General Preparations Prealiquoted reagents for each lab group – Stock tubes have been centrifuged prior to aliquoting – Use labeling scheme from lab instructions (student guide) Class sets of materials used throughout lab (one per group) – Pipettors (p20, p200) and tips – Tube rack containing full tube of dH2O – Microfuge tubes in baggie/tupperware – Waste container – Sharpie marker Each group should inventory materials at start of class
How do you begin to clone a gene? Isolate gene of interest – rfp from sea anemone Clone it into vector – pARA-R plasmid – Verify it (Lab 2a, 4a) Transform - insert gene of interest into cell for expression – Transformation of E. coli (Lab 5a) Express – transformed cells reproduce and make protein – Overnight Culture (TG E6) Purify protein of interest – Purifying the mFP (Lab 6)
How Do You Begin to Clone a Gene? Learning Goals – Describe the characteristics of plasmids – Explain how plasmids are used in genetic engineering – Describe the function of restriction enzymes – Explain how restriction enzymes are used to create a recombinant plasmid (rDNA)
Plasmid Features: Ori Promoter Antibiotic resistance Restriction sites
Restriction Enzymes “Cut & Paste” DNA Some concepts: In nature, used by bacteria to destroy phage DNA Recognize and bind to specific nucleotide sequences – Cut the phosphodiester backbone of DNA Off-center cuts produce single-stranded ends of DNA – “Sticky ends” Use the same restriction enzyme to cut DNA from two sources – Complimentary sticky ends will pair up to create rDNA
Modeling Activity: Choose the appropriate restriction enzyme to use to insert the insulin gene
pARA-R Why is this plasmid named pARA-R? Why is pARA-R an expression vector? What is the function of each region of pARA-R? Describe the products of digestion
BamH I Hind III BamH I Hind III Restriction digest of pARA-R Recombinant plasmid of interest pARA-R 5,302 bp P BAD -rfp 807 bp
Making Sure You’ve Got a Recombinant Plasmid Learning goals – Steps in a genetic engineering experiment are verified – Predict the migration speeds of plasmids and restriction fragments in a gel – Separate and identify plasmids and restriction fragments using gel electrophoresis
Early Steps in the Genetic Engineering Workflow Restriction Enzymes DNA Ligase restriction digestion of ligated plasmids verify the presence of the plasmid of interest Such as pARA-R
Separation of DNA by Size Using Gel Electrophoresis Why does DNA migrate from “-” to “+”? Why do some DNA molecules migrate farther/faster than others?
Sample Gel Diagram In this model of a gel, order the molecules (A-G) by size Which are the largest? Smallest? Explain how you know
Plasmid Configurations Why aren’t all plasmids the same shape and size? How will the plasmid shape and size affect movement through a gel? Why? How will undigested pARA-R be configured?
Lab 5A: Getting Recombinant Plasmids in Bacteria Learning Goals – Describe the role of transformation in the gene cloning process – Explain the purpose of each control in the transformation experiment – Explain how the information encoded in a gene is expressed as a trait
Why is this biotechnology? Manipulation of a natural process to produce something useful In nature, bacteria take up DNA from their environment and acquire novel traits (Griffith) In genetic engineering, bacteria are induced to take up DNA for traits of interest
How do we get the bacteria to take up the plasmid? How do we find the bacteria that have taken it up?
Transforming Bacteria with the pARA-R Plasmid Objective: –Transform E. coli with pARA-R containing the rfp gene of interest –Identify the bacteria that were successfully transformed with pARA-R
Preparing competent cells for transformation Lipid bilayer (inner) Lipid bilayer (outer) Peptidoglycan layer Adhesion zone Calcium ions Bruce Wallace
Transforming Escherichia coli with pARA-R Recombinant Plasmids Competent Cells pARA-R Bruce Wallace
Calcium ions pARA-R Transforming Escherichia coli with pARA-R Adhesion zone Lipid bilayer (inner) Lipid bilayer (outer) Peptidoglycan layer Bruce Wallace
Before you begin: Predict –Growth/No growth LB +/- LB/amp +/- LB/amp/ara –Characterize expected growth Lawns? Colonies? Color?
Lab 5a Protocol Keep everything on crushed ice Label tubes Aliquot E. coli cells Add plasmid to P+ 15 min. rest Label plates Heat shock Add LB 15 min. rest
Prep & Hints In advance: –Aliquot LB broth –Equilibrate water bath (42 deg C) and incubator (37 deg C) –Demonstrate aseptic technique and how to plate Day of lab: –Use crushed ice (get from athletic trainer, or use food processor/snow cone machine/etc.) –Thaw CC on ice 15 min. prior to lab; aliquot to each group During Lab –Bacterial waste goes in waste container/autoclave bag Tubes, tips, spreaders NO TRASH