1. AP Biology BACTERIAL TRANSFORMATION LAB

2. Lab Procedures, Rationale, and Protocol Please read the pdf file with the full lab description that can be found on the class website, in the AP Biology C Page http://merrillville.schoolwires.net/Page/5435 The full lab protocol is listed by the title “bacterial transformation lab” o An overview of the procedure and protocols begins on page 9 of the pdf file o The step by step procedure begins on page 12 of the pdf file o We will be using traditional procedures, not the Optional Rapid Transformation IT IS EXPECTED THAT ALL STUDENTS FULLY READ AND REVIEW THE PROCEDURES AS DESCRIBED IN THIS PDF FILE. IT IS YOUR RESPONSIBILITY TO UNDERSTAND WHAT YOU WILL BE EXPECTED TO DO AND WHY EACH STEP IS A NECESSARY PART OF THE PROCEDURE. A lab handout will be provided for you, but there is also a word file with the lab handout. The title, not surprisingly, is Transformation Lab Handout

3. Genetic Engineering Basics One of the most common applications of genetic engineering is the transformation of bacteria to create new strains of bacterial cells that will produce some useful protein. The simple nature of bacteria allows the cells to be cultured in large quantities using minimal space and resources. Also the nature of bacterial genetics simplifies the process of transformation and the ability to control the genetic activity of the transformed cells. ◦Bacteria commonly absorb and exchange plasmids, so the use of plasmids as a vector is fairly simple to accomplish ◦Bacterial genes are part of an operon which allows control of the expression of the gene. For example, associating the recombinant gene with the lac operon would allow you to control the expression of the gene by controlling the food supply in the culture. Providing the cells with lactose (and denying the cells a source of glucose) would maximize the activity of the operon and cause the cells to produce the desired protein in large quantities ◦Plasmids commonly contain genes for antibiotic resistance, which provides us with a simple method of isolating the transformed cells and eliminating all cells that were not transformed.

4. Producing Recombinant Bacteria 1.Choose an appropriate vector 2.Prepare the vector for receiving the donor gene 3.Isolate the donor gene 4.Insert the donor gene into the vector 5.Prepare the host cells to receive the vector 6.Introduce the vector & transform the host cells 7.Isolate the transformed cells

5. Donor DNA In our experiment, the donor gene will be a gene isolated from jellyfish. The gene is for fluorescence, so the transformed cells will gain the ability to produce GFP (green fluorescent protein). The cells will glow green when exposed to ultraviolet light

6. Preparing the Vector Both the plasmid and the donor DNA will be cut with the same restriction enzyme. This will leave both donor and vector DNA with complementary “sticky ends” The choice of an appropriate restriction enzyme is vital. A number of factors must be taken into consideration for success ◦The plasmid must have only one restriction site for the enzyme you use, more than one and the plasmid will not be “opened” it will be destroyed ◦The donor gene must be flanked on both ends by restriction sites so that it will “stick” to the plasmid at both ends, completing the circular nature of the plasmid ◦The donor gene must not have any restriction sites within it, or the gene itself will be destroyed Once the donor gene and the plasmid are cut and combined, they must be permanently attached by using DNA ligase (remember that DNA ligase serves the natural function of binding okazaki fragments during replication).

7. Transformation Even though bacteria will accept plasmids naturally, success is not guaranteed. A number of steps must be undertaken to maximize the rate of transformation First, bacteria are not “competent” to receive plasmids throughout their entire life cycle. Well established colonies of bacteria will not be transformed. Timing is of the utmost importance. The colonies will only be competent to receive plasmids if they are in the “logarithmic growth” phase of their life cycle. In other words, they will only be competent if they are in their phase of fastest growth and reproduction (which for our purposes is within 24 hours of the introduction of the bacteria to the culture media) Also, bacteria will have a higher rate of transformation if the colonies are stressed. We will introduce stress in 2 ways, chemical and temperature stress. ◦Chemical stress will be accomplished by adding a quantity of CaCl 2 to the cultures, affecting the permeability of the cell membranes ◦Temperature stress will be accomplished by heat shocking the cultures – moving them rapidly from and ice bath and a warm water bath

8. Isolating the Transformed cells Remember that plasmids generally contain genes that bacterial cells will use for protection against other microorganisms. The plasmid that we will use for a vector will contain a gene for ampicillin resistance. Ampicillin is a common antibiotic. Most students have been given a prescription for ampicillin to get rid of an infection at some point in their lives (UNLESS THEY ARE ALLERGIC TO IT, IN WHICH CASE THEY SHOULD NOT HANDLE ANY OF THE MATERIALS THAT CONTAIN THE AMPICILLIN – A REACTION IS UNLIKELY UNLESS THE AMPICILLIN IS ACTUALLY INGESTED, BUT WEAR THE GLOVES AND THE GOGGLES AND LET YOUR PARTNERS PLATE OUT THOSE CULTURES). Only the bacterial cells that successfully take up a plasmid will have the gene for antibiotic resistance, so all cells that have not been transformed will be killed by the ampicillin. Any cells that remain will have been transformed, and will be isolated in the “amp+” cultures

9. Procedure - Preparing the Culture Plates Each group will need 5 plates. A source plate for the stock culture and 4 plates for the actual experiment. Control Plates ◦will not receive the recombinant plasmids - DNA, - amp(no ampicillin) - DNA, + amp(get ampicillin) Experimental Plates ◦will receive the recombinant plasmids + DNA, - amp(no ampicillin) + DNA, + amp(get ampicillin) The plates need to be poured using melted nutrient agar. We will use sterile, disposable plastic petri dishes. They may have been poured ahead of time and provided for you, or you may be pouring them yourselves. https://www.youtube.com/watch?v=PiWwnBbCrNs Each plate will be labelled on the bottom as described, to show what treatment it will receive The plates that receive the ampicillin will also be “striped” – marked down the side of the lid to indicate that they have had ampicillin added to the agar

10. Procedure Day 1 - Prepare Source Plates Sterile technique is absolutely necessary. Transfer a “bacto-bead” to the petri dish, allow it to melt, and use the sterile inoculating loop to streak the sample. Rotate the dish 45 degrees and streak across the pattern you already made. The streak pattern should be similar to the sample shown on the right The following video gives a decent explanation of the process, but we will be using disposable, sterile plastic inoculating loops instead of re- usable metal loops, so we will NOT be flame sterilizing them. After use they go directly into the bleach bucket. Don’t set them on the table. https://www.youtube.com/watch?v=Ay2hhujTuvg Please note that this culture has had time to incubate. You will not see established colonies when you streak your plate!

11. Preparing Cultures for Transformation Materials: ◦Source plate with competent E. coli colonies ◦Ice Bath ◦Heat Shock bath (42 o C) ◦3 microcentrifuge tubes ◦1 with.50 mL CaCl 2 labelled –DNA ◦1 empty tube labelled + DNA ◦1 with the prepared recombinant plasmids (“pGFP”) ◦Appropriate sterile pipettes ◦4 petri dishes with nutrient agar ◦- DNA – amp ◦- DNA + amp(striped) ◦+ DNA – amp ◦+ DNA + amp(striped) 1.Using a sterile inoculating loop, transfer approximately 8-10 colonies from the source plate to the “- DNA” microtube with.50 mL of CaCl 2. (colonies only, not the agar!!) 2.Completely re-suspend the bacterial colonies into the CaCl 2. Don’t leave any clumps 3.Use a sterile pipette to transfer.25 mL from the “- DNA” microtube into the “+ DNA” microtube. Both tubes should now contain equal quantities of material 4.Use a sterile pipette to add the contents of the pGFP tube to the “+ DNA” microtube only 5.Place both microtubes into the ice bath for 10 minutes

12. Transformation: Chemical and Heat Shock The CaCl 2 in the microtubes serves to chemically shock the cells, increasing their competence to receive plasmids. The next step, heat shock, will further increase their competence. Timing and temperature control are very important for successful transformation. Here’s the routine: 1.Ice bath for 10 minutes 2.Heat shock at 42 o C for 90 seconds 3.Ice bath for 2 minutes 4.Add.25 mL of luria recovery broth to each microtube 5.Recovery in 37 o C bath for 30 minutes 6.Transfer to culture dishes For the heat shock you will need to maintain a water bath at 42 o C. It won’t need to be a large water bath, but you will need to carefully regulate the temperature. Keep a flask of hot water and a beaker of room temperature water handy, and add appropriate quantities of whichever you need to maintain the desired temperature. The microtubes will only be in the heat shock bath for 90 seconds, but it is important to keep the temp at 42 When you return the microtubes to the ice bath, add room temp. water to the bath to drop the temp to 37 o C. Before you put the microtubes in the recovery bath, remember to add the luria recovery broth! The microtubes will recover at 37 o C for 30 minutes before you plate the cultures out

13. Plating the Cell Cultures Materials: Sterile inoculating loops Sterile pipettes 4 petri plates, labelled appropriately 2 microcentrifuge tubes (after 30 minute recovery in 37 o C water bath) - DNA + DNA Bleach bath for used pipettes and inoculating loops All tools contacting the bacterial cultures must be sanitized in the bleach bucket. Don’t set used tools down on the lab table. Not ever. In the bleach bucket. No exceptions. Don’t be the one who infects another student with E. coli. Seriously. Bleach bucket. I’m not kidding. 1.Lay out your petri dishes, make sure that your – DNA plates are separated from your + DNA plates 2.Be careful to transfer the correct cultures to the correct dishes (- DNA to – DNA, and + DNA to + DNA), and make sure to use sterile transfer pipettes and inoculating loops. 3.Use sterile pipettes to transfer.25 mL of your cell cultures to the center of the petri dishes. 4.Use sterile inoculating loops to spread the cells over the entire plate, first outward from the center, then laterally, quarter turn, then laterally again (see pdf p. 13).

14. Upon successful completion... Stack your petri dishes and tape them together Make sure to label your group’s stack of cultures so you can get the correct one’s back after they incubate The dishes will incubate overnight at 37 o C 37 o C, by the way, is your body temperature. E. coli is an intestinal bacterium common to humans, so our optimum temperature is their optimum temperature We’ll observe the results the following day. Before you return to class to observe your results, you will need to complete your lab questions (side 1 of the handout only – just the rationale and the predictions) Here’s a pretty good video from University of Pennsylvania that describes the process. They’ve got better tools than we do, and they’re doing a follow-up activity that we won’t be doing, but it’s basically the same lab https://www.youtube.com/watch?v=iDfEVhePDPM And a link to Bozeman biology as well http://www.bozemanscience.com/ap-bio-lab-6-molecular-biology