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Transcript
Name ________________________________________________ Date ___________ Hour _______________ pGLO/ampR Bacterial Transformation Lab PURPOSE: To gain an understanding of the techniques of culturing E. coli bacteria and transforming E. coli bacteria using genetic engineering. INTRODUCTION: Escherichia coli: E. coli bacteria are the most common bacteria in the human gut. They helps 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! E. coli has all its required genes found in a single chromosome. Some E. coli cells also contain plasmids—small DNA molecules that carry genes for specialized functions— including resistance to specific drugs. Scientists have learned how to put new genes into a plasmid by cutting open the plasmid with restriction enzymes, inserting a new gene into plasmid, and then placing it into a bacterium. In this lab, you will be using already genetically altered plasmids 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 plasmids. NEW GENE PLASMID Resistance: 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 ampR gene, it will be called an “ –pGLO/ampR cell”, it will not be resistant to ampicillin and it will be killed by the antibiotic. If the bacterium has the ampR gene, it will be called a “ + pGLO/ampR cell”, it will be resistant to ampicillin and will survive to form colonies. Genetic transformation: Genetic transformation is a process where an organism will be forced to take into its genome a plasmid containing a foreign gene. In this lab, you will be adding a plasmid with the foreign gene, ampR, into E. coli bacteria that will transform them into ampicillin resistant bacteria. CHROMOSOME Ampicillin-sensitive E. coli cell PLASMID WITH +pGLO/ampR gene Ampicillin-resistant E. coli cell Competency: To transform bacteria cells, the cells need to be made competent or capable of taking up DNA plasmids. Bacteria will be more likely to take up plasmids if their cell walls are altered to allow the plasmids 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 bacteria: 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. 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 on the next page summarizes the basics of sterile procedure. ALWAYS NEVER Always wash your hands and work surface before beginning. 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 Bunsen burner flame unattended. PRELAB: KNOWLEDGE QUESTIONS 1. Describe E. coli. __________________________________________________________________________ __________________________________________________________________________________________ 2. What is a plasmid and what are humans using them for? __________________________________________ __________________________________________________________________________________________ 3. What genes have been put in the plasmids that we will be using and what is their function? __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ 4. What is meant by “antibiotic resistance”? _______________________________________________________ __________________________________________________________________________________________ 5. What is the difference between an “ +pGLO/ampR cell” and a “ – pGLO/ampR cell”? __________________________________________________________________________________________ 6. What is genetic transformation? ______________________________________________________________ __________________________________________________________________________________________ 7. What is meant by a bacterial cell becoming “competent”? __________________________________________ __________________________________________________________________________________________ 8. How will we make the bacterial cells “competent”? _______________________________________________ __________________________________________________________________________________________ 9. What is “culturing” bacteria? _________________________________________________________________ __________________________________________________________________________________________ 10. What is agar and what kind of agar will we be using in the lab? ____________________________________ __________________________________________________________________________________________ 11. What is a bacterial colony and how will it look? _________________________________________________ __________________________________________________________________________________________ 12. What is ampicillin? _______________________________________________________________________ __________________________________________________________________________________________ 13. What happens in a bacterium if the sugar arabinose is in its environment? __________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ 14. What happens in a bacterium if arabinose is not present? ________________________________________ __________________________________________________________________________________________ 15. If a bacterium does not pick up one of the plasmids, and it is placed on an agar that contains ampicillin, what should happen to the bacterium? _______________________________________________________________ __________________________________________________________________________________________ 16. When do sterile techniques need to be used? __________________________________________________ __________________________________________________________________________________________ PRELAB: 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: LAB OVERVIEW: You are going to be setting up four Petri dishes: One plate will have E. coli bacteria with no plasmids on ampicillin-free LB agar. This plate will be labeled: –pGLO/ampR plasmid + LB agar The second plate will have E. coli bacteria with no plasmids on LB agar with ampicillin in it. This plate will be labeled: –pGLO/ampR plasmid + LB/amp agar The third plate will have E. coli bacteria with ampR plasmids on ampicillin-free LB agar. This plate will be labeled: +pGLO/ampR plasmid + LB/amp agar The fourth plate will have E. coli bacteria with ampR plasmids on LB agar with ampicillin and arabinose. This plate will be labeled: +pGLO/ampR plasmid + LB/amp/ara agar. -pGLO/ampR + LB agar –pGLO/ampR + LB/amp agar +pGLO/ampR + LB/amp agar +pGLO/ampR + LB/amp/ara agar DATA: Describe and draw each bacterial plate. PLATE NAME PRE-DESCRIPTION - pGLO/ampR plasmid + LB agar Plate with E. coli bacteria with no plasmids on ampicillin-free agar. - pGLO/ampR plasmid + LB/amp agar +pGLO/ampR plasmid + LB/amp agar R +pGLO/amp plasmid + LB/amp/ara agar Plate with E. coli bacteria with no plasmids on agar with ampicillin. Plate with E. coli bacteria with +pGLO/ampR plasmids on agar with ampicillin Plate with E. coli bacteria with +pCLO/ampR plasmids on agar with ampicillin plus arabinose POST-DESCRIPTION RESULT 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—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/ampR plasmid + LB agar plate and why. __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ 5. Explain what happened on the - pGLO/ampR plasmid + LB/ampR agar plate and why. __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ 6. Explain what happened on the +pGLO/ampR plasmid + LB/amp agar plate and why. __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ 7. Explain what happened on the +pGLO/ampR plasmid + LB/amp/ara agar 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? ______________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________