BIO 109 General Biology I LaboratoryName: ____________________Transformation Introduction: In this lab, you will perform a procedure known as genetic transformation. A gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. Genetic transformation means change caused by genes, and involves the insertion of a gene into an organism in order to change the organism’s traits. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes disease. You will use a procedure to transform bacteria with a GFP gene that codes for Green Fluorescent Protein. The source of this gene is the bioluminescent jelly fish Aequorea victoria. After transformation, the bacteria will express their newly acquired gene and produce GFP, which causes them to glow green under ultraviolet light. You will learn about moving genes from one organism to another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Bacteria can transfer plasmids back and forth allowing them to share beneficial genes. The pGLO plasmid used in this experiment encodes the gene for GFP and a BLA gene that codes for Beta-lactamase which provides resistance to the antibiotic ampicillin. pGLO also incorporates a gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Procedure: BIO 109 General Biology I LaboratoryName: ___________________________ Transformation Week 1 1. What is Escherichia coli? 2. What is ampicillin and how does it affect Escherichia coli? 3. What is a plasmid? 4. What are the two genes and their resulting proteins on pGLO? 5. What is transformation? 6. What is LB Broth? 7. What is CaCl2 used for? 8. What is arabinose and how is it used in this experiment? 9. What is the difference between a colony and a lawn of bacterial growth? 10. Draw what you expect to happen (in terms of bacterial growth) to each of the following plates, and explain why you expect that to happen for each plate. Plate #1Plate #2Plate #3Plate #4 LB/AmpLB/Amp/araLB/AmpLB +pGLO+pGLO-pGLO-pGLO Transformation Week 2 10. Please draw the results you observed. Plate #1Plate #2Plate #3Plate #4 LB/AmpLB/Amp/araLB/AmpLB +pGLO+pGLO-pGLO-pGLO 11. Were these the results you expected? If you did not see the result you were expecting, explain a possible reason for the difference. 12. In which plate(s) did transformation occur? How do you know? 13. Which of the three non-ara plates could you add arabinose to and expect glowing to occur in about 24 hours? Explain why. 14. Why did the most growth (a lawn) appear on plate (-) LB? 15. Explain why bacteria is growing on plates (+) LB/AMP/ARA and (+)LB/AMP even though there is ampicillin present. 16. Explain why there isn’t any bacteria growing on the (-)LB/AMP plate. 17. Why are there just colonies and not a lawn of growth on plates (+) LB/AMP/ARA and (+)LB/AMP? 18. Why was there so much more growth on plate #4 than on plate #3? 19. I want to genetically transform an entire organism. To accomplish this do you think it is easier to work with an organism composed of many cells, or composed of a single cell? Why?

BIO 109 General Biology I LaboratoryName: Transformation Introduction: In this lab, you will perform a procedure known as genetic transformation. A gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. Genetic transformation means change caused by genes, and involves the insertion of a gene into an organism in order to change the organism’s traits. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes disease. You will use a procedure to transform bacteria with a GFP gene that codes for Green Fluorescent Protein. The source of this gene is the bioluminescent jelly fish Aequorea victoria. After transformation, the bacteria will express their newly acquired gene and produce GFP, which causes them to glow green under ultraviolet light. You will learn about moving genes from one organism to another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Bacteria can transfer plasmids back and forth allowing them to share beneficial genes. The pGLO plasmid used in this experiment encodes the gene for GFP and a BLA gene that codes for Beta-lactamase which provides resistance to the antibiotic ampicillin. pGLO also incorporates a gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Procedure: BIO 109 General Biology I LaboratoryName: _______ Transformation Week 1 1. What is Escherichia coli? 2. What is ampicillin and how does it affect Escherichia coli? 3. What is a plasmid? 4. What are the two genes and their resulting proteins on pGLO? 5. What is transformation? 6. What is LB Broth? 7. What is CaCl2 used for? 8. What is arabinose and how is it used in this experiment? 9. What is the difference between a colony and a lawn of bacterial growth? 10. Draw what you expect to happen (in terms of bacterial growth) to each of the following plates, and explain why you expect that to happen for each plate. Plate #1Plate #2Plate #3Plate #4 LB/AmpLB/Amp/araLB/AmpLB +pGLO+pGLO-pGLO-pGLO Transformation Week 2 10. Please draw the results you observed. Plate #1Plate #2Plate #3Plate #4 LB/AmpLB/Amp/araLB/AmpLB +pGLO+pGLO-pGLO-pGLO 11. Were these the results you expected? If you did not see the result you were expecting, explain a possible reason for the difference. 12. In which plate(s) did transformation occur? How do you know? 13. Which of the three non-ara plates could you add arabinose to and expect glowing to occur in about 24 hours? Explain why. 14. Why did the most growth (a lawn) appear on plate (-) LB? 15. Explain why bacteria is growing on plates (+) LB/AMP/ARA and (+)LB/AMP even though there is ampicillin present. 16. Explain why there isn’t any bacteria growing on the (-)LB/AMP plate. 17. Why are there just colonies and not a lawn of growth on plates (+) LB/AMP/ARA and (+)LB/AMP? 18. Why was there so much more growth on plate #4 than on plate #3? 19. I want to genetically transform an entire organism. To accomplish this do you think it is easier to work with an organism composed of many cells, or composed of a single cell? Why?

Human Anatomy & Physiology (11th Edition)

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ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...

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BIO 109 General Biology I LaboratoryName: ____________________
Transformation

Introduction:

In this lab, you will perform a procedure known as genetic transformation. A gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. Genetic transformation means change caused by genes, and involves the insertion of a gene into an organism in order to change the organism’s traits.

Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes disease.

You will use a procedure to transform bacteria with a GFP gene that codes for Green Fluorescent Protein. The source of this gene is the bioluminescent jelly fish Aequorea victoria. After transformation, the bacteria will express their newly acquired gene and produce GFP, which causes them to glow green under ultraviolet light.

You will learn about moving genes from one organism to another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Bacteria can transfer plasmids back and forth allowing them to share beneficial genes.

The pGLO plasmid used in this experiment encodes the gene for GFP and a BLA gene that codes for Beta-lactamase which provides resistance to the antibiotic ampicillin. pGLO also incorporates a gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates.

Procedure:

BIO 109 General Biology I LaboratoryName: ___________________________

Transformation Week 1

1. What is Escherichia coli?

2. What is ampicillin and how does it affect Escherichia coli?

3. What is a plasmid?

4. What are the two genes and their resulting proteins on pGLO?

5. What is transformation?

6. What is LB Broth?

7. What is CaCl2 used for?

8. What is arabinose and how is it used in this experiment?

9. What is the difference between a colony and a lawn of bacterial growth?

10. Draw what you expect to happen (in terms of bacterial growth) to each of the following plates, and explain why you expect that to happen for each plate.

Plate #1Plate #2Plate #3Plate #4

LB/AmpLB/Amp/araLB/AmpLB

+pGLO+pGLO-pGLO-pGLO

Transformation Week 2

10. Please draw the results you observed.

Plate #1Plate #2Plate #3Plate #4

LB/AmpLB/Amp/araLB/AmpLB

+pGLO+pGLO-pGLO-pGLO

11. Were these the results you expected?

If you did not see the result you were expecting, explain a possible reason for the difference.

12. In which plate(s) did transformation occur? How do you know?

13. Which of the three non-ara plates could you add arabinose to and expect glowing to occur in about 24 hours? Explain why.

14. Why did the most growth (a lawn) appear on plate (-) LB?

15. Explain why bacteria is growing on plates (+) LB/AMP/ARA and (+)LB/AMP even though there is ampicillin present.

16. Explain why there isn’t any bacteria growing on the (-)LB/AMP plate.

17. Why are there just colonies and not a lawn of growth on plates (+) LB/AMP/ARA and (+)LB/AMP?

18. Why was there so much more growth on plate #4 than on plate #3?

19. I want to genetically transform an entire organism. To accomplish this do you think it is easier to work with an organism composed of many cells, or composed of a single cell? Why?

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