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In nature genescan be transferredbetweenbacteriain three ways: conjugation, transduction, or transformation. Conjugation is a mating processduring which genetic material is transferred from one bacterium to another of a different mating type. Tfansduction requires the presenceof a virus to act as a vector (canier) to transfer small pieces of DNA from one bacterium to another. Bacterial transformation involves transfer of genetic information into a cell by direct uptake of the DNA. During gene transfer,the uptake and expressionof foreign DNA by a recipient bacterium can result in conferring a particular trait to arecipient lacking that trait. Transformation can occur naturally but the incidence is extremely low and is limited to relatively few bacterial strains.These bacteria can take up DNA only during the period at the end of logarithmic growth. At this time the cells are said to be competent. Competencecan be induced in E. coli with carefully controlled chemical growth conditions. Once competent,the cells are ready to accept DNA that is introduced from another source. Plasmidscan transfergenes(such as thosefor antibiotic resistance) that occur naturally within them, or plasmids can act as carriers (vectors) for introducing foreign DNA from other bacteria, plasmids, or even eukaryotesinto recipient bacterial cells. Restriction endonucleases be used can to cut and insert pieces of foreign DNA into the plasmid vectors (Figure 6.1). If theseplasmid vectors also carry genesfor antibiotic resistance,transformed cells containing plasmids that carry the foreign DNA of interest in addition to the antibiotic resistancegene can be easily selectedfrom other cells that do not carry the gene for antibiotic resistance.

Figur e 6. 1:

B a c te ri a l T ra n s fo rm a ti o n

U si ng a R estri cti on

E ndonucl ease

[. Createplasmid with geneof interest.

2. DNA of interest cleaved is

Ampicillin resistancegene (Amp r) ,/ by the samerestrictlon endonuclease -/

( \_-,/
1

Plasmid is cleaved by restriction endonuclease Amp r gene

D
DNA fragment with sticky ends

rm us
I

B"<Q! x'#4tz';*ffHJi*Ty (( ffi.

;"

NM.
B

.ou"*ra/

\o*oofinteresr

II. Transform recipient cells with plasmid DNA. III. Plate recipients on ampicillin plates and selectfor resistantcolonies. IV. Isolate colonies carrying the plasmid.

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13. Immediately spreadthe cells by using a sterile spreadingrod. Repeatthe procedurefor each plate. L4. Allow plates to set for severalminutes. Tape your plates together and incubate inverted overnightat3'7"C.

An a l y s is of Res ul ts L. Observethe colonies through the bottom of the culture plate. Do not open the plates. Counl the number of individual colonies; use a pefinanent marker to mark each colony as it is counted. If cell srowth is too denseto count individual colonies. record "lawn."
T

LB + Positive Control) LB/Amp + (Experimental)

LB - (Positive Control) LB/Amp - (Negative Control)

2. 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? a. LB + andLB -

b. LB/Amp - and LB/Amp +

c. LBiAmp + and LB +

3. Transformation efficiency is expressedas the number of antibiotic-resistantcolonies per microgram of pAMP. Becausetransformation is limited to only those cells that are competent, increasingthe amount of plasmid used does not necessarilyincreasethe probability that a cell will be transformed.A sample of competentcells is usually saturatedwith small amounts of plasmid, and excessDNA may actually interfere with the transformation process. a. Determine the total mass of pAMP used. (You used 10 pL of pAMP at a concentrationof 0.005 pg/pl-.) Total Mass = volume X concentration. b. Calculate the total volume of cell suspensionprepared. c. Now calculate the fraction of the total cell suspensionthat was spreadon the plate. Number of pL spread/totalvolume. d. Determine the mass of pAMP in cell suspension. Total mass of pAMP X fraction spread. e. Determine the number of colonies per mg of plasmid. Expressin scientific notation. pAMP spread[from calculation in Step 3.d] = Number of colonies observed/mass transformation effrciency.

67 trliiilI .$]6le ,6tig1 6ts( t4.a4.a a

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Figure 6.2a
Cleavageby Hae III producesblunt ends.

GGiC C
C C i GG

G GC C

Figure 6.2b
Cleavage by EcoRI produces sticky ends.

GIAAT TC ,/\

c T_TAAic
/\

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c T-f AAic

/\

|l

H
he

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C: E lec t r opho re s i s 1. Place the top on the electrophoresischamber and carefully connect the electrical leads to an approvedpower supply (black to black and red to red). Set the voltage to the appropriate level for your apparatus. When the cunent is flowing, you should seebubbles on the electrodes. 2. Allow electrophoresis proceeduntil the tracking dye has moved nearly to the end of the gel. to 3. After electrophoresisis completed, turn off the power, disconnectthe leads, and remove the cover of the electrophoresischamber. D : S t aining a n d V i s u a l i z a ti o n Note: Wear gloves. 1. Carefully remove the gel bed from the chamber and gently transfer the gel to a staining tray for staining. Use the scooperprovided with your kit or keep your hands under the gel during the transfer.You may wish to remove a small piece of gel from the upper right-hand corner to keep track of the gel's orientation. Do not stain in the electrophoresischamber. 2. Label the staining tray with your name and take it to your teacherfor staining. 3. Examine your stainedgel on a light box or overheadprojector. Compare your gel with the samplegel shownin Figure 6.3.

F igur e 6. 3:

Sa mp l e R e s tri c ti o n

D i g e st ot Lambda D N A

EcoRI

HindIII

No Enzvmes

Figure not to scale.

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Tabfe 6.1: Distance Hindlll Produced Fragments Migrate in Agarose Gel (cm)
Hindlll Measuled Dlstance (cm)

Actual bp 23,13O 9,416 6,557 4,361 2,322* 2,027* .,570*a 125 D

Table 6.2: Distance EcoRIProduced Fragments Migrate in Agarose Gel (cm)

EcoRl Measured
tlitlrnnc {^m}

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hh

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Band 1 Band 2 Band 3 Band4 Band 5 Band 6

E may form a single band A may not be detected

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4' For whicfr-llagment sizeswasyour graphmostaccurate? which fragmentsizes For wasit least accurate? what doesrhistell you aboutrheresolving ;i;;;;se_ger electrophoresis? "bility

Analysis
l' Discuss how each of the following factors would affect the results of electrophoresis: a. Voltage used

b. Runningtime

c. Amountof DNA used

d. Reversal of polarity

Two smallrestriction fragments nearry samebase of the pair sizeappear a singleband, as evenwhen the sampleis run to the very ind of the gel. what coutj'-deJone to resolvethe fragments? Why wouldit work?

Questions 1. Whatis a plasmid? How areplasmids usedin genetic engineering?

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7. Use the graph you prepared from your lab data to predict how far (in cm) a fragment of 8,000 bp would migrate.

8. How can a mutation that alteri a recognition site be detectedby gel electrophoresis?

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