Transformation & the Polymerase Chain Reaction Biology 61 Lab 5, 2013 Introduction In this weeks lab you will

carry out two techniques that are commonly used in many different types of biological research: transformation and the polymerase chain reaction (known as PCR). The first technique, transformation, describes the process in which bacterial cells can acquire pieces of DNA from their environment. These pieces of DNA can be inserted into the bacterial chromosome, permanently changing the bacterial genome, or they can replicate on their own if the piece of DNA is in the form of a plasmid, a bacterial mini-chromosome that can enter the bacterial cell and replicate independently of the bacterial chromosome. The second technique you will do in this weeks lab is the polymerase chain reaction, a technique that allows you to mimic DNA replication in a tube and make many copies of a particular sequence of DNA that you might want to study. Since transformation takes some time to carry out in the lab, we will start with a discussion of that topic since you will start this protocol first in lab. Next, we will introduce the technique of PCR and describe what we will be using the technique to discover about your genotype (the type of genes you have; or, in other words, what alleles of a particular gene you carry) and how this affects your phenotype (your physical traits). Transformation Many species of bacteria have evolved the ability to acquire pieces of DNA from their environment and use the genes on that DNA fragment, and we call this transformation. Other species, such as the E. coli cells we will work with this week, need to be washed in salts such as CaCl2 to make them competent to do this. You can imagine that DNA might be a good source of energy, but it might also help the bacteria if keeping and using the newly-acquired DNA somehow gives them a selective advantage over other populations of the same bacteria or other species. This is what can happen when a bacteria is transformed by a plasmid, a mini-chromosome (usually a few thousand base pairs in size) that can carry genes on it that direct the synthesis of proteins that might, for example, protect the bacteria from particular antibiotic compounds (keep in mind that natural antibiotics evolved as a mechanism for one microorganism to fight off another one, not as something to aid humans in fighting infections, and we sort of stole a good idea). Here is another neat example of real transformation. We have a lot of bacteria that live in our gut and in Japan one species of human gut bacteria has actually picked up a piece of DNA, containing a gene, from a marine bacterial species that lives on red algae (you know, the stuff that wraps up sushi!). As a result, the gut bacteria can now synthesize a protein that lets them metabolize a polysaccharide from red algae! They couldnt do this before, but now they can! Where did this DNA come from? From people eating red algae and its associated bacteria! Will this help the humans? Maybe it will be useful if their gut bacteria are happier and can give them more energy or vitamins or whatever they pay their human hosts as rent for living in their guts. (See Hehemann et al., 2010 if you want to learn more). For molecular biologists, transformation is a useful tool that we can use to put pieces of DNA inside bacterial cells and turn the bacteria into factories that synthesize the DNA, and the proteins encoded by the DNA, for us. In this weeks lab, you will be working with a strain of (safe) E. coli that has been treated with chemicals so that it will take up DNA that we add to it. We will be using a plasmid (pGLO from Biorad) that contains a gene to express green fluorescent protein or GFP, a protein first purified from a bioluminescent species of jellyfish 1

(Aequorea victoria), which can use cellular energy to power its fluorescence. Because the genetic code is the same for all organisms (mostly), if we transcribe this jellyfish GFP gene in bacteria, the bacteria can translate the gene into a functional protein that will fluoresce green inside the bacteria. Now, transformation is not a terribly common process, and most bacteria, even if treated with chemicals to make them competent to capture DNA, are not very efficient at it. As a result, we would expect very few bacteria (out of millions) to become transformed when we mix the cells with DNA. This is a problem, because we dont want to search through all these bacterial cells to find the ones that have captured our plasmid DNA. To make the identification of transformed cells easier, scientists have engineered plasmids to contain a marker that will allow us to detect the small number of transformed cells in the presence of many millions of untransformed cells. These markers are often genes whose products protect the cells from antibiotics, so we call them antibiotic resistance markers. One such gene, the gene that provides resistance to the antibiotic ampicillin, is found on the pGLO plasmid that you will use in your transformation. So, in order to tell transformed cells apart from the majority of untransformed cells, we will put our cells onto media that contains the antibiotic ampicillin, and this will kill off all the bacteria that have NOT been transformed with our plasmid DNA. One last feature of your plasmid is that the GFP gene is not allowed to be expressed as a protein until we say so! Lets say we want bacteria to make a protein for us, a human protein that we want to study, but the protein actually kills the bacteria if they just make it all the time at high levels (they are not used to having that protein in them, and it hurts them). What do we do? Well, we can control when the bacteria are allowed to express our protein! For example, in our case we do this by transcribing GFP under the control of a specific bacterial promoter, that promotes GFP gene expression only in the presence of a particular sugar (the sugar is arabinose, and we will add it to the bacterial growth media when we want to activate the expression of this gene). You will learn more about promoters and their regulation in lecture (Chapter 18). Procedure 1: Transformation 1) Add 2 L of your pGLO GFP plasmid DNA to 40 L of the competent E. coli cells in the microfuge tube provided. The cells will be in ice and should not be removed from the ice for too long. DO NOT mix by pipetting up and down!! Flick the tubes gently & quickly get them back into the ice. 2) Incubate on ice ~ 15-20 minutes (30 minutes is best). At this stage, the DNA associates with the bacterial cells. 3) Place the tube in a 42C water bath for 45 seconds no more & no less! This heat shock step helps to draw the plasmid DNA inside the cells. 4) Immediately transfer the tube back to the ice. Chill ~2 minutes. 5) Add 400 L of LB growth media (lacking ampicillin) to your tube. (LB is a simple solution of salts, proteins and yeast extract that supports bacterial growth).

6) Cap the tube tightly and place in a 37C shaker incubator for 30-40 minutes (45 minutes to 1 hour would be better, but we dont want you here all day). This gives the bacteria time to recover from your abuse and gives them time to transcribe and translate their new antibiotic-resistance gene, present on the plasmid, into a protein. 7) Spread your cells onto each of 4 labeled LB agar plates. Agar is a polysaccharide derived from algae, and when we mix it with LB, it solidifies into something like hard jello. To do this, start by lighting an alcohol lamp. Working one plate at a time, pipet 100L of your cells onto the center of the first plate. Dip the bacterial spreader into the ethanol and then into the flame of the alcohol lamp. Let the ethanol burn away; then count to10 to let the spreader cool. Before spreading the bacteria on the first of your four plates, further cool the spreader by touching it to the agar at the edge of the plate dont touch it directly to the cells or you will fry them! Now touch the spreader to the cell suspension in the middle of the plate and gently drag it back and forth three times. Rotate the plate 90 and repeat. Next, move on to the second plate start by adding 100L of your cells onto the center of the second plate, and re-sterilizing your spreader before you spread the cells. Repeat with the last two plates.

Plate #1

Plate #2

Plate #3

Plate #4

+ Amp

+Amp/+Ara

no antibiotic

+Kan

8) Incubate overnight at 37C. We will take the plates out of the incubator, and you will analyze
them during next weeks lab.

In the space below indicate with a (+) which plates had bacterial growth after 24 hours. Draw the appearance and coverage of bacterial colonies and explain possible reasons for growth and possible reasons for no growth.

+ Amp Reason for growth/GFP made:

+Amp/+Ara Reason for growth/GFP made:

no antibiotic Reason for growth/GFP made:

+Kan Reason for growth/GFP made:

Reason for no growth/no GFP:

Reason for no growth/no GFP:

Reason for no growth/no GFP:

Reason for no growth/no GFP:

PCR The polymerase chain reaction (PCR) is a technique for replicating DNA in a tube that results in the exponential amplification of a selected region of a DNA molecule. This technique allows us to make billions of copies of any gene or sequence we are interested in working with, and because PCR only requires a miniscule amount of starting material, it can be used to obtain sequences from trace amounts of DNA present in material left at crime scenes, such as hair or bloodstains, or from bones discovered from murder victims or those found at archaeological sites. PCR uses the basic principles of DNA replication that you will discuss in Chapter 16. Enzymes that replicate DNA are called DNA polymerases, and these enzymes all need certain things to carry out their job of building DNA polymers: 1) a template we need a piece of DNA to copy, and it has to be single stranded. 2) some deoxynucleotides (dNTPs) clearly we cant make any new DNA polymers without the nucleotide monomers used to build them. 3) primers you will learn that DNA polymerases are not capable of just copying DNA at random, but need to get started (primed) to initiate DNA synthesis at a particular place. We can select which regions of DNA get copied by giving the polymerase a set of primers that will determine which region of a large genome gets copied 4) A buffer solution that will help keep the enzyme happy at the proper pH, etc. 5) a polymerase we need to add purified DNA polymerase to our PCR reaction. We use a polymerase that is heat stable and doesnt mind being heated to almost boiling. During the PCR, we will heat up the DNA in order to denature it and make it singlestranded, then we cool it a bit to allow the primers to find their proper complementary DNA strands found within the DNA sequence you want to copy and base pair with them (so the polymerase can read the nucleotide sequence to copy it). Then, the polymerase is allowed to create new polymers of daughter DNA strands. We have to repeat this multiple times in order to create enough DNA to eventually analyze, which we will do in next weeks lab. The PTC gene and taste perception The plan for todays lab is to collect your DNA from cheek cells washed from your mouth. We have designed primers that will allow you to amplify DNA from a particular gene, called PTC (or TAS2R38), from your genome. There are two major alleles of this gene found within the human population, and which alleles you possess determine your ability to sense particular bitter tastes (Kim et al., 2003; Wooding et al., 2004). The term phenotype is used to describe the measurable traits of an organism, and an organisms phenotype is determined by its genotype, the collection of particular alleles that the organism contains. The ability to taste specific compounds is dependent on specialized taste cells that you have in your mouth, particularly on your tongue. These cells contain protein receptors in the plasma membrane can respond to certain chemicals in your food! For example, taste cells that help you detect sour flavors respond to levels of protons (H+), which would be found at high levels in acidic substances like lemon juice! We also have special protein receptors that sense salt, sweet, umami (savory), and bitter compounds. (Keep in mind that the compounds really dont taste like anything on their own, its how we perceive these flavors in our brains). Now, you might wonder why these kinds of receptors have evolved, especially receptors for bitter flavors. It turns out that these receptors are important 4

because they help organisms distinguish between compounds that are probably good to eat (sweet, salty & umami) and those that should be avoided because they are commonly associated with compounds that are poisonous or make us sick (sour and bitter). Some people are more sensitive to the taste of bitter compounds than others, and one commonly used compound used in taste studies is phenylthiocarbamide (abbreviated PTC). We can broadly classify some individuals as tasters of PTC and others as nontasters (the phenotype), and whether they can taste the PTC is dependent on what alleles of the PTC taste receptor gene they have (their genotype). The two very common alleles of the PTC taste receptor gene that determine sensitivity to PTC are designated PAV and AVI. People who are homozygous for the AVI allele tend to be nontasters, while those who are homozygous for the PAV allele, or heterozygous, tend to be tasters (Kim et al., 2003; Wooding et al., 2004). We are going to amplify a region of this PTC taste receptor gene in this weeks lab, and in next weeks lab you will carry out an analysis that will let you identify whether or not you are a taster or nontaster, then see which combination of alleles you possess. We will also discuss more about the function of this gene, the population biology of this gene and its significance during next weeks lab. Procedure 2: DNA Template Preparation 1. Rinse your mouth vigorously with the 10mL of 0.9% saline solution provided, and spit the solution back into the cup! It will taste bad. 2. Transfer 1.5 mL of the saline solution into a microfuge tube labeled with your initials (and any other information your TAs ask you for). 3. Spin the tube in a microfuge for 2 minutes at full speed. Place your tubes in the microfuge in a particular orientation so you know where to look for your pellet. You should be able to see a small pellet of cells at the bottom of the tube. 4. Gently remove the saline supernatant from the cells (if a tiny bit of liquid is left behind, that is OK). 5. Resuspend the pellet by vortexing or flicking the tube with your fingers. 6. Add 200 L of the InstaGene resin to your tube. Be sure the resin is resuspended before you pipet it!! 7. Incubate the DNA at 56C for 10 minutes, shaking the tube a couple of times during the incubation. 8. Remove the tube and place it at 100C for 5 minutes. Shake the tube vigorously and pellet the resin by spinning the tube in the microcentrifuge for 5 minutes at 6,000xg. 9. Transfer 20 L of your newly-purified DNA into each of 2 microfuge tubes labeled with your initials and #1 or #2 (and any other information your TAs ask you for). Please dont transfer any of the resin or it will inhibit your PCR reaction.

Procedure 3: PCR 1. Add the following components to microfuge tube #1 containing your template DNA: 10.0 L of 5x concentrated buffer (to provide the proper conditions for enzyme activity) 10.0 L of dNTPs (1 mM) a mix of dATP, dCTP, dGTP and dTTP monomers) 10.0 L of primers (forward & reverse are included at 2 M each) 2. The last step is to add 1.0 L of Taq polymerase enzyme. When you pipet this small of an amount, you will barely depress the plunger on your micropipettor you might not think you are even moving it, but be gentle and you will see that it will work. Your TA will have the enzyme, and you will just touch the very end of the pipet tip into the enzyme to remove this small volume (your TA will check to make sure you have the correct amount). Gently vortex the tube to mix. 3. You will then add 30 L of a PCR MasterMix (with everything already mixed in it) to microfuge tube #2. Gently vortex the tube to mix. 4. Finally, label 2 PCR tubes with your initials & #1 or #2. Transfer 45 L of your PCR reaction to the appropriate tube. Your TAs will place the tubes in the PCR machines and start the repeated polymerization reactions that will lead to amplification of your PTC gene (see page 403-4 in Ch.20 of your text). Questions for Review 1) What would happen if the plasmid lacked any antibiotic resistance marker? How could we tell if a bacteria was transformed with this plasmid? 2) Explain what might happen if you added the LB media to your cells and immediately spread them on the LB agar plates + ampicillin? 3) What would happen to our PCR if we replaced Taq polymerase with a polymerase isolated from humans? 4) Taq polymerase can function at high temperatures, so you decide to just carry out the reactions at high temperature, but as a result the primers never bind to the DNA template. How might this affect your reaction? 5) happen as a result? You forget to add any dNTPs into your reaction mix. What will

References Hehemann, J-H., G. Correc, T. Barbeyron, W. Helbart, M. Czjzek and G. Michel. (2010) Nature 464:908-914. Kim, U-K., E. Jorgenson, H. Coon, M. Leppert, N. Risch and D. Drayna. (2003) Science 299:1221-1225. 6

Wooding, S., U-K. Kim, M. Bamshad, J. Larsen, L. Jorde and D. Drayna. (2004) Am. J. Hum. Genet. 74:637-646.