pglo/amp R Bacterial Transformation Lab

Transcription

1 pglo/amp R Bacterial Transformation Lab Name: Date: Purpose: To gain an understanding of the techniques of culturing E. coli bacteria and transforming E. coli bacteria using genetic engineering. Introduction: E. coli bacteria are the most common bacteria in the human gut. They help us digest food and create Vitamin K. E. coli has been extensively studied in the laboratory and is an important research organism, mainly because it reproduces very rapidly---a single bacterium can divide and form millions of bacteria overnight! E. coli has all its required genes found in a single chromosome. Some E. coli cells also contain s small DNA molecules that carry genes for specialized functions including resistance to specific drugs. Scientists have learned how to put new genes into a by cutting open the with restriction enzymes, inserting a new gene into, and then placing it into a bacterium. In this lab, you will be using already genetically altered s that contain a new gene for resistance to the antibiotic ampicillin and a second gene called pglo that will enable us to recognize bacteria that have picked up the s. New Gene PLASMID Resistance is the ability of bacteria and other organisms to not be affected or killed by certain drugs or chemicals. If the bacterium does not have the amp R gene, it will be called an pglo/amp R cell, it will not be resistant to ampicillin and it will be killed by the antibiotic. If the bacterium has the amp R gene, it will be called a pglo/amp R cell, it will be resistant to ampicillin and will survive to form colonies. Genetic transformation is a process where an organism will be forced to take into its genome a containing a foreign gene. In this lab, you will be adding a with the foreign gene, amp R, into E. coli bacteria that will transform them into ampicillin resistant bacteria. CHROMOSOME PLASMID WITH gene Ampicillin-sensitive E. coli cell Ampicillin-resistant E. coli cell To transform bacteria cells, the cells need to be made competent or capable of taking up DNA s. Bacteria will be more likely to take up s if their cell walls are altered to allow the s in more easily. The bacteria cells will be made competent by a process that uses calcium chloride and heat shock. Bacteria cells are also more competent if they are in a rapid growth stage, so the timing of the transformation will be critical. Culturing is the process of growing bacteria in Petri dishes on a gelatin-like substance called agar. Agar contains nutrients and moisture for bacterial growth and reproduction. In this lab, you will be using Luria Broth (LB) agar. The bacteria will grow in small piles called colonies since they contain millions of individual bacteria cells. Some of the agar will be laced with the antibiotic ampicillin, to determine if the bacteria are resistant or killed by the antibiotic. Arabinose (ara) is a simple sugar and is a source of energy for bacteria. If arabinose is present in the bacterium s environment, it allows the bacterium to turn on genes, to produce the enzymes, to digest the arabinose. If arabinose is not present, the bacterium does not turn on these genes, and thus doesn t waste energy producing an enzyme that is not needed.

2 Sterile Procedures: The techniques of sterile procedure apply to any activity in which you work with bacteria or fungi. Since you are working with E. coli bacteria in this lab, it is important that you not contaminate your work with any foreign bacteria or expose yourself to potentially hazardous bacteria. The chart below summarizes the basics of sterile procedure. ALWAYS Always wash your hands and work surface before beginning. NEVER Never have food on your work surface. Always keep the lid of the Petri dish on it or over it at all times. Microbes are everywhere! Never lay the lid of the Petri dish or culture tube on the lab bench. Always open all sterile tools carefully. Never touch the end of a tool that touches bacteria. Always keep hair pulled back and use goggles when flame is present. Never throw biohazard materials in the regular trash. Always wash your hands thoroughly with soap and hot water before leaving the lab. Never leave a burner flame Bunsen unattended.

3 PreLab Knowledge Questions: 1. Describe E. coli. 2. What is a and what are humans using them for? 3. What genes have been put in the s that we will be using and what is their function? 4. What is meant by antibiotic resistance? 5. What is the difference between an cell and a pglo/amp R cell? 6. What is genetic transformation? 7. What is meant by a bacterial cell becoming competent? How will we make the bacterial cells competent? 8. What is culturing bacteria? 9. What is agar and what kind of agar will we be using in the lab? 10. What is a bacterial colony and how will it look? 11. What is ampicillin?

4 12. What happens in a bacterium if the sugar arabinose is in its environment? 13. What happens in a bacterium if arabinose is not present? 14. If a bacterium does not pick up one of the s, and it is placed on an agar that contains ampicillin, what should happen to the bacterium? 15. When do sterile techniques need to be used? PreLab Observations: Recall that the goal of genetic transformation is to change an organism s traits (phenotypes). Before any change in a trait can be detected, a thorough examination of its natural phenotype must be made. Look at the colonies of E. coli on your starter plate. List all observable traits that can be described. Number of colonies: Size of largest colony (mm): Size of smallest colony (mm): Color of colonies: Distribution of colonies (locations on plate): Appearance of colonies under ultraviolet light:

5 Lab Overview: You will be setting up four Petri dishes: 1. One plate will have E. coli bacteria with no s on ampicillin-free LB agar. This plate will be labeled: pglo/amp R LB agar 2. The second plate will have E. coli bacteria with no s on LB agar with ampicillin in it. This plate will be labeled: pglo/amp R LB/amp agar 3. The third plate will have E. coli bacteria with amp R s on ampicillin-free LB agar. This plate will be labeled: LB/amp agar 4. The fourth plate will have E. coli bacteria with amp R s on LB agar with ampicillin and arabinose. This plate will be labeled: LB/amp/ara agar. -pglo/amp R LB agar pglo/amp R LB/amp agar LB/amp agar LB/amp/ara agar

6 Data: Describe and draw each bacterial plate. Plate Name Pre-Description Post-Description Result - pglo/amp R LB agar Plate with E. coli bacteria with no s on ampicillin-free agar - pglo/amp R LB/amp agar Plate with E. coli bacteria with no s on agar with ampicillin LB/amp agar Plate with E. coli bacteria with s on agar with ampicillin LB/amp/ara agar Plate with E. coli bacteria with s on agar with ampicillin plus arabinose

7 Conclusions and Interpretations: 1. To genetically alter an entire organism, you must insert the new gene(s) into every cell in the organism. Which organism is better suited for total genetic transformation worm, fish, bacteria, or mouse? Why? 2. Which plate in your experiment was the control? What is the purpose of a control? 3. Of the E. coli traits you originally noted, which now seem to be significantly different after performing the transformation procedure? 4. Explain what happened on the - pglo/amp R LB agar plate and why. 5. Explain what happened on the - pglo/amp R LB/amp R agar plate and why. 6. Explain what happened on the LB/amp agar plate and why. 7. Explain what happened on the LB/amp/ara agar plate and why. 8. What two factors need to be present in the environment for the bacteria to glow green under the UV light? 9. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain environmental conditions?