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 1 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 extrachromosomal 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 2 3 4 E coli with no plasmid and no ampicillin present in the medium LB E coli with no plasmid and ampicillin present in the medium LB Amp E coli with a plasmid and no ampicillin preset in the medium LB 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 2 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 3 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 250 L 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 plasmid DNA are referred to as competent cells 4 Place both tubes on ice 5 Using one side of a plastic sterile loop transfer a cell mass of E coli cells from the starter plate into the DNA tube by sweeping the cells superficially a Note You only need a small quantity of bacteria Do NOT completely fill the loop Notes Be careful not to transfer any agar from the plate along with the cell mass Immerse the cells on the loop in the calcium chloride solution in the DNA tube and vigorously tap the loop against the wall of the tube to dislodge the cell mass Hold the tube up to the light to observe that the cell mass has fallen off the