Transformation-Wk2-Efficiency-Calculations-InPerson

0Transformation Efficiency Calculation – Part 2 Please highlight all your answers with a yellow background or use a different color font that is easy to read. LEARNING OBJECTIVES 1. Calculate transformation efficiency from pGLO transformation experiment. 2. Communicate central dogma/gene transformation information to various target audiences. 3. Develop an oral and written presentation of a semester-long experiment of your design. INTRODUCTION In the previous lab, you learned how to transform bacteria with the pGLO gene (using pGLO plasmid), which encodes the green fluorescent protein (GFP) of the bioluminescent jellyfish Aequorea victoria . In this lab, you will begin investigations with the bacteria you genetically transformed with the plasmid, pGLO. You will learn to multiply bacteria (containing the pGLO plasmid) for producing large quantities of GFP. One of the basic tools of modern biotechnology is DNA splicing: cutting DNA and linking it to other DNA molecules. The basic concept behind DNA splicing is to remove a functional DNA fragment-let's say a gene-from one organism and combine it with the DNA of another organism in order to make the protein that gene codes for. The desired result of gene splicing is for the recipient organism to carry out the genetic instructions provided by its newly acquired gene. For example, certain plants can be given the genes for resistance to pests or disease, and in a few cases to date, functional genes can be given to people with nonfunctional or mutated genes, such as in a genetic disease like cystic fibrosis. Genes can be cut out of human, animal, or plant DNA and placed inside bacteria (using plasmids). For example, a healthy human gene for the hormone insulin can be put into bacteria. Under the right conditions, these bacteria can make authentic human insulin. When allowed to multiply in gigantic vats (fermenters) these bacteria can be used to mass produce the human insulin protein. This genetically engineered insulin is purified using protein chromatography and used to treat patients with the genetic disease diabetes, whose insulin genes do not function normally. A common problem in purifying genetically engineered "designer" proteins from transformed bacteria is contamination by endogenous bacterial proteins. Chromatography is a powerful method used in biotechnology industry for separating and purifying proteins of interest from other proteins (e.g., bacterial proteins). Proteins purified by chromatography can be used as medicine to treat human disease, or for household agents such as natural enzymes to make better laundry detergents. The cloning and expressing of the GFP gene, followed by the purification of its protein, is completely analogous to the processes used in the biotechnology industry to produce and purify proteins with commercial value. The pGLO plasmid that contains the GFP gene also contains the gene for beta-lactamase, a protein that provides bacteria with resistance to the antibiotic ampicillin. The beta-lactamase protein is produced and secreted by bacteria that contain the plasmid. The secreted beta-lactamase inactivates ampicillin, allowing only pGLO cells to grow in its presence. The bacteria that were transformed with the pGLO plasmid were plated onto LB/amp and LB/amp/ara agar plates. Expression of the GFP gene is under the regulatory control of the arabinose promoter. Thus, when the bacteria were grown on LB agar containing arabinose (LB/amp/ara), GFP was expressed and the colonies

appeared bright green. Conversely, when the bacteria were grown on LB agar that did not contain arabinose (LB/amp), the gene was turned off and the colonies appeared white. ACTIVITY 1: DATA COLLECTION FROM TRANSFORMATION PLATES Observe the results you obtained from the previous week's transformation lab under normal room lighting. Then turn out the lights and hold the ultraviolet light over the plates. NOTE: To prevent damage to your skin or eyes, avoid exposure to the UV light. Never look directly into the UV lamp. Wear safety glasses for prolonged viewing. 1. Observe carefully and sketch what you see on each of the four plates. Record your data to allow you to compare observations of the Transformed bacterial cells (that incorporated the pGLO Plasmid with those you record for the Non-Transformed E. coli . Write down the following observations for each plate. Under UV light, you may need to use a Sharpie marker to place a dot on the bottom of each agar plate where you see a transformed (glowing) colony in order to count each new colony. Table 1. Observed results. E. coli Transformation with the pGLO Plasmid -Plasmid LB -Plasmid LB/amp +Plasmid LB/amp +Plasmid LB/amp/ara # of colonies 0 0 1 24 Amp resistance? No No Yes Yes White or green under normal light? White White White White White or green under UV light * White White White Green GFP expressed? No No No Yes 2. What two factors must be present in the bacteria's environment for you to see the green color? Plasmid and Arabinose 3. If you picked a colony from the fourth column of the table above (+Plasmid LB/amp) and plated it on a new dish containing LB/amp/ara, what would happen? Would it grow? Would it glow? Justify your answer. It would grow and glow because it would contain the arabinose it needs. 4. If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what might be inferred about the other genes on the plasmid that you used in your transformation procedure? The other genes are still present, they are just not being expressed. 5. From the results you obtained, how would you know these changes were due to the procedure that you performed?

Be sure to include the units as you set up each calculation and in each of your answers below . Plasmid (μg) = (concentration of Plasmid) x (volume of Plasmid μL) 1. In this experiment you used 10 μL of pGLO at a concentration of 0.03 μg/μL. This means that each microliter of solution contained 0.03 µg of pGLO Plasmid. Calculate the Total Amount of Plasmid used in this experiment. Show the calculation: 10 µµL x 0.03 µg Enter that total here: 0.3 µg 2. Since not all the Plasmid that you added to the bacterial cells will be transferred to the agar plate, you need to find out what fraction of the Plasmid was actually spread onto the LB/amp/ara plate. To do this, divide the volume of Plasmid you spread on the LB/amp/ara plate by the total volume of liquid in the test tube containing the Plasmid. A formula for this statement is: Fraction of DNA used = ( Volumespread on LB / amp / ara plate )/( Total sample volume ∈ tube ) You spread 100 μL of cells containing Plasmid from a test tube containing a total volume of 510 μL of solution. (Look in the laboratory procedure and locate all the steps where you added liquid to the reaction tube.) What is in this 510 µL of solution? List each component and corresponding volume : Components: total plasmid = 0.3 µg Use the above formula to calculate the Fraction of Plasmid you spread on the LB/amp/ara plate. Show the calculation: 100/510 = 10/51= Enter that total here: 0.19608 3. Determine how many micrograms of Plasmid you spread on the LB/amp/ara plates. To answer this question, you will need to multiply the Total Amount of Plasmid Used in this experiment by the Fraction of Plasmid you spread on the LB/amp/ara plate. pGLO Plasmid spread ( μg ) = Total amount of Plasmid used (μg) x fraction of Plasmid Show the calculation: 0.3µg x (10/51) = Enter that total here: 0.0588 4. Using these calculations from above:

Transformation efficiency = Total numberof colonies growing onthe + Plasmid LB / amp / ara plate Amount of Plasmid spreadonthe plate Show the calculation: 24/ 0.0588 Enter that total here: 408 Transformation efficiency = ___ 408 _______ transformants / μg Transformation efficiency written in scientific notation: _ 4.08x10 2 _________ transformants / μg Record your data in scientific notation along with the class data in Table 2 and answer the questions at the end of this unit. Table 2. Class results. # of Transformed Colonies on the LB/amp/ara Plate Calculated Transformation Efficiency Group 1 14 2.38x10 2 Group 2 9 1.5306x10 2 Group 3 11 1.87x10 2 Group 4 43 7.31x10 2 Group 5 8 1.36x10 2 Group 6 24 4.08x10 2 DISCUSSION QUESTIONS TRANSFORMATION EFFICIENCY CALCULATION 1. How does your transformation efficiency compare with the above ideal range of efficiency? It is half of what is expected/ideal. 2. How does your transformation efficiency compare with other groups in your class? It is higher than the majority but not the highest. 3. What might account for these differences? ( Hint: Look to the protocol) Using the pipes might have accounted for the differences, some groups might have added too much or not enough of something. Before you leave lab, complete the Transformation lab handout for both “weeks” (Part 1& 2) of Transformation and tum it in to your TA along with the DNA Fingerprinting lab handouts (including the completed Crown Jewel activity) via Moodle.