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BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 EXPERIMENT 1: CREATING RECOMBINANT DNA MOLECULES DAY ONE: INTRODUCTION TO RECOMBINANT DNA TECHNOLOGY AND THE RIVER SCIENCE PROJECT...

BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 EXPERIMENT 1: CREATING RECOMBINANT DNA MOLECULES DAY ONE: INTRODUCTION TO RECOMBINANT DNA TECHNOLOGY AND THE RIVER SCIENCE PROJECT OBJECTIVES: Today's laboratory will introduce you to some of the basic equipment used in molecular genetics research (micropipettes, microcentrifuges, and vortexers) and address how to create a recombinant DNA molecule. By the end of today’s laboratory you should be able to:  Accurately use a micropipette to measure volumes ranging from 1 uL to 1 mL.  Safely operate a microcentrifuge and vortexer.  Define the term “vector” and list the qualities that make a good vector.  Explain how restriction enzymes are utilized for the creation of recombinant DNA molecules.  Explain how enzymes can be used to ligate a foreign piece of DNA into a vector.  List some of the practical applications for recombinant DNA technology.  Explain why we are “cloning” a portion of a bacterial ribosomal gene from soil samples in areas where urban outflow waters enter the Sacramento River INTRODUCTION: 1. EQUIPMENT Molecular geneticists (and molecular biologists in general) often work with very small volumes, and special equipment and supplies have been created specifically for this purpose. A whole new set of equipment and plastic ware has grown out of molecular biology and biotechnology explorations. These include micropipettes, which deliver down to 1/1000 of a ml (1 μL) and microcentrifuge tubes, which hold a total volume of 0.2- 2.0 mL for reactions and centrifugation. Other commonly used equipment includes vortexers, for rapid mixing of small volumes, and microcentrifuges for rapid separation of small quantities of cells and large molecules and/or precipitates. Below is some information that you may find useful as a reference when pipetting small volumes of liquid with micropipettes. 1. VOLUME CONVERSIONS: 1L (liter) = 103 mL (milliliter or “mil”) = 106 µL (microliter) 2. VOLUME RANGE OF MICROPIPETTES: Micropipette Size Volume range p20 1-20 uL p200 20-200 uL p1000 200-1000 uL 1 | Page BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 EXPERIMENT 1: CREATING RECOMBINANT DNA MOLECULES DAY ONE: INTRODUCTION TO RECOMBINANT DNA TECHNOLOGY AND THE RIVER SCIENCE PROJECT OBJECTIVES: Today's laboratory will introduce you to some of the basic equipment used in molecular genetics research (micropipettes, microcentrifuges, and vortexers) and address how to create a recombinant DNA molecule. By the end of today’s laboratory you should be able to:  Accurately use a micropipette to measure volumes ranging from 1 uL to 1 mL.  Safely operate a microcentrifuge and vortexer.  Define the term “vector” and list the qualities that make a good vector.  Explain how restriction enzymes are utilized for the creation of recombinant DNA molecules.  Explain how enzymes can be used to ligate a foreign piece of DNA into a vector.  List some of the practical applications for recombinant DNA technology.  Explain why we are “cloning” a portion of a bacterial ribosomal gene from soil samples in areas where urban outflow waters enter the Sacramento River INTRODUCTION: 1. EQUIPMENT Molecular geneticists (and molecular biologists in general) often work with very small volumes, and special equipment and supplies have been created specifically for this purpose. A whole new set of equipment and plastic ware has grown out of molecular biology and biotechnology explorations. These include micropipettes, which deliver down to 1/1000 of a ml (1 μL) and microcentrifuge tubes, which hold a total volume of 0.2- 2.0 mL for reactions and centrifugation. Other commonly used equipment includes vortexers, for rapid mixing of small volumes, and microcentrifuges for rapid separation of small quantities of cells and large molecules and/or precipitates. Below is some information that you may find useful as a reference when pipetting small volumes of liquid with micropipettes. 1. VOLUME CONVERSIONS: 1L (liter) = 103 mL (milliliter or “mil”) = 106 µL (microliter) 2. VOLUME RANGE OF MICROPIPETTES: Micropipette Size Volume range p20 1-20 uL p200 20-200 uL p1000 200-1000 uL 1 | Page BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 2. OVERVIEW OF RECOMBINANT DNA TECHNOLOGY Recombinant DNA technology is a set of tools that allows molecular biologists to create novel DNA molecules that do not exist in nature, and to produce large quantities of “recombinant DNA” molecules for research and therapeutic purposes. The basic tools, which include bacterial plasmid “vectors”, restriction enzymes, and ligases, were developed by Stanly Cohen and Herbert Boyer in the early 1970s. Boyer (who is now a multi-millionaire) then went on to become one of the founders of Genentech, the first company to apply recombinant DNA technology, and the company that is credited with starting the biotechnology “boom”. In 1978, Genentech made the first important contribution of recombinant DNA technology to medicine: the company succeeded in cloning the human insulin gene into a bacterial plasmid vector that allowed the human insulin protein to be “grown” or propogated within E. coli. This accomplishment provided an important breakthrough in treating diabetes, since isolating insulin protein from the pancreases of slaughtered cows was laborious and gave rise to a product that had to undergo significant modifications before it could be injected into patients without subsequent immune rejection. In 1982, recombinant human insulin, or humulin, became the first recombinant DNA product approved by the FDA. The basic process by which recombinant DNA molecules are made is quite simple. First, it is important to have a DNA molecule called a vector that can be manipulated in vitro so that a foreign piece of DNA can be inserted into the vector. The recombinant plasmid can then be introduced into a living organism (usually bacteria) to replicate it along with its own DNA and then studied. In some cases, the protein encoded by the foreign DNA within the vector is the desired product (e.g. humulin) and can be isolated from the bacterial host in large quantities by purification processes. However, in our case, the desired outcome is to recover the recombinant plasmid from the bacterial host so the foreign insert can be analyzed, potentially by DNA sequencing the insert to determine the source of the DNA. The foreign DNA we are interested in sequencing is a portion of the 16s ribosomal RNA gene that is common to all bacteria which can be used to determine the taxonomic identity of the organism. Our goal is to determine the diversity of microbes that are influencing the water quality of the Sacramento River which is part of the River Science Project (multi-grant project within the Biological Sciences Department designed to integrate authentic research experiences into the classroom). Microbes are so prevalent and influence much of our environment including our own health. Thus, it is important to understand the role bacteria play in critical issues such as water quality. The Sacramento River is the largest river and watershed system in California, and the water runoff from urban, agricultural and other sources deposited into the river are a considerable source of contamination (e.g. pesticides, methylmercury neurotoxin, antibiotics, and pathogenic bacteria). Microbes can be a source of contaminants but also can be beneficial to water quality such as by detoxifying pollutants. Thus, it is important to understand the diversity of the microbial populations at sites that contribute to the water quality of the Sacramento River since the particular species that are found will be indicators as to the health of the watershed system. Our role in this project is “clone” the 16s rRNA gene fragment that was previously PCR amplified by students in our Biology department as part of their Bio 2 laboratory course. With respect to the context of the word “clone”, it means to insert the PCR fragment into a vector and recover this recombinant plasmid from a colony of bacterial cells that are identical with respect to their DNA since they were derived from a single ancestral cell. The source of the soil sample that was originally used for PCR amplification by Bio 2 students will be communicated to you by your genetics laboratory instructor so that you have context as to the location where the bacteria were found. The goal of the experiment is to isolate recombinant plasmid DNA from different bacterial colonies and subsequently analyze it by enzymatically digesting the DNA using two different restriction enzymes. After performing the restriction digest, the DNA fragments will be separated and analyzed by agarose gel electrophoresis to see if these recombinant 2 | Page BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 plasmids have different restriction fragment length patterns from other clones indicating that these 16s rRNA gene fragments have different DNA sequences. 3. RECOMBINANT DNA METHODOLOGY In our experiment, we will be working with the bacterium Escherichia coli because it is easy to culture and is not usually harmful to humans making it an ideal organism for growing up recombinant DNA molecules. In fact, it is an essential component of our intestinal flora. E. coli cells are rod-shaped, with about 1/500th the volume of a typical eukaryotic cell. Like other bacteria, it has an outermost wall of carbohydrate and protein. The specific composition of some bacterial cell walls allow them to be stained (Gram-positive) or not (Gram-negative) by a combination of compounds formulated by the microbiologist Gram. E. coli is a Gram-negative bacterium which means it has less peptidoglycan content within its outer cell wall than Gram-positive types. Within the cytoplasm, there are no membrane-bound organelles, yet all the basic life machinery is present such as ribosomes for protein synthesis. In addition, there is a single, circular, double-stranded DNA molecule which carries the bacterium's genetic information. Although bacteria have common structures, different species of bacteria differ in what proteins they can produce and what nutrients they can utilize. E. coli is a rather versatile bacterium that can grow at a range of temperatures (optimum is body temperature, 37°C), utilize a variety of sugars as carbon and energy sources, and manufacture most of the other organic molecules it requires if provided with sugar, salts, and water. At first, E. coli was used in studies because it was easy to isolate and not especially infectious or pathogenic (although certain strains of E. coli can cause “food poisoning” and should be handled with caution). Later it was found to be suitable for genetic studies because it is easily grown through asexual cycles in as little as 20 minutes, generally has just one gene per trait, can have many different colonies displayed on a single Petri plate, can participate in gene exchange by conjugation, transformation, and transduction, and serves as host to numerous bacteriophages (bacterial viruses) and plasmids. As previously mentioned, plasmids (as well as viral DNA) make excellent vectors for carrying foreign genes into bacteria, so E. coli was an obvious choice as an experimental organism to Cohen and Boyer. Plasmids are small double-stranded circles of DNA, which are naturally carried by some bacteria in addition to their larger circular genome. In nature, plasmids carry extra information not normally required for survival of the cell, such as genes for antibiotic resistance or toxic proteins that enable the bacterium to better invade its host. In addition, they are self-replicating. Moreover, many bacteria will spontaneously “take up” plasmid DNA from the surrounding environment through a process called transformation. Together, these characteristics of plasmids make them ideal as vectors for recombinant DNA technology. bacterial cell wall and cell membrane transformation E. coli chromosome (4.6 million bp) plasmid (~4,000 bp) Producing a recombinant plasmid for studies involves several steps: 1. A bacterial plasmid is manipulated with enzymes outside the cell in a test tube. First, a double stranded break in the plasmid DNA molecule is made using a restriction enzyme. Next, 3 | Page BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 the gene of interest (e.g. 16s rRNA) is introduced to the test tube, along with an enzyme (typically ligase but other similar enzymes can be used) that seals the DNA molecule back together. The resulting molecule is called a recombinant plasmid. Ligation of foreign DNA sequence into cut plasmid (“paste”) Restriction enzyme cuts plasmid open in plasmid one spot recombinant foreign gene plasmid 2. The recombinant plasmid is introduced into E. coli cells by transformation. Some bacterial cells take up plasmids naturally, but E. coli must be “coaxed” to do so. There are several methods for making the cells “competent” to take up plasmids. In this lab, we will treat the cells with calcium chloride, followed by a brief exposure to elevated temperature (“heat shock”). 3. As the transformed E. coli cells replicate, the plasmid is also replicated and can produce recombinant protein. Thus, a large amount of the desired recombinant DNA and/or expressed protein (e.g. humulin) is produced as the E. coli culture grows (doubling time ∼20min). 4. The cells are lysed open and the plasmid DNA or protein of interest is purified. There are many plasmid vectors that are commercially available. All are derived from plasmids that were originally isolated from bacterial cells in nature. However, commercial plasmids differ from natural plasmids because they have been modified by molecular biologists in clever ways that make them particularly useful for the creation of recombinant DNA molecules. All commercial plasmids have at least three components in common: a. An origin of replication (Ori). This DNA sequence ensures that the plasmid will be recognized by the bacterial replication machinery and replicated along with the bacterial chromosome. The plasmid remains an autonomous unit and generally replicates to very high copy number within a single bacterial cell (1,000 or more copies/cell). b. A selectable marker. Even though the efficiency of transformation can be greatly increased by making them competent (as described above), the efficiency is still low. The best methods yield approximately one transformant per 1,000 cells. Because of this limitation, the plasmid must carry a selectable marker that allows only those cells that actually pick up the plasmid to be selected for against the background of all the cells that did not. The most common selectable markers are antibiotic resistance genes. For example, many commercial cloning plasmids contain the kanr (kanamycin resistance) gene. After the bacterial cells are transformed with the plasmid, the cells are grown on media containing the antibiotic kanamycin. All cells that failed to take up the plasmid (the vast majority of cells) will die. Only those cells that were actually transformed will grow (i.e. are selected) by this method. 4 | Page BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 Cells “take up” plasmid via Cells are plated on transformation LB-agar + kanamycin LB-agar + kanamycin Plasmids Kanamycin resistant colony containing E. coli culture recombinant plasmid c. A cloning site. Restriction enzymes will only cut DNA at specific sites. For example, the restriction enzyme “Eco RI” cuts DNA only at the sequence: 5’-GAATTC-3’. (Note the complementary strand will have the same sequence with the opposite polarity.) A single site where foreign DNA can be specifically ligated into the plasmid is essential since it would be difficult to incorporate the DNA if the enzyme cut at multiple locations. Most plasmids are engineered to have the option to choose from multiple restriction enzyme sites at this location (often termed a cloning cassette) to provide flexibility to the researcher for their experimental strategy. In today’s experiment, we will begin this process by creating a recombinant DNA molecule by performing a ligation using a plasmid that has been previously cut with a restriction enzyme and has been engineered to have a “T” overhang to match the “A” overhang that the PCR enzyme Taq typically leaves at the end of the PCR product when synthesized. We will be using the TA Cloning™ Kit (Thermofisher) which uses the pCR™2.1 Vector and the enzyme ligase for inserting foreign DNA, in our case this is the 16s rRNA PCR product from a American River soil sample. In the upcoming classes, we will perform the subsequent cloning steps (transformation, extraction of plasmid DNA and restriction enzyme digestion of purified plasmid DNA) to see if we successfully cloned the PCR product. http://www.slideshare.net/minhdaovan/cloning-7980218 5 | Page  BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 http://www.slideshare.net/thelawofscience/biotechnology-gene-cloning General scheme for creating a recombinant DNA molecule. The desired foreign DNA molecule is ligated into a cut vector (linearized). The recombinant vector is then used to introduce the foreign DNA molecule into E. coli or another host bacteria by transformation. Transformation is inefficient, so transformed bacteria are selected for by plating on agar plates infused with antibiotic since the plasmid has the particular antibiotic resistance gene. If the transformed cells were plated without the antibiotic infused into the agar, all cells including the non-transformed cells would grow and form a lawn of cells on the agar THINGS TO DO: plates. THINGS TO DO: Part I. Equipment 1. Practice using the micropipettes as directed by your instructor. The steps are listed below for your reference. a. Place a tip of the appropriate size on the end of the micropipette. The p20 and p200 micropipettes use the smaller tips. The p1000 uses the larger (blue) tips. b. Use the dial on the micropipette to set the desired volume. c. Depress the plunger until initial resistance is met. d. Place the tip below the surface of the liquid. 6 | Page BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 e. Slowly release the plunger, making sure that the tip remains below the surface of the liquid. f. Remove the tip from the liquid. The volume of liquid being pipetted should now be inside the tip. (Always check visually to make sure this is the case.) g. Place the tip in the container (e.g. microcentrifuge tube, plate) into which the liquid is to be transferred. h. Depress the plunger until resistance is met and then continue to depress the plunger all the way. All of the liquid in the tip should now leave the tip. (Check visually; sometimes a very small bead of liquid may remain.) i. Dispose of the pipette tip in the biohazard container on the bench top.  Micropipettes should always be used with a disposable tip and held upright, to prevent contamination of the barrel. 2. Prepare two 1.5-ml microcentrifuge tubes into which you will pipette a total of 500 uL of water into each tube (your instructor will provide you with the exact volumes you are to pipette). Cap them tightly and then vortex them for several seconds. Experiment with how the vortexers work. On one setting, you can simply push down on the tube to start the vortexer, while on the other setting the vortexer runs continuously. The third setting is “off.” 3. Place the tubes in one of the microcentrifuges, making sure they are balanced. Label them on the cap; ink on the side of the tube is easily rubbed off. When microcentrifuge tubes are placed in the centrifuge, the hinge attaching the cap should be placed outermost. This will help you find a very small pellet, which should be in line with the hinge, since the region farthest from the rotor axle is the point of greatest centrifugal force. The microcentrifuges have a knob with which to set the duration of centrifugation. Their covers will not open when the rotor is spinning. The grey models have a second knob to control rotor speed. Grey models also have a small central pulsing button, which you can depress and hold down for a few seconds. Practice using the microcentrifuges and familiarize yourself with these various options. You will be using them later. Part II. Creating a Recombinant DNA Molecule (note: all reagents should be found at your lab bench in an ice bucket) Materials: i) Vector tube (labeled as "Lig" on top lid) ii) PCR product tube (labeled "P" on lid top) iii) Salt solution (labeled as "S" on lid top) 1. The “Lig” tube contains 3uL of vector DNA. This vector is extremely heat sensitive and should be kept on ice. Label the top and side of the "Lig" tube with your initials to identify your lab group. 2. Transfer 3 uL of solution from the "P" tube to the "Lig" tube. Transfer 1 uL of solution from the “S” tube to the “Lig” tube. Mix contents in the "Lig" tube by gently pipetting up and down, trying to avoid creating air bubbles or froth. If liquid lines the tube wall or lid, use the “pulse” option on a microcentrifuge (about 5 sec) to collect the solution at the bottom of the tube. Incubate the "Lig" tube in the tube rack provided by your instructor. 7 | Page BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 3. Label the “P” tube with your group’s initials and return this labeled tube to the same rack provided by your instructor. 8 | Page BIO 184 Laboratory Manual California State University, Sacramento Version: 8/7/2023 3. Label the “P” tube with your group’s initials and return this labeled tube to the same rack provided by your instructor. 8 | Page