Pglo tranformation manual 1660033_edu

Lesson 1 Introduction to Transformation
In this lab you will perform a procedure known as genetic transformation. Remember that a gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. Genetic transformation literallymeans “change caused by genes,” and involves the insertion of a gene into an organismin order to change the organism’s trait. Genetic transformation is used in many areas ofbiotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseasescaused by defective genes are beginning to be treated by gene therapy; that is, by geneticallytransforming a sick person’s cells with healthy copies of the defective gene that causesthe disease.
You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfishAequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow inthe dark. Following the transformation procedure, the bacteria express their newly acquiredjellyfish gene and produce the fluorescent protein, which causes them to glow a brilliantgreen color under ultraviolet light.
In this activity, you will learn about the process of moving genes from one organism to another with the aid of a plasmid. In addition to one large chromosome, bacteria naturallycontain one or more small circular pieces of DNA called plasmids. Plasmid DNA usuallycontains genes for one or more traits that may be beneficial to bacterial survival. In nature,bacteria can transfer plasmids back and forth allowing them to share these beneficialgenes. This natural mechanism allows bacteria to adapt to new environments. The recentoccurrence of bacterial resistance to antibiotics is due to the transmission of plasmids.
Bio-Rad’s unique pGLO plasmid encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin. pGLO also incorporates a special gene regulation system, which canbe used to control expression of the fluorescent protein in transformed cells. The gene for GFPcan be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrientmedium. Selection for cells that have been transformed with pGLO DNA is accomplished bygrowth on ampillicin plates. Transformed cells will appear white (wild-type phenotype) onplates not containing arabinose, and fluorescent green under UV light when arabinose isincluded in the nutrient agar medium. You will be provided with the tools and a protocol for performing genetic transformation. LESSON 1 STUDENT MANU
2. Determine the degree of success in your efforts to genetically alter an organism. Lesson 1 Focus Questions
There are many considerations that need to be thought through in the process of planning a scientific laboratory investigation. Below are a few for you to ponder as you takeon the challenge of doing a genetic transformation. Since scientific laboratory investigations are designed to get information about a question, our first step might be to formulate a question for this investigation.
Consideration 1: Can I Genetically Transform an Organism? Which Organism?
1. To genetically transform an entire organism, you must insert the new gene into every cell in the organism. Which organism is better suited for total genetic transformation—one composed of many cells, or one composed of a single cell? 2. Scientists often want to know if the genetically transformed organism can pass its new traits on to its offspring and future generations. To get this information, which would be abetter candidate for your investigation, an organism in which each new generation develops and reproduces quickly, or one which does this more slowly? 3. Safety is another important consideration in choosing an experimental organism. What traits or characteristics should the organism have (or not have) to be sure it will notharm you or the environment? 4. Based on the above considerations, which would be the best choice for a genetic transformation: a bacterium, earthworm, fish, or mouse? Describe your reasoning. STUDENT MANU LESSON 1
Consideration 2: How Can I Tell if Cells Have Been Genetically Transformed?
Recall that the goal of genetic transformation is to change an organism’s traits, also known as their phenotype. Before any change in the phenotype of an organismcan be detected, a thorough examination of its natural (pre-transformation) phenotypemust be made. Look at the colonies of E. coli on your starter plates. List all observabletraits or characteristics that can be described: The following pre-transformation observations of E. coli might provide baseline data to make reference to when attempting to determine if any genetic transformation hasoccurred.
d) Distribution of the colonies on the plate e) Visible appearance when viewed with ultraviolet (UV) light f) The ability of the cells to live and reproduce in the presence of an antibiotic such as 1. Describe how you could use two LB/agar plates, some E. coli and some ampicillin to determine how E. coli cells are affected by ampicillin. LESSON 1 STUDENT MANU
2. What would you expect your experimental results to indicate about the effect of Consideration 3: The Genes
Genetic transformation involves the insertion of some new DNA into the E. coli cells. In addition to one large chromosome, bacteria often contain one or more smallcircular pieces of DNA called plasmids. Plasmid DNA usually contains genes for morethan one trait. Scientists use a process called genetic engineering to insert genes codingfor new traits into a plasmid. In this case, the pGLO plasmid has been genetically engineered to carry the GFP gene which codes for the green fluorescent protein, GFP,and a gene (bla) that codes for a protein that gives the bacteria resistance to an antibiotic.
The genetically engineered plasmid can then be used to genetically transform bacteriato give them this new trait.
pGLO plasmid DNA
Flagellum
Beta-lactamase
(antibiotic resistance)
Bacterial
chromosomal
Consideration 4: The Act of Transformation
This transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells toexpress their newly acquired genes.
To move the pGLO plasmid DNA through the cell membrane you will:
1. Use a transformation solution containing CaCl (calcium chloride).
2. Carry out a procedure referred to as heat shock.
For transformed cells to grow in the presence of ampicillin you must:
3. Provide them with nutrients and a short incubation period to begin expressing their STUDENT MANU LESSON 1
LESSON 2 STUDENT MANU
Lesson 2 Transformation Laboratory
Workstation (✔) Checklist
Your workstation: Materials and supplies that should be present at your workstation prior
to beginning this lab are listed below.
Student workstation
Material
Quantity
Poured agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara) Container (such as foam cup) full of crushed ice (not cubed ice)1 Common workstation. A list of materials, supplies, and equipment that should be present
at a common location to be accessed by your team is also listed below.
Material
Quantity
(optional, see General Laboratory Skills–Incubation)2–20 µl adjustable volume micropipets Transformation Procedure
1. Label one closed micro test tube +pGLO and another -pGLO. Label both tubes with
your group’s name. Place them in the foam tube rack.
STUDENT MANU LESSON 2
2. Open the tubes and, using a sterile transfer pipet, transfer 250 µl of transformation Transformation Solution
LESSON 2 STUDENT MANU
4. Use a sterile loop to pick up 2–4 large colonies of bacteria from your starter plate.
Select starter colonies that are "fat" (ie: 1–2 mm in diameter). It is important to take
individual colonies (not a swab of bacteria from the dense portion of the plate), since the
bacteria must be actively growing to achieve high transforation efficiency. Choose only
bacterial colonies that are uniformly circular with smooth edges. Pick up the +pGLO tube
and immerse the loop into the transformation solution at the bottom of the tube. Spin the
loop between your index finger and thumb until the entire colony is dispersed in the
transformation solution (with no floating chunks). Place the tube back in the tube rack in
the ice. Using a new sterile loop, repeat for the -pGLO tube.
5. Examine the pGLO DNA solution with the UV lamp. Note your observations. Immerse a new sterile loop into the pGLO plasmid DNA stock tube. Withdraw a loopful. There
should be a film of plasmid solution across the ring. This is similar to seeing a soapy
film across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of
the +pGLO tube. Optionally, pipet 10 µl of pGLO plasmid into the +pGLO tube & mix.
Do not add plasmid DNA to the -pGLO tube. Close both the +pGLO and -pGLO tubes
and return them to the rack on ice.
pGLO Plasmid DNA
6. Incubate the tubes on ice for 10 min. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice.
STUDENT MANU LESSON 2
7. While the tubes are sitting on ice, label your four LB nutrient agar plates on the bottom Label one LB/amp plate:
Label the LB/amp/ara plate:
Label the other LB/amp plate:
Label the LB plate:
LB/amp/ara
8. Heat shock. Using the foam rack as a holder, transfer both the (+) pGLO and
(-) pGLO tubes into the water bath, set at 42oC, for exactly 50 sec. Make sure to
push the tubes all the way down in the rack so the bottom of the tubes stick out and
make contact with the warm water. Double-check the temperature of the water bath
with two thermometers to ensure accuracy.
When the 50 sec are done, place both tubes back on ice. For the best transformationresults, the transfer from the ice (0°C) to 42°C and then back to the ice must be rapid.
Incubate tubes on ice for 2 min.
Water bath
42°C for 50 sec
LESSON 2 STUDENT MANU
9. Remove the rack containing the tubes from the ice and place on the bench top.
Open a tube and, using a new sterile pipet, add 250 µl of LB nutrient broth to thetube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the 10. Gently flick the closed tubes with your finger to mix and resuspend the bacteria. Using a new sterile pipet for each tube, pipet 100 µl of the transformation and control suspensionsonto the appropriate nutrient agar plates. Transformation plates
Control plates
11. Use a new sterile loop for each plate. Spread the suspensions evenly around the
surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loopback and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR.
Uncover one plate at a time and re-cover immediately after spreading the suspension ofcells. This will minimize contamination.
STUDENT MANU LESSON 2
12. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack of plates upside down in the 37°C
incubator until the next day. The plates are inverted to prevent condensation on the
lid which may drip onto the culture and interfere with your results.
LESSON 2 STUDENT MANU
Lesson 2 Review Questions
Name ___________________
Before collecting data and analyzing your results answer the following questions.
1. On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observed? Explain your predictions.
2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions.
3. Which plates should be compared to determine if any genetic transformation has 4. What is meant by a control plate? What purpose does a control serve? Lesson 3 Data Collection and Analysis
A. Data Collection
Observe the results you obtained from the transformation lab under normal room lighting.
Then turn out the lights and hold the ultraviolet light over the plates. Alternatively the protocolcan incorporate digital documentation of the plates with Vernier’s Blue Digital BioImagingSystem (Appendix E).
1. Carefully observe and draw what you see on each of the four plates. Put your drawings in the data table below. Record your data to allow you to compare observations of the
“+ pGLO” cells with your observations for the non-transformed E. coli. Write down the
following observations for each plate.
2. How much bacterial growth do you see on each plate, relatively speaking? 4. How many bacterial colonies are on each plate (count the spots you see). STUDENT MANU LESSON 3
Observations
Transformation
Observations
B. Analysis of Results
The goal of data analysis for this investigation is to determine if genetic transformation 1. Which of the traits that you originally observed for E. coli did not seem to become altered? In the space below list these untransformed traits and how you arrived at thisanalysis for each trait listed.
Original trait
Analysis of observations
LESSON 3 STUDENT MANU
2. Of the E. coli traits you originally noted, which seem now to be significantly different after performing the transformation procedure? List those traits below and describe thechanges that you observed.
New trait
Observed change
3. If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic ampicillin, then what might be inferred about the other genes on the plasmid thatyou used in your transformation procedure? 4. From the results that you obtained, how could you prove that the changes that occurred were due to the procedure that you performed? Lesson 3 Review Questions
Name _____________________
What’s Glowing?
If a fluorescent green color is observed in the E. coli colonies then a new question might well be raised, “What are the two possible sources of fluorescence within thecolonies when exposed to UV light?” 1. Recall what you observed when you shined the UV light onto a sample of original pGLO plasmid DNA and describe your observations.
2. Which of the two possible sources of the fluorescence can now be eliminated? STUDENT MANU LESSON 3
3. What does this observation indicate about the source of the fluorescence? 4. Describe the evidence that indicates whether your attempt at performing a genetic transformation was successful or not successful.
Lesson 3 Review Questions
Name ____________________
The Interaction between Genes and Environment
Look again at your four plates. Do you observe some E. coli growing on the LB plate thatdoes not contain ampicillin or arabinose? LESSON 3 STUDENT MANU
1. From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB plate? Explain your answer.
2. How would you change the bacteria’s environment—the plate they are growing on—to best tell if they are ampicillin resistant? 3. Very often an organism’s traits are caused by a combination of its genes and its environment.
Think about the green color you saw in the genetically transformed bacteria: a. What two factors must be present in the bacteria’s environment for you to see the green color? (Hint: one factor is in the plate and the other factor is in how you lookat the bacteria).
b. What do you think each of the two environmental factors you listed above are doing to cause the genetically transformed bacteria to turn green? What advantage would there be for an organism to be able to turn on or off particulargenes in response to certain conditions? Lesson 4 Extension Activity: Calculate Transformation Efficiency
Your next task in this investigation will be to learn how to determine the extent to which you genetically transformed E. coli cells. This quantitative measurement is referred to asthe transformation efficiency. In many experiments, it is important to genetically transform as many cells as possible. For example, in some types of gene therapy, cells are collected from the patient, transformed inthe laboratory, and then put back into the patient. The more cells that are transformed to pro-duce the needed protein, the more likely that the therapy will work. The transformation efficien-cy is calculated to help scientists determine how well the transformation is working.
You are about to calculate the transformation efficiency, which gives you an indication of how effective you were in getting DNA molecules into bacterial cells. Transformation effi-ciency is a number. It represents the total number of bacterial cells that express the greenprotein, divided by the amount of DNA used in the experiment. (It tells us the total numberof bacterial cells transformed by one microgram of DNA.) The transformation efficiency is calculated using the following formula: Transformation efficiency = Total number of colonies growing on the agar plate Amount of DNA spread on the agar plate (in µg) Therefore, before you can calculate the efficiency of your transformation, you will need (1) The total number of green fluorescent colonies growing on your LB/amp/ara
(2) The total amount of pGLO plasmid DNA in the bacterial cells spread on the
LB/amp/ara plate.
STUDENT MANU LESSON 4
1. Determining the Total Number of Green Fluorescent Cells
Place your LB/amp/ara plate near a UV light. Each colony on the plate can be assumed to
be derived from a single cell. As individual cells reproduce, more and more cells are formed
and develop into what is termed a colony. The most direct way to determine the total number
of bacteria that were transformed with the pGLO plasmid is to count the colonies on
the plate.
2. Determining the Amount of pGLO DNA in the Bacterial Cells Spread on the
LB/amp/ara Plate
We need two pieces of information to find out the amount of pGLO DNA in the bacterialcells spread on the LB/amp/ara plate in this experiment. (a) What was the total amount ofDNA we began the experiment with, and (b) What fraction of the DNA (in the bacteria) actually got spread onto the LB/amp/ara plates. Once you calculate this data, you will multiply the total amount of pGLO DNA used in this
experiment by the fraction of DNA you spread on the LB/amp/ara plate. This will tell you
the amount of pGLO DNA in the bacterial cells that were spread on the LB/amp/ara plate.
a. Determining the Total Amount of pGLO plasmid DNA
The total amount of DNA we began with is equal to the product of the concentra- LESSON 4 STUDENT MANU
(DNA in µg) = (concentration of DNA in µg/µl) x (volume of DNA in µl) In this experiment you used 10 µl of pGLO at concentration of 0.08 µg/µl. This
means that each microliter of solution contained 0.08 µg of pGLO DNA. Calculate
the total amount of DNA used in this experiment.
Total amount of pGLO DNA (µg)
How will you use this piece of information? b. Determining the fraction of pGLO plasmid DNA (in the bacteria) that actually got spread
onto the LB/amp/ara plate: Since not all the DNA you added to the bacterial cells will be trans-
ferred to the agar plate, you need to find out what fraction of the DNA was actually spread onto
the LB/amp/ara plate. To do this, divide the volume of DNA you spread on the LB/amp/ara plate
by the total volume of liquid in the test tube containing the DNA. A formula for this statement is
You spread 100 µl of cells containing DNA from a test tube containing a total volume of 510 µl of solution. Do you remember why there is 510 µl total solution? Look in the laboratoryprocedure and locate all the steps where you added liquid to the reaction tube. Add the volumes.
Use the above formula to calculate the fraction of pGLO plasmid DNA you spread on
How wil you use this piece of information? So, how many micrograms of pGLO DNA did you spread on the LB/amp/ara plates?
To answer this question, you will need to multiply the total amount of pGLO DNA used
in this experiment by the fraction of pGLO DNA you spread on the LB/amp/ara plate.
pGLO DNA spread in µg = Total amount of DNA used in µg x fraction of DNA used STUDENT MANU LESSON 4
Look at all your calculations above. Decide which of the numbers you calculated belong in the table below. Fill in the following table.
Micrograms of pGLO DNAspread on the plates Now use the data in the table to calculate the efficiency of the pGLO transformation Transformation efficiency = Total number of colonies growing on the agar plate Amount of DNA spread on the agar plate (in µg) Analysis
LESSON 4 STUDENT MANU
Transformation efficiency calculations result in very large numbers. Scientists often use a mathematical shorthand referred to as scientific notation. For example, if the calculatedtransformation efficiency is 1,000 bacteria/µg of DNA, they often report this number as: 103 transformants/µg
(103 is another way of saying 10 x 10 x 10 or 1,000) How would scientists report 10,000 transformants/µg in scientific notation? Carrying this idea a little farther, suppose scientists calculated an efficiency of 5,000 bacteria/µg of DNA. This would be reported as: 5 x 103 transformants/µg
How would scientists report 40,000 transformants/µg in scientific notation? One final example: If 2,600 transformants/µg were calculated, then the scientific notation 2.6 x 103 transformants/µg
How would scientists report 960,000 transformants/µg in scientific notation? Report your calculated transformation efficiency in scientific notation.
Use a sentence or two to explain what your calculation of transformation efficiencymeans.
Biotechnologists are in general agreement that the transformation protocol that you have just completed generally has a transformation efficiency of between 8.0 x 102 and 7.0 x 103 transformants per microgram of DNA.
How does your transformation efficiency compare with the above? In the table below, report the transformation efficiency of several of the teams in theclass.
Efficiency
STUDENT MANU LESSON 4
How does your transformation efficiency compare with theirs? Calculate the transformation efficiency of the fol owing experiment using the informationand the results listed below.
DNA plasmid concentration: 0.08 µg/µl
250 µl CaCl transformation solution
10 µl pGLO plasmid solution
250 µl LB broth
100 µl cells spread on agar
227 colonies of transformants
Fill in the following chart and show your calculations to your teacher: If a particular experiment were known to have a transformation efficiency of 3 x 103 LESSON 4 STUDENT MANU
bacteria/µg of DNA, how many transformant colonies would be expected to grow on theLB/amp/ara plate? You can assume that the concentration of DNA and fraction of cellsspread on the LB agar are the same as that of the pGLO laboratory.
Our bodies contain thousands of different proteins which perform many different jobs.
Digestive enzymes are proteins; some of the hormone signals that run through our bodiesand the antibodies protecting us from disease are proteins. The information for assemblinga protein is carried in our DNA. The section of DNA which contains the code for making aprotein is called a gene. There are over 30,000–100,000 genes in the human genome.
Each gene codes for a unique protein: one gene, one protein. The gene that codes for adigestive enzyme in your mouth is different from one that codes for an antibody or the pigment that colors your eyes. Organisms regulate expression of their genes and ultimately the amounts and kinds of proteins present within their cells for a myriad of reasons, including developmental changes,cellular specialization, and adaptation to the environment. Gene regulation not only allows foradaptation to differing conditions, but also prevents wasteful overproduction of unneeded proteins which would put the organism at a competitive disadvantage. The genes involved inthe transport and breakdown (catabolism) of food are good examples of highly regulatedgenes. For example, the sugar arabinose is both a source of energy and a source of carbon.
E. coli bacteria produce three enzymes (proteins) needed to digest arabinose as a foodsource. The genes which code for these enzymes are not expressed when arabinose isabsent, but they are expressed when arabinose is present in their environment. How is thisso? Regulation of the expression of proteins often occurs at the level of transcription from DNA into RNA. This regulation takes place at a very specific location on the DNA template,called a promoter, where RNA polymerase sits down on the DNA and begins transcription of APPENDIX D
the gene. In bacteria, groups of related genes are often clustered together and transcribedinto RNA from one promoter. These clusters of genes controlled by a single promoter arecalled operons. The three genes (araB, araA and araD) that code for three digestive enzymes involved in the breakdown of arabinose are clustered together in what is known as the arabinoseoperon.3 These three proteins are dependent on initiation of transcription from a single promoter, P . Transcription of these three genes requires the simultaneous presence of the DNA template (promoter and operon), RNA polymerase, a DNA binding protein called araCand arabinose. araC binds to the DNA at the binding site for the RNA polymerase (the beginning of the arabinose operon). When arabinose is present in the environment, bacteriatake it up. Once inside, the arabinose interacts directly with araC which is bound to the DNA.
The interaction causes araC to change its shape which in turn promotes (actually helps) thebinding of RNA polymerase and the three genes araB, A and D, are transcribed. Threeenzymes are produced, they break down arabinose, and eventually the arabinose runs out.
In the absence of arabinose the araC returns to its original shape and transcription is shut off.
The DNA code of the pGLO plasmid has been engineered to incorporate aspects of the ) and the araC gene are present. However, the genes which code for arabinose catabolism, araB, A and D, have been replaced by the single gene which codes for GFP. Therefore, in the presence of arabinose, araC protein promotes the binding of RNA polymerase and GFP is produced. Cells fluoresce brilliantgreen as they produce more and more GFP. In the absence of arabinose, araC no longerfacilitates the binding of RNA polymerase and the GFP gene is not transcribed. When GFPis not made, bacteria colonies will appear to have a wild-type (natural) phenotype—of whitecolonies with no fluorescence. This is an excellent example of the central molecular framework of biology in action: arabinose
arabinose
APPENDIX D

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