MOLECULAR GENETICS: TRANSFORMATION AND CLONINGadapted by Dr. D. L. Vogelien

Introduction

The field of molecular genetics has resulted in a number of practical applications thathave been of tremendous benefit to us. One such benefit is the ability to produce largequantities of biological materials that were previously difficult to obtain. These newproduction methods involve isolating the gene for the needed product and placing thatgene into a rapidly reproducing organism such as a bacterium, which will thenmanufacture large amounts of the desired substance in a relatively short period of time.This process of altering the production capabilities of living organisms by theintroduction of new genes into those organisms is commonly referred to as geneticengineering. The most common way of introducing these genes and producing thesealtered organisms is through the use of plasmids.

Plasmids are small circular molecules of DNA that exist apart from the singlechromosome present in many bacterial species. Such plasmids are usually notessential to the survival of the bacteria, but may contain genes that encode productsthat are useful to the bacteria. Many plasmids carry one or more genes that conferresistance to various antibiotics. A bacterium carrying such a plasmid can live andmultiply in the presence of that antibiotic. Plasmids can be introduced into bacteria ifthe bacteria are first treated with calcium chloride. Treatment with calcium chloriderenders the bacteria competent to take up foreign plasmid DNA. This process (uptakeof plasmid DNA) is referred to as transformation. As the bacterial populationincreases, the number of plasmid molecules increases. Following growth of thebacteria in the presence of the antibiotic, the plasmid DNA can be easily isolated fromthe bacterial culture.

Plasmids are useful tools for the molecular biologist because they can serve as gene-carrier molecules, or cloning vectors. A gene of interest is joined to this vectorplasmid to from a hybrid or recombinant plasmid that is able to replicate and beexpressed in bacteria. In order to prepare recombinant plasmids, it is necessary to cutthe plasmid, and the gene of interest to be inserted, at precise locations and then to join(or ligate) the plasmid and the gene together.

The cutting is accomplished by the use of enzymes called restriction endonucleaseswhich recognize a specific sequence of 4-8 nucleotides (a restriction site) in the targetDNA molecule and cut at specific locations within that site. The restriction site must bepresent in both the plasmid and the gene to be inserted. The

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restriction site of one such enzyme (EcoR1) is given below:

cut ....GAATTC.... -----> ....G

AATTC........CTTAAG.... ....CTTAA

G.... cut

Since any given enzyme recognizes and cuts at a unique sequence, the number ofcuts in any given DNA molecule is limited. Typically, the restriction sites for a givenenzyme are hundreds to thousands of base paris apart. The staggered cut of theenzyme illustrated above generates sticky, cohesive, single-strand ends. These areimportant in recombinant DNA work because they enable any two DNA fragments to belinked together by complementary base pairing at their ends, provided they weregenerated with the same restriction enzyme. One basic procedure for generatingrecombinant DNA molecules is illustrated on the next several pages.

A plasmid that carries a gene that renders its host bacterium resistant to someantibiotic is cleaved at a single restriction site by EcoR1. The gene to be inserted isalso cut with the same enzyme (Figure 1). The resulting fragments are then reannealedby complementary base pairing and the newly formed joints are sealed by the action ofanother enzyme, namely DNA ligase. This enzyme normally performs this fragmentsealing operation during DNA replication. Because a mixture of fragments exists, it ispossible that some of the plasmids will reanneal without the gene being inserted, whileothers will contain the foreign gene (Figure 2). The recombinant molecules thus formedare introduced into a suitable bacteria, such as Escherichia coli, by transformation.The bacteria are then grown in the presence of the designated antibiotic. Thosebacteria containing the plasmid (e.g. transformed) will grow, while those not containingthe plasmid (untransformed) will be killed by the antibiotic.

In this experiment you will use the plasmid pUC18 as a cloning vector in order toamplify segments of the genome of the bacteriophage lambda. Plasmid pUC18 is asmall plasmid (2686 bp) that contains an ampicillin-resistance gene. This plasmid alsocontains part of the lac Z gene that encode for the first 146 amino acids of the enzymebeta-galactosidase. If the bacteria used for transformation contain the remainder ofthe gene, complementation occurs and the complete enzyme is produced. When thecomplete enzyme is produced, the bacteria are able to metabolize the enzyme'ssubstrate, X-gal. If the nutrient agar on which the bacteria are grown contains X-gal,the bacterial colonies will be blue. However, if the inserted lambda gene happens to beinserted in the middle of the lac Z gene, then the active enzyme cannot be produced bycomplementation, the bacteria will not be able to use X-gal, and the resulting colonieswill be white instead of blue (Figure 3).

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This exercise will allow you to perform transformation and cloning using materials thathave already been prepared for you. E. coli cells will have been made competent bytreatments with CaCl2. You will receive a sample labeled A, B, or C. One of these willcontain the plasmid pUC18, one will contain the plasmid pUC18 into which a foreigngene has been inserted (fragment of lambda genome), and one will contain no plasmid.Samples will be incubated with the competent E. coli and plated onto nutrient agarcontaining the antibiotic ampicillin and the substrate X-gal. Observation of the colonieswhich grow on these plates will enable you to correctly identify the three samples. Youridentification will be verified by electrophoresis in a subsequent experiment.

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Figure 1. Restriction Endonuclease Digestion

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Figure 2. Ligation of Foreign DNA into a Plasmid

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LambdaDNA

Lambda fragment

Figure 3. Transformation of E. coli

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The entire protocol is provided for your reference. You will actually begin with step B-3(**). Gloves and protective glasses should be worn at all times. Dispose of all usedmaterials that have come in contact with or may be contaminated by the plasmidsand/or E. coli (e.g. pipet tips, pipets, microfuge tubes, gloves) in biohazard bags.

A. Preparation of Competent Cells

Procedure

1. Place the vial of CaCl2 and the tube of E. coli in an ice bath and invertthe tube several times to mix the e. coli.

2. Transfer the bacteria to the CaCl2. Transfer 0.5 ml from the vial to thetube and back again.

3. Mix by gentle tapping.

4. Incubate 1 hour or more on ice.

B. Transformation and Incubation

Procedure

1. Obtain three sterile microcentrifuge tubes and label them A, B and C.

2. Using a sterile micropipet, place 10 l of the sample (A, B or C) into theappropriate tube.

** 3. Using a micropipet with a sterile disposable tip, add 100 l of competentE. coli to each tube. Use a new sterile tip for delivery to each tube. Thiswill prevent contamination of the E. coli stock with plasmid samples as wellas contamination between sample tubes.

4. Mix by gently tapping and store on ice for 30 minutes. This allows theplasmid DNA to adsorb to the surface of the E. coli.

5. Incubate @ 37 °C (in water bath, oven, or incubator) for 5 minutes. Thisallows the adsorbed plasmid DNA to be transported across the membraneinto the E. coli.

6. Using a sterile 1 ml pipet, add 0.7 ml of nutrient broth (without ampicillin)to each tube and incubate at 37 °C for 30 minutes. Use a different sterilepipet for delivery to each tube. This incubation period allows the bacteria torecover from the CaCl2 treatment and also allows them to begin to expressthe ampicillin resistance gene (if present).

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7. Obtain 3 plates of nutrient agar containing ampicillin and X-gal.

8. Using a sterile transfer pipet, transfer 100 l of sample from each tubeonto one of the nutrient agar plates and spread evenly with a bent steriletransfer needle. Again, use a different sterile pipet to delivery eachsample.

9. Incubate the plates at room temperature for a few minutes until the liquidhas been absorbed.

10. Invert plates and incubate @ 37 °C for 24-48 hours. Place plates in therefrigerator until the next lab period.

11. Examine plates for colony growth. Record observations (e.g. are therecolonies growing, what color are they). Dispose of cutlures in a biohazardbag.

LAB ASSIGNMENT

1. Give the identity of plasmid samples A, B and C.

2. Explain the basis for your identification.

3. Two different selection methods were used to determine the identity of plasmidsA, B and C. One was ampicillin resistance and the other was the ability tometabolize X-gal. Why were both necessary? (i.e. Why wouldn't either of themhave been sufficient by itself?)

4. In a similar type of experiment you want to transform E. coli, but you will use adifferent plasmid. The available plasmid has been genetically engineered tocontain resistance genes to both tetracycline and ampicillin. An EcoR1restriction site lies within the gene encoding resistance to tetracycline. Assumeyou restrict the plasmid and lambda DNA with EcoR1 and mix the two DNA'stogether and allow for reannealing of fragments. You then use this DNA sampleto transform another sample of E. coli. How might you 1) determine iftransformation was successful and 2) determine if the transforming plasmid wasrecombinant?

MOLECULAR GENETICS: ANALYSIS OF PLASMIDS BY RESTRICTION DIGESTION

Introduction

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Large organic molecules can be separated by a variety of techniques.Chromatography makes use of either paper or specially coated glass plates toseparate proteins, amino acids, carbohydrates or other molecules with the aid oforganic solvents. Electrophoresis is a technique that relies on differences in sizeand/or overall electrical charge to separate molecules in an electric field. It is mostcommonly used to separate either proteins or nucleic acids (both DNA and RNA).Support media most often used include cellulose acetate, starch agarose, andpolyacrylamide. A liquid solution composed of one of these chemicals, when properlytreated, will solidify to form a semi-solid support matrix in the shape of a slab known asa gel.

In this exercise, a separation procedure involving agarose gel electrophoresis willbe performed to separate DNA fragments. Since all DNA has basically the sameoverall charge, the gel electrophoresis used here will essentially separate fragmentsbased on differences in size. The agarose gel, in this case, acts like a molecular sieve.Smaller molecules will be able to travel more quickly through the gel. Aselectrophoresis continues, smaller molecules will be located towards the bottom of thegel, while larger ones will be found closer to the loading wells.

The fragments to be analyzed by agarose gel electrophoresis are the plasmids used inthe Transformation and Cloning exercise. In this exercise you will attempt to confirmyour tentative sample identification from the previous week's lab exercise by reactingthe remainder of the three samples (A, B, and C) with the restriction endonucleaseEcoR1 and analyzing the resultant DNA fragments by agarose gel electrophoresis. Forexample, the sample with no plasmid should produce no DNA fragments, whereas thesample containing only pUC18 should produce a single DNA fragment of 2686 basepairs because the plasmid has a single cut site for EcoR1. The closed circular form willbe converted to a linear form by enzymatic cleavage. By running a mixture of DNAfragments of known size (known as a standard) it should be possible to determine thesize of the inserted foreign DNA

While the entire procedure has been provided for you, you will actually beginwith Sample Preparation and Analysis step B-5(**).

Procedure

A. Preparation of Agarose Gel

1. Seal the ends of the casting tray with masking tape.

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2. Prepare electrophoresis buffer (TAE: Tris-acetate-EDTA, pH 8.0) byadding 33 ml of the 100X concentrate to 3.3 liters of deionized water.

3. Weigh 1.2 g of agarose and mix it in a flask with 100 ml of TAE buffer.

4. Dissolve the agarose by heating it in a boiling water bath. Heat untilboiling and no solids are observed.

5. Pour the warm agarose solution into the casting tray and place the combat one end of the tray.

6. Allow the gel to cool until it is firm (about 15 - 30 minutes).

7. Remove tape from the ends of the tray.

8. Place the gel tray into the horizontal unit so that the wells are at thecathode end (the black electrical connection).

9. Fill the buffer reservoir with TAE buffer, covering the gel to a depth ofabout 2 mm. Remove the comb carefully.

B. Sample Preparation and Analysis

1. Place 10 ul of EcoR1 into three tubes (labeled A, B, C).

2. Add 5 ul of the corresponding sample to the tubes and mix gently.

3. Incubate for 40 minutes at 37 °C.

4. Add 5 ul of electrophoresis sample buffer (blue in color) to each tube.

**5. Load 15 - 20 ul of each of the digested samples into the sample wells.Also load 10 ul of a DNA standard into a separate well. The molecularweight standard contains 4 fragments: 3621, 2040, 1120 and 784 bp.

6. Electrophorese at 170 volts until the blue tracking dye has migrated towithin 1 cm of the end of the gel. Note: this voltage is for mini gels; 4 gelsper rig.

7. At the end of the run, transfer gel to a dish containing an ethidiumbromide solution and soak gel for 15 - 30 minutes. Caution: ethidiumbromide is a carcinogen! Wear gloves at all times when working with

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ethidium bromide or any materials that have been in contact with it.Remove the gel from the ethidium bromide solution and place it on asheet of plastic wrap. Analyze on an ultraviolet transilluminator. BESURE TO WERE PROTECTIVE EYEWARE WHEN USING UV LIGHT.Ethidium bromide attaches to DNA and causes it to fluoresce under UVradiation. DNA should be visible as distinct fluorescent bands.

8. Use a metric ruler to measure the distance migrated (from the loadingwells) by each fragment in the standards and samples. Note: recorddistance in mm.

9. Construct a standard curve from the DNA fragments in the markersamples:

a) Plot fragment size/length (in base-pairs) vs distance migrated for allfragments (from both sets of markers) as one data set.

b) Draw a straight line that best fits all data points; do not connect thedots.

10. Do your results in this part of the experiment confirm your tentativesample ID from the transformation experiment? Explain. Identify thecontents of samples A, B and C.

11. Using the standard curve, estimate the size of the pUC18 plasmid. Doesit match the expected value of 2686 bp?

12. Estimate the size(s) of the foreign DNA inserts(s) from those samples thathave them.

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LAB ASSIGNMENT

1. Note whether the results obtained from this laboratory exercise confirm yourtentative sample ID from the transformation exercise. Explain why/why not (bevery specific as to why).

2. Submit your standard curve (title the graph; include lab partner's name ongraph).

3. Provide your size estimate for pUC18 generated using the submitted standardcurve.

4. Provide size estimate(s) for lambda DNA insert(s) using the submitted standardcurve. Referring to Figure 1 in the Transformation and Cloning exercise, whichof the fragments generated by EcoR1 digestion of lambda DNA did you have asan insert in your sample?

5. Explain why more than one insert might have been detected.

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