1. Learning Targets “I Can...” -Explain what it means for a gene to be expressed. -Explain the role of plasmids. -Define bacteria “transformation.” -Insert a plasmid into bacteria to observe the process of transformation as an important biotechnology process. -Define bacteria “transformation.”

2. Bacterial Transformation Using the pGLO gene A Study of Gene Expression

3. Biology and Honors Biology: Start reading through the experiment procedures on your tables so that you are familiar with what we will be doing today.

4. Background and Review A gene is a piece of DNA that contains the instructions for making a particular protein; this protein gives an organism a certain trait A gene is a piece of DNA that contains the instructions for making a particular protein; this protein gives an organism a certain trait Some genes can be inserted into organisms to change their traits; this is called... Some genes can be inserted into organisms to change their traits; this is called... TRANSFORMATION

5. Anatomy of a Bacteria Cell Bacteria contain bits of circular DNA called plasmids Bacteria contain bits of circular DNA called plasmids Bacteria can exchange plasmids freely with one another and with other sources Bacteria can exchange plasmids freely with one another and with other sources

6. Background and Review You will be working with a bacteria called E. coli You will be working with a bacteria called E. coli In order to transform E. coli, you need to grow LOTS of it In order to transform E. coli, you need to grow LOTS of it Today we will be placing E. coli on a petri dish and incubating it at body temperature (37◦C) to grow colonies. Today we will be placing E. coli on a petri dish and incubating it at body temperature (37◦C) to grow colonies. Each 1mm colony consists of millions of bacteria cells originating from a single cell that has divided through mitosis for several generations Each 1mm colony consists of millions of bacteria cells originating from a single cell that has divided through mitosis for several generations

7. What is a colony?

8. How do we get colonies? By Preparing A Streak Plate

9. Purpose of a Streak Plate

10. What is aseptic technique? Be as sterile as possible! *Wipe the surface you are working on before and after the lab *Sterilize your instruments with extreme heat (or use disposable instruments) *Open the petri dish lid slightly; do NOT lay the lid on the table

11. What is aseptic technique?

12. Why aseptic technique?

13. What will you do in lab? Green Fluorescent Protein (GFP) causes the jellyfish species Aequorea victoria to glow in the dark in the presence of UV light. Green Fluorescent Protein (GFP) causes the jellyfish species Aequorea victoria to glow in the dark in the presence of UV light. This gene will be inserted into bacteria, in the form of a plasmid, to cause them to glow as well This gene will be inserted into bacteria, in the form of a plasmid, to cause them to glow as well

14. What is a plasmid? A plasmid is … A plasmid is … a small circular DNA strand in the cytoplasm of a bacterium that can be moved around from one cell to another. a small circular DNA strand in the cytoplasm of a bacterium that can be moved around from one cell to another. Plasmids allow bacteria transformation to occur.

15. What is a plasmid?

16. What will you do in lab? The pGLO plasmid contains two genes: The pGLO plasmid contains two genes: 1. The gene for GFP (glowing gene) 2. The gene to be resistant to ampicillin (an antibiotic) Two pGLO tubes in lab: -pGLO= no pGLO plasmid +pGLO = pGLO plasmid For the GFP to actually glow, the sugar arabinose must be present.

17. LB Petri Dish (-pGLO) LB is a nutrient that allows E. coli to grow LB is a nutrient that allows E. coli to grow

18. LB/amp Petri Dish (-pGLO) LB is a nutrient that allows E. coli to grow LB is a nutrient that allows E. coli to grow Amp stands for ampicillin, an antibiotic Amp stands for ampicillin, an antibiotic What do antibiotics do to bacteria growth? What do antibiotics do to bacteria growth?

19. LB/amp Petri Dish (+pGLO) LB is a nutrient that allows E. coli to grow LB is a nutrient that allows E. coli to grow Contains ampicillin Contains ampicillin Contains plasmid with two genes Contains plasmid with two genes

20. LB/amp/ara Petri Dish (+pGLO) LB is a nutrient that allows E. coli to grow LB is a nutrient that allows E. coli to grow Contains ampicillin, an antibiotic Contains ampicillin, an antibiotic Also contains arabinose, a sugar Also contains arabinose, a sugar Contains plasmid with two genes Contains plasmid with two genes

21. Review Questions 1. On which of the plates would you expect to find bacteria most like the original non- transformed E. coli on your starter plates? Explain your predictions. LB –pGLO LB/amp –pGLO LB/amp +pGLO LB/amp/ara +pGLO

22. Review Questions 2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions. LB –pGLO LB –pGLO LB/amp –pGLO LB/amp +pGLO LB/amp/ara +pGLO

23. Review Questions 3. Which plates should be compared to determine if any genetic transformation has occurred? Why? LB –pGLO LB/amp –pGLO LB/amp +pGLO LB/amp/ara +pGLO

24. Review Questions 4. What is meant by a control plate? What purpose does a control serve?

25. Hypothesis:

26. References https://sarahinbiology.wordpress.com/2012/05/04/pglo-transformation/ https://sarahinbiology.wordpress.com/2012/05/04/pglo-transformation/ https://sarahinbiology.wordpress.com/2012/05/04/pglo-transformation/ http://www.bio-rad.com/en-us/sku/166-0405edu-pglo-plasmid http://www.bio-rad.com/en-us/sku/166-0405edu-pglo-plasmid http://www.bio-rad.com/en-us/sku/166-0405edu-pglo-plasmid https://stonybrook.digication.com/mariam_shareef/Effects_of_Temperatur e_on_Bacteria/published https://stonybrook.digication.com/mariam_shareef/Effects_of_Temperatur e_on_Bacteria/published https://stonybrook.digication.com/mariam_shareef/Effects_of_Temperatur e_on_Bacteria/published https://stonybrook.digication.com/mariam_shareef/Effects_of_Temperatur e_on_Bacteria/published http://lem.ch.unito.it/didattica/infochimica/2008_GFP/immagini/Jellyfish.j pg http://lem.ch.unito.it/didattica/infochimica/2008_GFP/immagini/Jellyfish.j pg http://lem.ch.unito.it/didattica/infochimica/2008_GFP/immagini/Jellyfish.j pg http://lem.ch.unito.it/didattica/infochimica/2008_GFP/immagini/Jellyfish.j pg https://www.pinterest.com/pin/153826143494514820/ https://www.pinterest.com/pin/153826143494514820/ https://www.pinterest.com/pin/153826143494514820/ http://dentlight.com/faqs/doe-work/ http://dentlight.com/faqs/doe-work/ http://dentlight.com/faqs/doe-work/ http://proteopedia.org/wiki/index.php/Image:GFP_mice.png http://proteopedia.org/wiki/index.php/Image:GFP_mice.png http://proteopedia.org/wiki/index.php/Image:GFP_mice.png