lab #06

1Copyright © 2013 Quality Science Labs, LLC

Big Idea 3: Genetics and Information Transfer

How can we use genetic engineering techniques to manipulate heritable information?

Please be sure you have read thestudent intro packet before you do this lab.

(If needed, the student intro packet is available at www.qualitysciencelabs.com/AdvancedBioIntro.pdf)

Lab Investigations SummaryPre-lab: Genetic Engineering

Genetic Engineering: Recombinant DNA: A Bacterial Plasmid Transformation Model

Lab Investigation 7.1: Bacterial Transformation Part 1 - Bacterial Transformation of the Jellyfish Gene for Green Fluorescent Protein (GFP)Part 2 - Student Guided Inquiry

Lab Investigation 7.2: Calculating Transformation Efficiency

LAB 7Biotechnology:

Bacterial Transformation

2 Copyright © 2013 Quality Science Labs, LLC

LAB 7 - Biotechnology: Bacterial Transformation

Big Idea 3: Genetics and Information Transfer

How can we use genetic engineering techniques to manipulate heritable information?

BACKGROUND

Introduction to TransformationTransformation occurs when a cell takes up (takes inside) and expresses a new piece

of genetic material — DNA. This new genetic information often provides the organism with a new trait which can be identified after transformation has occurred. Genetic transformation literally means change caused by genes and involves the insertion of one or more gene(s) into an organism in order to change the organism’s traits.

Have you heard of genetically modified foods (GMF)? The classic example is Bt corn. A gene from the bacteria Bacillus thuringiensis has been inserted into the corn gene. When expressed, the Bt gene produces a toxin that kills caterpillars and controls earworm that damage corn. The safety of GMFs is a controversial issue. Are the new toxins in corn safe for human consumption? In agriculture, genetic engineering is genetically transforming genes coded for traits such as frost, pest, or drought resistance into plants.

Another classic example is the application of genetic engineering in high production of insulin for diabetes patients. Because insulin is a protein coded for by a gene, Genentech developed a process in 1978 of inserting the healthy gene for the hormone insulin into a bacterial plasmid, resulting in bacteria growing large volumes of insulin for diabetic patients worldwide. The Pre-lab will model this process. In other areas of medicine, diseases caused by defective genes are beginning to be treated with gene therapy by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes their disease (examples include hemophilia, muscular dystrophy, cystic fibrosis, and leukemia).

Future applications of genetic engineering using transformation into bacteria and viruses include making vaccines, making enzymes, engineering cells for cleaning up oil and chemical spills, production of environmentally friendly fuels, and storing excess carbon dioxide to help slow global climate change. Genetic engineering and human manipulation of DNA raises several ethical, social, and medical issues upon which policy decisions will be made in the near future.

Biotechnology transformation uses plasmids, which are double-stranded, circular DNA molecules found in many strains of bacteria. Plasmids allow us to move DNA from one bacterium to another relatively easily. In Lab Investigation 7.1, you will transform a piece of engineered supercoiled DNA plasmid that has two markers: one selective marker for ampicillin resistance and one color marker that will change the color of the bacteria. Lab Investigation 7.2 will enable you to calculate that transformation efficiency of Lab Investigation 7.1. Transformation typically involves a very small number, compared to the total amount of cells. Usually one in every 10,000 cells transforms external DNA into their Plasmid.


PREPARATION

Materials and Equipment are listed with each lab separately.

Timing and Length of Lab Activities Pre-lab is a paper modeling of recombinant DNA — splicing insulin DNA from

a human genome and inserting it into a bacteria plasmid DNA. Recommended time is one lab period. Lab Investigation 7.1 is the actual transformation lab using a plasmid DNA designed with two markers: a color marker that changes the color of the bacteria and a selective marker, ampicillin resistance, for isolating only the transformed colonies on ampicillin agar. E.coli bacteria is used to take up the DNA plasmid during the transformation process. A total of three lab periods are recommended for preparations, preview, transforming cells and streaking plates, growing bacteria 24–36 hours, and calculating transformation efficiency with Lab Investigation 7.2. Student guided inquiry Lab Investigation 7.3 will require two to three lab periods for planning, prep, conducting, collecting data, and presentation. Data analysis and evaluation of results can be done outside of the lab.

Learning objectives aligned standards and science practices (SP)

•To demonstrate the universality of DNA and its expression (3C1 and SP 1.4)

•To explore the concept of phenotype expression in organisms (3C1 and SP 6.4, 7.2)

•To explore artificial selection and how genetic information can be transferred from one organism to another (3A1 and SP 6.4)

•To investigate how horizontal gene transfer is a mechanism by which genetic variation is increased in microorganisms (1A2 and SP 7.1)

•To explore the relationship between environmental factor and gene expression (3A1 and SP 6.4)

•To investigate the connection between the regulation of gene expression and observed differences between individuals in a population of organisms (1A2 and SP 7.1)

General Safety Precautions Basic sterile technique — Although the strain of E.coli and the DNA plasmid

used in this investigation are not pathogenic or disease-causing, it is important not to introduce other contaminating bacteria, molds, or fungi into the experiment. Since microorganisms are found everywhere, basic sterile technique requires avoiding all contaminating surfaces. Here are some guidelines to follow:


•Wash hands when entering and leaving the lab area.

•Do not eat, drink, apply cosmetics, or use personal electronic devices in the lab area.

•Cover your sneezes.

•When working with inoculation loops, pipets, and agar plates, do not touch the tips or the agar surface or place them directly onto contaminating surfaces.

•Work surfaces should be decontaminated before and after lab procedures with a 10% household bleach solution at least once a day and after any spill of viable material.

•All contaminated liquid or solid wastes are decontaminated before disposal — either in an autoclave or soaking in a 10% bleach solution for 20 minutes.

•Ampicillin may cause allergic reactions or irritation to the eyes, respiratory system, and skin. In case of contact with the eyes, rinse immediately with plenty of water and seek medical advice. Wear suitable protective clothing.

Caution: Anyone with penicillin allergies (ampicillin is a member of the penicillin family of antibiotics) should avoid contact with ampicillin.

Working with E.coli The strain of Escherichia.coli that you are being provided with is not pathogenic.

There are many naturally occurring strains of E.coli that inhabit the lower intestinal tracks of many animals and humans. The strains found in different animals vary genetically. The strain used in this lab is a weakened strain of the normal E.coli of the gut and does not cause disease.

The pathogenic strains contain genes not found in harmless organisms. These genes encode for toxins and proteins that enable the organism to invade cells within the body. Some E.coli have genes for an enterotoxin, which causes the travelers’ diarrhea often called “Montezuma’s revenge.”

Laboratory strains of E.coli used in molecular biology research do NOT contain any of these disease genes and are harmless under normal conditions. However, standard microbiological practices should be followed when working with any microorganism. Every day, hundreds of scientists and their students handle these organisms without consequence.

In these investigations, everything that comes into contact with the E.coli has been pre-sterilized: transformation solution (CaCl2), LB Broth, transfer pipets, inoculating loops, micro centrifuge tubes, and petri dishes. Always open sterile transfer pipets and loops so that you do not touch the tip ends. Open a pipet from the bulb end and open a loop from its pointed end. DO NOT REUSE pipets or transfer loops.


Pre-lab: Genetic Engineering:

Genetic Engineering: Recombinant DNA: A Bacterial Plasmid Transformation Model

This lesson introduces the process of using recombinant DNA by using paper models to represent how a human gene such as the gene for insulin can be inserted into a bacterial plasmid. It is a great introduction to recombinant DNA technology, especially before doing a real transformation in Lab Investigation 7.1. There are two themes to this activity: understanding the techniques used in recombinant DNA technology and understanding how bacteria are used to produce insulin for diabetics.

Recombinant DNA refers to DNA of one organism inserted into the DNA of another. Transformation refers to the process of creating recombinant DNA. The major tools of recombinant DNA technology are bacterial enzymes called restriction enzymes. Each enzyme recognizes a short, specific nucleotide sequence in DNA molecules, and cuts the backbones of the molecules at that sequence. The result is a set of double-stranded DNA fragments with single-stranded ends, called “sticky ends.” Sticky ends are not really sticky; however, the bases on the single stranded ends do easily form base pairs with the complementary bases on other DNA molecules. Thus, the sticky ends of DNA fragments can be used to join DNA pieces originating from different sources.

In order to be useful, the recombinant DNA molecules have to be made to replicate and function genetically within a cell. One method for doing this is to use plasmid DNA from bacteria. Plasmids are relatively small circular pieces of double-stranded DNA found in bacteria. Small DNA fragments can be inserted into the plasmids, which are then introduced into bacterial cells. As the bacteria reproduce, so do the recombinant plasmids. The result is a bacterial colony in which the foreign gene has been cloned.

In 1978 Genentech in South San Francisco used this process to create recombinant insulin known as rInsulin, or Humulin®. Insulin is a hormone produced by the pancreas

TA

TA

GC

CG

CG G G C

C C G G

C

GC

CG

TA

ATT

ATA

GC

CG

CG

CG

CG

GC

GC

GC

CG

TA

AT

Cutting DNA StrandsEnzyme Recognition Site

Sticky Ends

Creating Recombinant Plasmids

Recombinant Plasmid

Cut Plasmid

Sticky Ends

Plasmid

EnzymeRecognition

Site

Foreign DNA


used to digest sugars. Diabetes occurs when the body does not produce enough, or properly responds to, insulin. Because insulin is a protein coded for by a gene, Genentech developed a process of inserting the gene for insulin into a bacterial plasmid, facilitation the production of insulin for diabetic patients worldwide. This discovery made Genentech one of the first biotech companies in the world, and one of the biggest and most influential even to this day.

Materials: •Scissors*•Tape* •Handouts:

•Blue colored Plasmid Sequence Strips•Pink colored Human DNA Base Sequence Strips•Yellow colored Restriction Enzyme Sequence Cards

*item not included

Procedures:

1. Review the following procedures and create a flow chart of the steps you will take in this experiment before starting.

2. Cut out the Plasmid (blue) strips. The shaded area on the strip is the replication site. Tape the strips end to end to form a ring of paper. You have now formed a model of a plasmid, which is a ring of DNA. The blue strips can be in any order, but it needs to be a circle. This is the bacterial plasmid.

3. Cut out the Cell DNA (pink) strips. The pink strips need to be in order (match the numbers) and should be one long strip. They must be taped together in the order indicated at the bottom. That is, strip 2 is taped to the bottom of strip 1; strip 3 is attached to the bottom of strip 2, etc. This is the human DNA with the code for making insulin.

4. Create a Plasmid Map (see Pre-lab Table 1). Copy down the full plasmid sequence, and map the relative locations of the genes for antibiotic resistance that your plasmid contains highlight and label all antibiotic resistant genes. See the figures to the left for the antibiotic resistant genes.

AGCCGTA

TCGGCAT

Ampicillan Resistance gene sequence

CCCAGAG

GGGTCTC

Kanamycin Resistance gene sequence

CCTAGGA

GGATCCT

Tetracycline Resistance gene sequence


Pre-lab Table 1: PLASMID MAP Write out the sequence of your plasmid. Highlight the locations of your

antibiotic resistant genes and replication gene; and label them. Be sure that your base pairs line up.

5. You are now ready to begin testing the various restriction enzymes that you have in your “lab.” The yellow squares are the restriction enzymes. They will need to match the bases of the restriction enzymes with the same base sequences on the blue and pink strips. Whenever they find a matching sequence they need to draw the cut points on the strips and label them.Cut out the enzymes (yellow sheet). Find a restriction enzyme that will cut open your plasmid at ONE site only. The same enzyme should be able to cut your cell DNA at TWO sites, ONCE on EACH side of the protein gene (we will imagine that this is the gene for the protein insulin). Try to cut the cell DNA as close to the insulin gene as possible.You have eight enzymes to choose from. Some of the enzymes cannot cut open your plasmid, some can. Some of the enzymes cannot cut your cell DNA at two sites. You cannot use these enzymes. For example: Take an enzyme, Ava II, and check the plasmid for a location(s) which can be cut by this enzyme. Use a pencil to mark on the plasmid where the enzyme will cut. Write the name of the enzyme that would make the cut next to your mark. See how many times the enzyme will cut your plasmid. REMEMBER, YOU WANT TO FIND AN ENZYME THAT WILL ONLY MAKE ONE CUT IN YOUR PLASMID. Continue this procedure until all eight enzymes have been tried.

6. Check the eight enzymes for possible cut sites on the cell DNA. REMEMBER, THE GOAL IS TO FIND AN ENZYME THAT WILL MAKE CUTS CLOSE TO THE INSULIN GENE, ONE ON EITHER SIDE. Use a pencil to mark on your DNA where the enzymes will cut; write the name of the enzyme next to each mark.

7. After testing all eight enzymes, decide which ONE enzyme you will use to cut the plasmid and cell DNA. Explain in Pre-lab Table 2 why you will use this enzyme, and not the other enzymes. Use a pair of scissors to make the cut in your plasmid and cell DNA in the staggered fashion made by the actual enzyme.

5' 3'3' 5'

5' 3'3' 5'


8. Use tape to splice your insulin gene into the plasmid. In a real experiment you would add the enzyme Ligase, the repair enzyme. Ligase “glues” the sticky ends together, so in this case as you tape the plasmid you are acting like Ligase. You have now created a recombinant plasmid.

9. Write out the plasmid map with the inserted inulin gene in Pre-lab Table 3. Highlight the newly added gene.

Pre-lab Table 3: RECOMBINANT PLASMID MAP

Pre-lab Table 2 RESTRICTION ENZYMES

Restriction Enzyme Used Not

used Reason why or why not chosen

1. Ava II

2. Hind III

3. Bam HI

4. Bgl II

5. Hpa II

6. Eco RI

7. Sac I

8. Xma I

9. LIGASE

5' 3'3' 5'

5' 3'3' 5'

5' 3'3' 5'

5' 3'3' 5'

5' 3'3' 5'


In an actual lab, you would now mix your recombinant plasmid with host bacteria and grow the bacteria on petri dishes. Some of the host bacteria would take in your recombinant plasmids, multiply, and begin producing human insulin. The product Humulin® is made this way.

After mixing your recombinant plasmid with host bacteria, you must test to see if the plasmid has been taken into the host cells. Antibiotics such as tetracycline, kanamycin, and ampicillin normally kill the host bacteria. Any host bacteria that survive and reproduce in a petri dish containing one or more of those antibiotics must have taken up the plasmid containing genes for antibiotic resistance. These host bacteria reproduce and should begin to produce the gene product, insulin in this case.

Analysis Questions: Thinking Like a Biotechnician

1. Which antibiotics could you use in your petri dishes to see if bacteria have taken in your plasmid? Why?

2. Which antibiotics would you not use? Why?

3. What would happen if you used an enzyme that cut the plasmid in two places?

4. How do you think this process is important in our everyday life?

5. How else could this process be used?


LAB Investigation 7.1: Bacterial TransformationPart 1 - Transformation Lab

Bacterial Transformation of the Jellyfish Gene for Green Fluorescent Protein (GFP)

BACKGROUNDGFP

In 1994 GFP was cloned. Now GFP is found in laboratories all over the world where it is used in every conceivable plant and animal. Flatworms, algae, E.Coli bacteria, frogs, and pigs have all been made to fluoresce with GFP.

The importance of GFP was recognized in 2008 when the Nobel Committee awarded Osamu Shimomura, Marty Chalfie and Roger Tsien the Chemistry Nobel Prize “for the discovery and development of the green fluorescent protein, GFP.”

Why is it so popular? GFP is the “molecular microscope” of the twenty-first century. Using GFP we can see when proteins are made, and where they can go. This is done by joining the GFP gene to the gene of the protein of interest so that when the protein is made it will have GFP hanging off it. Since GFP fluoresces, one can shine light at the cell and wait for the distinctive green fluorescence associated with GFP to appear.

This is an amazing new area of biotechnology that might help fight cancer, create new products, improve agriculture, and even combat terrorism. Imagine a glowing gene that lights up in the presence of anthrax spores and other chemical warfare agents. Imagine a gene that causes crops to glow, indicating they need more water.

Artificial TransformationThe techniques required for gene transfer in higher plants and animals are

complex, costly, and difficult even in the research laboratory. However, the techniques of gene transfer in E.coli bacteria are simple and appropriate for the learning lab. One common technology, bacterial plasmid-based genetic transformation that you practiced in the Pre-lab, allows manipulation of genetic information to understand more fully how DNA operates.

In the engineered plasmid DNA, there are two main characteristics that have been designed into the DNA. There is coding in the DNA for a selective survival advantage (resistance to an antibiotic called ampicillin) and there is a special color marker gene (from a bioluminescent jellyfish) that fluoresces green under UV light. The strain of E.coli has no antibiotic resistance to ampicillin.

Transformation is not naturally or spontaneously accomplished in E.coli bacteria. For bacterial transformation to occur, bacterial cells must be in a physiological state to accept the external DNA. This is referred to as competency, where the bacterial cell wall is rendered permeable to large DNA molecules. This is accomplished by treating the cells with chloride salts and metal ions of calcium like calcium chloride


(CaCl2). Additionally, heat and cold shocking can also help induce competency. Once cells are in a competent state, they allow DNA molecules to pass through their cell walls and cell membranes, but are very fragile and must be treated carefully.

Since plasmid is used that contains genes with an enzyme that destroys ampicillin (β-lactamase); transformed cells are selected by growing them on agar containing the antibiotic ampicillin. Only transformed cells will grow in the presence of ampicillin. (Agar without ampicillin will grow both transformed and non-transformed bacteria, but usually the cells that do not transform have population growth rates that overcome and mask the transformed cells).

Sometimes, satellite colonies appear around the green fluorescent transformed colony that do not fluoresce and are not transformed. Why do you think they are able to grow in the presence of ampicillin? The enzyme that destroys ampicillin is from the transformed bacteria. This allows limited growth of cells that have not been transformed and are normally inhibited by the ampicillin.

There is also a small DNA sequence that codes for a protein (IPTG) called a promoter that you will add to the agar. It will facilitate the “turning on” process of the cloned GFP gene in the engineered recombinant plasmid. The strain of E.coli you are using has been genetically engineered to contain an enzyme RNA polymerase, which is under the control of the promoter protein and can be turned on (or induced) in the presence of IPTG (isopropyl-beta-D-thiogalactopyranoside), a small molecule that binds and inhibits the lac repressor protein, thus allowing expression of the GFP protein. Depending on the engineered E.Coli strain, arabinose may be used as a promoter.

This lab is amazing because it clearly shows that creature traits deriving from genes are like an artistic mosaic picture. When a functioning gene is properly inserted the “mosaic picture” changes.

Materials: •Procedure Booklet•Bacterial transformation kit•Thermometer•Stopwatch

Supplies NOT in the kit:• Incubator oven at 37 °C (optional)• Ice water bath (crushed ice)•Warm water baths (Styrofoam cups) at 42 °C and 37 °C•Distilled water (if making agar from a powder)•Microwave oven or heat source for hot water•Marking pens (red and green)•Masking tape


Procedures:

Refer to instructions in the lab kit.

Note: Part 1 and Part 2 will be conducted simultaneously or back-to-back. Review Part 2 and select your inquiry driving question and plan appropriately as you make preparations.


Data Collection and Analysis: Experimental Design of Controls, Predictions, and Results

Observe and record your results from each plate – on the next two pages draw and describe what you observe. For each of the plates, record the following:

•Count the number of colonies or label “lawn” if massive growth is observed and individual colonies cannot be identified (take photos if possible);

•The color of the colonies;•Compare transformation numbers with other classmates,

if possible, and explain the differences, if any;• If you did not get good results, what variables could be

responsible?


Control #1: E.coli in the presence of the DNA plasmid is cultured on LB Agar with no ampicillin antibiotic.

Is this a positive or negative control?

What do you expect E.coli to do on this plate?

Actual Results:

Control #2: E.coli goes through the transformation procedures in the absence of the plasmid and is cultured on the LB agar plate.



Actual Results:

LB+ DNA Plasmid

- Ampicillin

Control #1

LB- DNA Plasmid

- Ampicillin

Control #2


Control #3: E.coli goes through transformation procedures in the absence of the plasmid and is plated on LB/Amp agar.



Actual Results:

Experimental Plate: LB/Amp agar with E.coli transformed with plasmid.What do you expect E.coli to do on this plate?

Actual Results:

LB- DNA Plasmid

+ Ampicillin

Control #3

LB+ DNA Plasmid

+ Ampicillin

Control #4


Discussion Questions 1. Compare and contrast the number of colonies on each of the

following pairs of plates. What does each pair of results tell you about the experiment? •LB + Plasmid and LB –Plasmid

•LB/Amp –Plasmid and LB –Plasmid

•LB/Amp +Plasmid and LB/Amp –Plasmid

•LB/Amp +Plasmid and LB +Plasmid

2. What are you selecting for in this experiment?

3. What does the phenotype of the transformed colonies tell you?

4. What one plate would you first inspect to conclude that the transformation occurred successfully? Why?

5. What is your transformation efficiency? To answer this question proceed to Lab Investigation 7.2 to determine your transformation efficiency.


Part 2 - Student Guided Inquiry

Here are some questions to consider in developing a transformation investigation:

•Did you notice any satellite colonies growing on the LB/Amp +Plasmid plate? Satellites are smaller colonies that grow around the larger transformed colony. What observations can you make about the satellites? Do they look like transformed bacteria? How can you tell if they contain the plasmid? If they do not, what allows them to grow in the presence of ampicillin? Is ampicillin still in the agar surrounding the transformed bacteria where the satellites are growing? Design and conduct an experiment to determine if the E.coli satellite colonies from your genetic transformation experiment in 7.1 Part 1 are transformed too.

•What about mutants? What are the effects of mutations on gene expression? Do the mutations affect the plasmid? Some postulated mutagens that you can handle safely include: dilute hydrogen peroxide (H2O2); caffeine; UV light source (the bacteria must be kept in the dark to prevent DNA repair and you must wear UV goggles); potassium nitrate (a food preservative). Design and conduct an experiment to determine the results of mutagens on the transformed and/or non-transformed colonies from 7.1 Part 1.

•Design and conduct an experiment to determine if having this plasmid gave the transformed bacteria in the colonies from 7.1 Part 1 an advantage other than antibiotic resistance? You might try mixing equal amounts of transformed bacteria with untransformed bacteria and plate them together on one plate. Which colonies are bigger after 24 hours? Which colonies are more numerous?

•Can a genetically transformed organism pass its new traits on to it offspring? Are transformation markers carried to the next generation bacteria? Each colony represents one bacterium that multiplies into billions. Design and conduct an experiment to determine what would happen if you grew a sample colony that had been transformed from 7.1 Part 1 in LB broth and plated? Would all offspring carry the markers?

Procedures:

Step 1. Choose one of these driving issues and design a transformation experiment.

Step 2. Design your experiment following the guidelines of the ExD (Experimental Design) form.


Experimental Design (ExD) FormComplete this ExD pre-lab planning form before beginning your lab

1. Independent variable: (What is the cause agent? What are you changing?)

2. Dependent variable: (What is being measured?)

3. Lab set-up:

ExperimentalGroups

Number of Trials

4. Control: (What is the experimental group being compared to?)

5. Hypothesis: (Use an “if ” [Independent Variable], “then” [Dependent Variable] format. State the cause and e�ect relationship between the I.V. and the D.V. It must be testable.)

6. Lab title: (�e e�ect of ____[I.V.] ____on ____[D.V.]____.)

7. Experimental constants: (Name at least six variables NOT altered during the experiment.)

8. Sketch of experimental set up with labels:

9. Detailed procedure:


Transformation efficiency =

Total number of colonies growing on the LB/Amp plate (Experimental Plate)Amount of DNA (μg) spread on the

LB/Amp plate

LAB Investigation 7.2: Calculating Transformation Efficiency

Amount of DNA (μg) spread on the LB/Amp plate: •How do you determine quantitatively the extent to which you genetically

transformed the E.coli cells or what is the transformation efficiency? •How competent are the bacteria cells in taking up the plasmid DNA?•What is the importance of quantifying how many cells have been transformed?

Gene therapy in chronic leukemia, cystic fibrosis, hemophilia, and muscular dystrophy (to name a few) patients are examples of how cells that are collected from the patient are transformed in the lab and then put back into the patient. The more cells that are transformed to produce the needed protein, the more likely the therapy will work. Gene therapy is being used in many ways:

•Replace missing or defective genes; •Deliver genes that speed the destruction of cancer cells; •Supply genes that cause cancer cells to revert back to normal cells; •Deliver bacterial or viral genes as a form of vaccination; •Provide genes that promote or impede the growth of new tissue; and; •Deliver genes that stimulate the healing of damaged tissue.

Gene delivery can be used in cells that have been removed from the body or in cells that are still in the body. Genes can be delivered into cells in different ways.

•Genes can be carried into cells by bacteria and viruses; •Genes can be delivered within tiny synthetic “envelopes” of fat molecules; and

recently,•Genes can gain entrance into cells when an electrical charge is applied to

the cell to create tiny openings in the membrane that surrounds a cell. This technique is called electroporation. A new “bionic chip” has been developed to help gene therapists using electroporation to slip fragments of DNA into cells. It contains a single living cell embedded in a tiny silicon circuit.

Calculating transformation efficiency gives you an indication of how effective you were in getting plasmids carrying new information into host bacterial cells. In this example, transformation efficiency is expressed as the number of antibiotic-resistant colonies per μg of plasmid DNA used in the experiment. The transformation efficiency is calculated using the following formula:


Procedures:

1. Calculate the total number of transformed cells.•Calculate the amount of plasmid DNA in the bacterial cells

spread on the LB/Amp agar plate (Experimental Plate).•Calculate the total mass of plasmid DNA (total mass=

concentration X volume) Remember you used 10 μL of plasmid at a concentration of 0.005 μg/μL.

DNA plasmid (μg) = (concentration of DNA of μg/μL) × (volume of DNA in μL)

2. Calculate the fraction of plasmid DNA that actually got spread onto the LB/Amp agar plate (since not all of the DNA you added to the bacterial cells will be transferred to the agar plate).

(Hint: Refer to the procedure and your notes. How many μL of cells containing DNA did you spread onto the plate? What was the total volume of solution in the test tube? Did you add ALL the volumes?)

3. Calculate the μg of plasmid DNA that you spread on the LB/Amp plate. To answer this question, you multiply the total mass of plasmid DNA used times the fraction of plasmid DNA you spread on the LB/Amp plate.

DNA spread (μg) = Total amount of DNA used (μg) × fraction of DNA used

What does this number tell you?

4. Calculate the transformation efficiency.Look at your calculations. Fill in the blanks with the correct numbers:Number of colonies on the LB/Amp plate __________________ Amount of plasmid DNA (μg) spread on the plate ____________

5. Rewrite your transformation efficiency number to scientific notation (for example: 12,000 = 1.2 X 104).

Fraction of DNA used = Volume spread on the LB/Amp agar plate (μL)Total sample volume in test tube (μL)

Transformation efficiency =

Total number of colonies growing LB/Amp plate

Amount of DNA (μg) spread on the LB/Amp plate


Evaluating Results of Transformation Efficiency

1. What does your calculation of transformation efficiency mean?

2. Biotechnologists have rated the transformation protocols that you just completed as having transformation efficiency between 8.0 × 102 and 7.0 × 103 transformants per μg of DNA. How does your transformation efficiency compare?

3. What factors could explain a transformation efficiency that was either greater or less than predicted?

4. What are some challenges you had in performing your investigation?

5. Did you make any incorrect assumptions?


6. What are some possible sources of error in the transformation procedure? What are ways to minimize these potential sources of error?

7. Does a bacterial cell take in a plasmid with a gene the cell already possesses? If so, would this affect your calculations of transformation efficiency?

8. Transformation efficiency actually decreases if there is an over saturation of the DNA plasmid. Why do you think this occurs?

9. What advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions?

lab #06

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