1 GENERAL BIOLOGY LAB 1 (BSC1010L) Lab #10: Biotechnology The data collected in this lab will be used for lab report #2 ______________________________________________________________________________ OBJECTIVES: • Understand the principles of bacterial transformation and related techniques. • Transform Escherichia coli into an ampicillin resistant strain. ______________________________________________________________________________ NOTES: • E. coli is a potentially dangerous microorganism! o Wear protective equipment and clothing. All rules regarding lab safety and hygiene will be rigorously observed in today’s lab. o Clean your workstation with bleach, followed by ethanol, at the end of Tasks 1 and 2. o Place all used disposable materials in the biohazard containers. o Wash your hands thoroughly with soap and warm water before leaving the lab. • Make sure that ice is present at your table before proceeding with the experiment. • Timing is EXTREMELY important in this experiment. Be as focused as possible and read ahead so that you are aware of what is to happen next and are able to plan accordingly. ______________________________________________________________________________ INTRODUCTION: Biotechnology is the technological application of biological systems or their derivatives to make/modify products or processes for a specific use. Biotechnology plays a vital role in health care to manufacture hormones (i.e. insulin), antibiotics and vaccines, in agriculture to produce disease resistant crops, and in environmental preservation to make biodegradable products. Today, the most commonly used form of biotechnology is genetic engineering. This field involves the direct manipulation of genes (i.e., movement of genes from one organism to another and even from one species to another) to impart a particular characteristic (e.g. pesticide/herbicide resistance, a longer shelf life, and increased nutritional value of agricultural crops) to an organism of interest. Genetic engineering includes techniques such as transformation and cloning. While the latter process creates multiple copies of a desired gene, transformation alters the genetic code of a cell through the uptake, incorporation and expression of a foreign gene provided by a “donor” cell. In order for transformation to be successful, three conditions are required: 1) a host into which the foreign DNA can be inserted, 2) a means of delivering the DNA into the host cells, and 3) a way to identify and select for the transformed cells.2 You will use the bacterium Escherichia coli as the host organism for the current experiment. E. coli are gram-negative bacteria that form the normal flora of the gut. In the digestive system, E. coli benefit their host by producing vitamin K as well as preventing other pathogenic bacteria from establishing residence. However, certain strains of E. coli are pathogenic and result in food poisoning, gastrointestinal and urinary tract infections, neonatal meningitis and pneumonia. E. coli is used extensively in biotechnology because it has only one chromosome composed of 5 million base pairs (less than 0.2% of the human genome), a short reproduction time (cell division every 20 minutes) and a fairly rapid growth rate. Delivery of foreign DNA into the host cell is mediated by a vector. Commonly used vectors in genetic engineering include viruses and plasmids. In today’s experiment you will use a plasmid to transport the gene of interest into the host E. coli cells. In general, a plasmid is a small circular DNA molecule that exists separately from that of the host hence, it is termed extra-chromosomal. Because the chances of a successful transformation are small, an experimental setup that will allow researchers to identify transformed cells is crucial. One way to separate the transformed from non-transformed cells is by “tagging” the plasmid with a selectable marker. This is done by adding a gene to the plasmid that confers some type of selective advantage (e.g. antibiotic resistance) to the host cells. In today’s lab you will use a plasmid containing a gene for ampicillin resistance (pAMP) to transform the E. coli bacterium into an ampicillin resistant strain. Through the acquisition of this gene, E. coli will become resistant to ampicillin (an antibiotic similar to penicillin capable of killing the bacteria) enabling the bacterial cells to grow in its presence. ______________________________________________________________________________ TASK 1 - Transformation of E.coli (Adopted from Carolina Biological Supply Company - Tranformations: A Teacher’s Manual) NOTE: As you proceed with this task, make sure to use sterile techniques, i.e., wear gloves at all times and use sterile equipment where appropriate. In addition, do NOT reuse pipette tips or plastic transfer loops. At the end of this task, you will plate the following combinations on media infused with Luria Broth (LB): 1. E. coli with no plasmid and no ampicillin present in the medium (LB -) 2. E. coli with no plasmid and ampicillin present in the medium (LB/Amp -) 3. E. coli with a plasmid and no ampicillin preset in the medium (LB +) 4. E. coli with a plasmid and ampicillin present in the medium (LB/Amp +) Provide your expectations for each of the 4 aforementioned conditions and explain why you think these results will be observed in Table 1. Note: Remember that under normal circumstances E. coli will not grow in the presence of ampicillin.3 Table 1: Condition Expected result (growth or no growth) Reason for expectation LB - LB/Amp - LB + LB/Amp + The circles below represent each of the four petri dishes listed in Table 1. Based on your predictions, draw what you expect to occur in each.4 Develop a Hypothesis: Based on your expectations (Table 1), state null and alternative hypotheses regarding what you expect to see if the bacteria plated on an ampicillin rich medium were successfully transformed. Procedure: 1. Obtain two sterile Eppendorf tubes. a. Mark one tube “+ DNA” – this tube will receive the plasmid. b. Mark the other “- DNA”. 2. On the side of both tubes, write your group number. 3. Add 250µL of ice-cold calcium chloride (CaCl2) to each tube using a sterile transfer pipette. Addition of CaCl2 to the host cells, followed by heat shock (see step 14), increases the likelihood of plasmid uptake. Cells that are able to take up the