Bacterial Transformation Lab - pglo Name: Date: Pre-Lab Score: Lab Overview: In this investigation, you will gain an understanding of the techniques of culturing E. coli bacteria and transforming using genetic engineering. Background Escherichia coli (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 over night! a 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. Antibiotic 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 that will transform them into ampicillin resistant bacteria. 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 contains nutrients and moisture for bacterial growth and reproduction. In this lab, you will be using Luria Broth (LB). The bacteria will grow in small piles called colonies since they contain millions of individual bacteria cells. Some of the 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.
Sterile Procedures The techniques of sterile procedure apply to any activity in which you work with bacteria or fungi. Since you are working with 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 wash your hands thoroughly with soap and hot water before leaving the lab. Never throw biohazard materials in the regular trash.
Pre-Lab 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: Overview of Lab Procedure You are going to be setting up four Petri dishes: 1. One plate will have with no s on ampicillin-free LB. This plate will be labeled: pglo/amp R LB. 2. The second plate will have with no s on LB with ampicillin in it. This plate will be labeled: pglo/amp R LB/amp. 3. The third plate will have with ampr s on ampicillin-free LB. This plate will be labeled: pglo/amp R LB/amp. 4. The fourth plate will have with ampr s on LB with ampicillin and arabinose. This plate will be labeled: pglo/amp R LB/amp/ara. pglo/amp R LB pglo/amp R LB/amp pglo/amp R LB/amp pglo/amp R LB/amp/ara
Post-Lab Observations Describe and draw each bacterial plate. Plate Name Pre-Description Post-Description Result -pglo/amp R LB with no s on ampicillin-free. -pglo/amp R LB/amp with no s on with ampicillin. pglo/amp R LB/amp with pglo/amp R s on with ampicillin pglo/amp R LB/amp/ara with pclo/amp R s on with ampicillin plus arabinose
Analysis Questions 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 earthworm, fish, bacteria, mouse, daisy? 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 plate and why. 5. Explain what happened on the - pglo/amp R LB/amp plate and why. 6. Explain what happened on the pglo/amp R LB/amp plate and why. 7. Explain what happened on the pglo/amp R LB/amp/ara plate and why. 8. What two factors need to be present in the bacteria s environment for them 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?