Transformation Intro to Lab #8

Figure 20.2 Bacterium 1 Gene inserted into plasmid Cell containing gene of interest Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome (“foreign” DNA) 2 Plasmid put into bacterial cell Recombinant bacterium 3 Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed from gene of interest Gene of interest Copies of gene Protein harvested 4 Basic research and various applications Figure 20.2 A preview of gene cloning and some uses of cloned genes. Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hormone treats stunted growth

Transformation Identify a restriction enzyme that will cut out gene of interest and cut open the plasmid Isolate DNA from 2 sources (making sure the at the plasmid has a genetic marker) Cut both types of DNA with the same restriction enzyme Mix the 2 types of DNA to join them through complementary base pairing Add DNA ligase to bond DNA covalently producing Recombinant DNA Incubate bacteria at 42 C with calcium chloride; bacteria become competent / permeable - so that the bacteria will take in the plasmid (TRANSFORMATION) Use a genetic marker to identify bacteria with the recombinant plasmid Clone bacteria

Pre-Lab Inquiry You work on a team in a research lab. You have three separate plasmids in your lab, but the labels have come off of the tubes and you no longer know which plasmid is which. For the time being, you will refer to the plasmids are plasmids 1, 2, and 3. You know the following: One plasmid has kanamycin-resistant gene. Two plasmids have an ampicillin-resistance gene. In addition to having an antibiotic-resistance marker, one plasmid also codes for the gene for green fluorescent protein (GFP), a protein from the bioluminescent jellyfish Aequorea victoria.

Pre-lab Inquiry Bacteria that produce the green fluorescent protein look green under white light and fluoresce under ultraviolet light Assume that each research team has access to the following materials and is responsible for identifying one of the three plasmid. (Note: you will not actually use any materials during this pre-lab inquiry). Before planning your experiment, answer the following questions to ensure that you understand some of the basic concepts involved in the lab. Some of these questions are specifically designed to help you think about setting up your experiment.

Materials Available Starter plates (containing E. coli in rapid growth) Kanamycin plates Ampicillin plates LB plates (plates contain Luria Broth/no antibiotic) Plasmids 1, 2, and 3 Transformation agents, tubes, and equipment (inoculating loops, calcium chloride, ice, 42C water bath, incubator)

Questions 1. What is a plasmid? 2. What is transformation? Small, usually circular, extra-chromosomal piece of DNA that exits in nature in some bacteria and yeasts. They can be transferred between organisms. In the lab they can be used to manipulate and introduce DNA of interest into bacterium. 2. What is transformation? The uptake of exongenous, naked DNA by a cell. The newly adopted DNA can become a heritable part of the cell’s genetic material.

Questions 3. Why is naturally occurring transformation beneficial to bacteria? It provides them with access to new genetic material, which increases their chances of being able to adapt to the environment. 4. Why is transformation useful to research scientists? It enables them to introduce foreign DNA into bacteria. This allows them to further study and work with genes on the plasmid DNA and the proteins that the genes code for.

Questions 5. Should you plate some of your transformed bacteria onto plates with antibiotic? Why or Why not? Yes – you will select for/isolate bacteria that have been transformed with the plasmid with the resistance gene 6. What would you expect to see if you plated some of your transformed bacteria onto a plate without antibiotic? Would there be an advantage to doing this? Explain. You would see a lawn of bacteria (the plate would be covered). This tells you that the bacteria survived the transformation process. When compared to the antibiotic plates it gives an indication of how many bacteria were transformed

Questions 7. To transform bacteria with plasmids, technicians first make the bacteria competent (capable of taking up DNA) by placing them in calcium chloride and chilling them. Plasmid is then added to the competent bacteria and the plasmid/bacteria combination is taken through a few more steps to make the bacteria take up DNA. In your experiment, should you treat a tube of bacteria that you do not add plasmid to exactly as you do the tube of bacteria that you will transform? Why or why not? This provides a control demonstrating that the antibiotic- resistance colonies that appear on the plate are a result of the plasmid being taken up by the cells. If no plasmid, then there should be no growth on antibiotic plates.

What would you expect? LB AMP plates LB Kan plates LB plates plasmid + plmd - plmd plmd Color when present Amp Kan Amp & GFP

What would you expect? LB AMP plates LB Kan plates LB plates plasmid + plmd - plmd plmd Color when present Amp 25 -100 colonies 0 colonies colonies Lawn yellow Kan 25-100 colonies Yellow Amp & GFP Yellow/green or green

What you need… 2 LB plates 2 LB/Kan plates 2 LB/Amp plates 2 sterile transformation tubes & rack Container with crushed ice 3 sterile inoculating loops 6 sterile transfer pipets Waste container 3 mL vial of LB 3 mL vial of CaCl2 (on ice)

Basic Outline of the Lab Label 1 tube + / 1 tube – Add CaCl2, place on ice Use sterile loop to transfer E. coli to both tubes (no agar!) return to ice Use sterile loop to add loopful of plasmid (1, 2, or 3) to + tube Ice for 15 min & label 1of each plate “+” and 1 of each “-” Heat shock (42C for 90 sec) Add luria broth & sit at room temp 5 to 15 minutes Spread bacteria on plates