A cell with DNA containing the gene of interest Plasmid E. coli bacterium 2 The cell’s DNA is isolated. Bacterial chromosome 1 A plasmid is isolated. Gene of interest DNA 3 The plasmid is cut with an enzyme. 4 The cell’s DNA is cut with the same enzyme. Gene of interest Figure 12.1B_s2 An overview of gene cloning (part 1, step 2) 9

A cell with DNA Plasmid containing the gene of interest E. coli Figure 12.1B_s3 A cell with DNA containing the gene of interest Plasmid E. coli bacterium 2 The cell’s DNA is isolated. Bacterial chromosome 1 A plasmid is isolated. Gene of interest DNA 3 The plasmid is cut with an enzyme. 4 The cell’s DNA is cut with the same enzyme. Gene of interest 5 The targeted fragment and plasmid DNA are combined. Figure 12.1B_s3 An overview of gene cloning (part 1, step 3) 10

A cell with DNA Plasmid containing the gene of interest E. coli Figure 12.1B_s4 A cell with DNA containing the gene of interest Plasmid E. coli bacterium 2 The cell’s DNA is isolated. Bacterial chromosome 1 A plasmid is isolated. Gene of interest DNA 3 The plasmid is cut with an enzyme. 4 The cell’s DNA is cut with the same enzyme. Gene of interest 5 The targeted fragment and plasmid DNA are combined. Figure 12.1B_s4 An overview of gene cloning (part 1, step 4) 6 DNA ligase is added, which joins the two DNA molecules. Recombinant DNA plasmid Gene of interest 11

The recombinant plasmid is taken up by a bacterium Figure 12.1B_s5 Recombinant DNA plasmid Gene of interest 7 The recombinant plasmid is taken up by a bacterium through transformation. Recombinant bacterium Figure 12.1B_s5 An overview of gene cloning (part 2, step 1) 12

The recombinant plasmid is taken up by a bacterium Figure 12.1B_s6 Recombinant DNA plasmid Gene of interest 7 The recombinant plasmid is taken up by a bacterium through transformation. Recombinant bacterium 8 The bacterium reproduces. Figure 12.1B_s6 An overview of gene cloning (part 2, step 2) Clone of cells 13

The recombinant plasmid is taken up by a bacterium Figure 12.1B_s7 Genes may be inserted into other organisms. Recombinant DNA plasmid Gene of interest 7 The recombinant plasmid is taken up by a bacterium through transformation. 9 Recombinant bacterium Harvested proteins may be used directly. 8 The bacterium reproduces. Figure 12.1B_s7 An overview of gene cloning (part 2, step 3) Clone of cells 14

animation file

How can cut gene of interest out? How can cut plasmid to insert? Restriction Enzymes: natural defense (a.k.a. restriction endonucleases) Enzymes that recognize a particular sequence in DNA and cut there Sequence must be “symmetrical” in that it is a “complementary palindrome” Area to be cut = restriction site animation file

Restriction Enzymes Details Usually cut in a staggered format Results in “sticky ends” Allows to hydrogen bond to other restriction fragments Fragments of DNA cut by restriction enzymes Connections made permanent by DNA ligase How to ensure insertion? Waitaminute! What is problem?

Transforming Prokaryotes vs. Eukaryotes Difficulty in inserting genes from eukaryotes into prokaryotes (and vice versa) in terms of getting past plasma membrane = transformation How to do this? What can use (from learned?) Viruses (retrovirus), heating, electroporation getting them to function…why? Different regulation mechanisms Prokaryotes = operons Eukaryotes = enhancers, etc. Often use expression vectors segments of DNA/RNA that will make sure gene is turned on after transformation and insertion

Let’s do an activity to showcase recombinant DNA! Will come back to PPT in last 20 minutes of class

Genomic Libraries: Storing genes Can store genes in a plasmid library or in BAC (Bacterial artificial chromosomes) Can use reverse transcriptase to make cDNA (complementary)  cDNA library Different because doesn’t have introns

Directly Identifying Genes (Probes) With library, when look at a new organism’s genome, we can identify what genes it has using: nucleic acid hybridization Construct a nucleic acid probe: short single-stranded DNA complement to gene of interest  tagged fluorescent

Nucleic Acid Probe Hybridization When mix probe (that came from gene of interest) with a library, can identify gene! Why not sequence? More modern (problems with variation)

Polymerase Chain Reaction (PCR) Amplifying DNA directly Instead of putting gene of interest in bacteria, and having WHOLE cells reproduce to reproduce DNA… Isolate DNA within a cell and make it PCR can produce billions of copies of a target DNA segment in a few hours Bacteria would take days to make the same amount (reproduce whole cell)

PCR Steps 1) Denaturation: Heat DNA = (unzip) 2) Annealing: cool to allow (DNA) primers to attach (primers specific for target sequence) 3) Elongation: allow DNA polymerase to work (denature?) Repeat Cycle (exponential)

Biotechnology at its best PCR machines automate the cycles Insert DNA with target gene Insert primers Machine runs automatically thru cycles Was designed/engineered by Kary Mullis (won Nobel Prize) http://www.youtube.com/watch?v=x5yPkxCLads http://www.youtube.com/watch?v=CQEaX3MiDow http://www.youtube.com/watch?v=8k_WgseFy6Q