Laboratory #1: Transformation of Bacteria with DNA

Laboratory #1: Transformation of Bacteria with DNA

Introduction

Without a doubt, the workhorse of biotechnology and recombinant DNA technology is the bacterial cell. Bacteria are easily manipulated in the laboratory and grow to incredible numbers in short amounts of time. Molecular biologists genetically alter the genome of bacteria in order to study how a gene functions.

The genome of bacteria is comprised of a large circular chromosome (also called a genophore) and several smaller, circular pieces of double-stranded DNA called plasmids. Because these plasmids are not part of the chromosome, they are called extra-chromosomal.These plasmids have a variety of functions, including playing roles in conjugation and providing antibiotic resistance. While these plasmids are not part of the chromosome, they are replicated along with the chromosome so that the daughter bacteria that result from binary fission receive the same kinds of plasmids originally found in the “parental” bacteria.

The presence of these plasmids and the fact that they are replicated along with the chromosome provides scientists with a wonderful opportunity to genetically manipulate bacteria. Today, modified plasmids can be obtained commercially and genetically engineered through the insertion of target genes to produce a recombinant plasmid. This recombinant plasmid can be re-introduced back into bacteria. When the bacteria express the genes from their endogenous plasmids, they will also express the gene artificially introduced via the recombinant plasmid. Grow enough bacteria, isolate the engineered recombinant plasmid and you now have a significant amount of DNA that can be used for research.

Introducing DNA into bacteria is a straightforward process known as transformation.

Bacterial transformation is one of the ways that bacteria can acquire genetic variability. In transformation, a naked piece of DNA, such as a plasmid, is taken up by bacteria. In the lab, the DNA used to transform bacteria is frequently a modified plasmid that contains a specific gene of interest.

In bacterial transformation, the DNA must cross the bacterium’s plasma membrane. The ability to take up extracellular/foreign DNA is referred to as competent. Some strains of bacteria are naturally competent. Others must be treated in order to make them competent. There are two ways of doing this –chemically and electrically. Either way requires the preparation of bacteria so that their plasma membrane is weakened. In this way, it is easier to either chemically or electrically induce transformation. Typically, the bacteria are prepared by treating them with ice-cold solutions of salt to weaken their plasma membranes. Once done, the bacteria are referred to a “competent”.

To transform competent bacteria chemically, you simply mix a small volume (i.e. an aliquot) of competent bacteria with the DNA you wish to introduce and “heat shock” the bacteria by suddenly transferring them to a different temperature. This heat shock “opens” small “holes” in the plasma membrane and the DNA diffuses into the cell. If the DNA is a plasmid, then the plasmid will be introduced into the bacteria.

The pGLO Bacterial Transformation Kit

The kit you will be using in this exercise is from Bio-Rad (cat#166-0003EDU). This kit will allow you to transform bacteria with a plasmid called pGLO. Within this plasmid is a gene called GFP – Green Fluorescent Protein. The real-life source for this GFP is the bioluminescent jellyfish Aequoreavictoria. The pGLO plasmid is shown here. A more detailed map of this plasmid is available to you from your professor. The pGLO plasmid has three crucial areas. One area is a gene called bla, which codes for an enzyme (-lactamase) that can break down the antibiotic ampicillin. Expression of this gene will allow for growth in media containing ampicillin. Exposing the transformed bacteria to ampicillin as they are replicating in this media should allow only those bacteria containing the ampicillin resistance gene (from the introduced plasmid) to survive. This is known as antibiotic selection.

The second area is an operon called araC. This operon will become turned on in the presence of the sugar arabinose so that the bacteria can make the enzymes to break arabinose down for energy production. The arabinose operon has a gene that codes for a regulator protein called araC. When arabinose is present in the growth medium, it binds the araC regulator protein. This complex can bind the operator region and allow for the binding of the RNA polymerase at the promoter and operon transcription. In molecular biology, we say that the expression of the genes in the operon are “under the control of the araC promoter”.

The third region is the GFP gene. This gene replaces the genes of the arabinose operon. Therefore, it is under the control of arabinose, which means it will be expressed in the presence of arabinose. Exposure of bacteria expressing the GFP protein results in their ability to glow.

Safety considerations

In this lab, there are a few things to consider. First is safety. You are working with a strain of bacteria known as Escherichia coliHB101 K-12. This K-12 strain is not considered pathogenic. It has been genetically modified to prevent its growth unless grown on an enriched medium. However, you should handle any material contaminated with this E. coli strain using Standard Microbiological Practices. These practices include: decontaminating your work surface after use and after any spills with a disinfectant; washing your hands with soap and water after working with the bacteria and before exiting the laboratory; pipetting only with approved devices (no mouth pipetting); no eating, drinking or smoking in the lab and the wearing of appropriate PPE (Personal protective equipment such as gloves and eyeware). Before you begin this lab, be sure to read the lab’s safety procedures and any other safety documents. These are contained within a binder that is stored in the lab.

In addition to exercising Standard Microbiological Practices, special considerations come into play when working with bacteria. Materials such as pipettes, loops, pipette tips that have come in contact with E.coli are to be disposed of in the appropriate waste containers. Growth plates with E.coli colonies never go in the trash but are disposed of in separate waste containers. Your professor will review these containers before you begin the lab.

Ampicillin is a member of the penicillin family of antibiotics. As such, it may cause allergic reactions or irritation to the eyes, respiratory system and skin. In case of contact with eyes, rinse immediately with plenty of water and inform your professor. Those with extreme allergies to penicillin or any other member of the penicillin family should avoid contact with ampicillin. Inform your professor of this before you begin the lab.

The UV lamps that you will be using are a source of longwave UV light. Longwave UV light is less dangerous than shortwave but proper precautions should be observed. Any UV light can cause damage to the skin and eyes. Do not expose these surfaces to UV light. If provided, wear UV-rated safety glasses.

Sterile technique

With any type of microbiology procedure (i.e. working with bacteria, fungus, yeast), it is important not to contaminate your samples with any other microbiological agent. Because bacteria and yeast are found everywhere – on your fingers, benchtops etc…. Therefore, it is very easy to contaminate a bacterial sample. To prevent contamination, you will use sterile technique.

In sterile technique, you must ensure that the round circle at the end of your inoculating loop, the tip of your pipette, the surface of the bacterial agar plate does not touch anything or it may become contaminating with unwanted bacteria. Your professor will outline proper sterile technique to you before you start the experiment.

In preparation for your experiment, write out a protocol for sterile technique in your lab manual.

Use of the Kit Provided Transfer Pipet

This kit uses plastic transfer pipettes with graduations on them. While you know they are not the most accurate way of transferring a volume of liquid, precision is not important in this procedure. As a result, you can use the provided transfer pipette. You should also familiarize yourself with the use of these transfer pipettes before you start.

The pipette has 100 uL, 250 uL and 1 mL marks on the barrel. These will be used as units of measurement throughout the lab. Be sure you use sterile technique when handing this pipette. If you touch the tip of the pipette to any surface, discard it immediately and acquire a new one. When using a transfer pipette to transfer bacteria to your growth plates, be especially careful not to contaminate your pipette because throwing this away will result in the discarding of your bacteria and the end of your experiment!!!

Experimental Considerations

E.coli plates: Your professor has prepared starter plates of E. coli K-12. These were streaked out onto LB agar plates containing the selection antibiotic ampicillin, 24 hrs before this protocol and grown at 37°C. You will pick individual colonies and use these to transform with the pGLO plasmid. You will pick 2 to 4 large colonies for each transformation. While you would normally only choose one bacterial colony in a typical molecular bio experiment using bacteria, we want to ensure that you have enough bacteria to transform. So you will pick more than one colony. It is important to take individual colonies and not a swab from a dense portion of the plate. This is because you want bacteria that are actively growing in order to improve your transformation results. Bacteria in colonies represent actively growing bacteria, while bacteria in dense areas have ceased to grow.

Heat shock: Increasing the efficiency of DNA uptake by a bacterium can be accomplished by rapidly changing the temperature of its environment. This can destabilize the cell’s membrane and allow for the entrance of DNA. The best way to do this is to transfer between very cold (i.e. ice) to 42°C – a heat shock. The absence of a heat shock will decrease the number of resulting bacterial transformants ten-fold!! Increasing the length of time in the water back can also decrease transformation efficiency (e.g. a 90 sec heat shock will decrease efficiency by 50%).

Spreading transformants: You will transfer and spread 100uL of transformed bacterial suspension. Any more than this will increase the length of time it will take for the solutions to be absorbed by the agar and will not result in a significant increase in bacterial transformants.

LB media and LB agar:LB media is an acronym for Luria and Bertani, the two scientists who came up with its composition. It is a nutrient broth for the growth of bacteria. It can be used as a liquid to grow bacteria or combined with agar and solidified into LB agar plates. It can also be combined with antibiotics to select for the growth of transformed bacteria. A reminder – agar is derived from algae. It melts when heated but solidifies when cooled to room temperature. It functions as a solid support for bacterial growth.

Antibiotic selection: The pGLO plasmid contains the gene for beta-lactamase (i.e. bla). Beta-lactamase is an enzyme that can break down ampicillin, a member of the penicillin family. Its presence is necessary for E.coli strain K-12 to grow in environments containing ampicillin since this bacteria is not naturally resistant to this antibiotic.

Transformation solution: It is thought that the Ca2+ cation in the 50 mMCaCl2 solution (pH 6.1) you use in this kit neutralizes the repulsive negative charges between the phosphate backbone of DNA and the phospholipids of the cell membrane and allows the DNA to enter the bacteria.

Recovery period: The ten minute recovery period following heat shock/transformation is thought to allow for the bacterial membrane to stabilize and for the beta-lactamase gene to begin expression. While this protocol only uses a recovery time of 10 minutes at room temperature, typical transformation protocols use a 1 hour recovery time at 37°C (with gentle shaking).

Sterility: There will be many common stock solutions available at the common work station. USE STERILE TECHNIQUE when obtaining aliquots from these stock solutions so as not to contaminate the work of your classmates.

Required Materials for Lab

Common workstation:

LB nutrient broth

Plastic Inoculation loops

1 ml sterile Transfer pipets

Foam microtube holders

1.5 mLmicrofuge tubes

K-12 E.coli starter plates

Poured agar plates – LB only, LB+amp, LB+amp+ara

Rehydrated pGLO plasmid DNA

Crushed ice

Containers for crushed ice

Permanent markers

Equipment needed

P20 micropipettes

P200 micropipettes

P1000 micropipettes

Pipette tips

32°C incubator

42°C water bath

UV light lamps

Pre-lab activities

Before you start, describe the purpose of this experiment in your lab notebook and the expected end results. Based on what your professor will tell you in the introductory lecture, make predictions about what you expect to find on each of the four LB agar plates at the end of this two-day experiment.

READ THE PROTOCOL. Be sure you understand what each step in this protocol is for.

List the reagents involved in this experiment in your notebook. Describe the purpose for these reagents.

Observe a starter plate of E.coli strain K-12. List all observable traits or characteristics, such as color of colonies, number of colonies, size of colonies.

Transformation protocol: Day 1

1. Obtain two microfuge tubes, close them and label one +pGLOand the other –pGLO. Label them also with the initials of one member of your group. Place them into the foam holder.

1. Obtain four LB agar plates (1 LB only, 2 LB+amp & 1 LB+amp+ara) from the common workstation. Carefully label your four LB nutrient agar plates on the bottom (not the lid) as follows:
2. LB/amp: +pGLO
3. LB/amp/ara: +pGLO
4. LB/amp: -pGLO
5. LB: -pGLO

CAUTION: Be sure to correctly label each plate. Do not mix up these plates or your experiment will not turn out properly!

1. Open the tubes and with a sterile transfer pipette, transfer 250ul of transformation solution (50 mMCaCl2) into each tube. Remember that the transfer pipettes you are using have gradation lines. The first line represents 250 uL.

1. Place the tubes on ice.

1. Use a sterile plastic inoculation loop to pick2 to 4 large E. coli colonies from the starter plate. Choose colonies that are uniformly circular with smooth edges and are well separated from surrounding colonies. Pick up your tube marker +pGLO and immerse the loop in the liquid. Spin the loop in the solution to release the bacteria into the solution. Place the tube back into the rack and discard your inoculation loop.

1. Repeat step 5 with the tube marked –pGLO.

1. Examine the tubes under UV light and record your observations in your notebook

1. Obtain a new inoculation loop and immerse it into the stock solution of pGLO plasmid at the common workstation. There should be a film of solution across the ring when you withdraw it (not unlike a film of soapy water across a ring when you blow soap bubbles). Immerse the wet inoculation loop in the tube marked +pGLO. Close the tube and return it to the ice

OPTION: If micropipettes are provided by your professor, pipette 10uL of pGLO plasmid solution into the tube marked +pGLO.

1. Do NOT add any DNA into the tube marked –pGLO

1. Incubate both tubes on ice for 10 minutes. While you are incubating, clean up your station and make any notes in your lab notebook about the procedure thus far.

Laboratory Technique: When incubating tubes on ice, remove the tubes from the holder and press them halfway down into the ice, so that the tubes make good contact with the ice.

1. After 10 minutes on ice, heat shock your bacterial samples. Using the foam rack as a holder, return your tubes to the foam rack and transfer the rack with your two tubes into the 42C water bath for exactly 50 seconds.

Laboratory Technique: Make sure that your tubes make good contact with the water. Do NOT immerse your tubes completely in the water because you may contaminate them with the unsterile water of the water bath.

1. When the 50 seconds are done, place the tubes back onto the ice and incubate on ice for another 2 minutes.

1. Remove the tubes from the ice and open them. With a sterile transfer pipette, transfer 250 ul of LB broth to both tubes using proper sterile technique.

Laboratory Technique: If you are confident about your technique, you may use the same pipette to transfer LB to both bacterial tubes. If you have contaminated your pipette, discard it and acquire a new sterile on before continuing.

1. Incubate the tubes for 10 minutes at room temperature.

1. Gently flick the tubes with your finger to re-suspend the bacteria. With a sterile transfer pipette (or a P200 micropipette if provided), transfer 100 uL of the bacterial solution to the appropriate LB agar plates. If using a P200 micropipette, be sure to change tips as you pipette the +pGLO and – pGLO solutions.