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Lab 6A Bacterial Transformation

Introduction:
Genes are transferred between bacteria by way of conjugation, transduction, or
transformation. Conjugation takes place when the genetic material is transferred from one
bacterium to another of a different mating type. Transduction requires the presence of a
virus to act as a vector, or a carrier to transfer small pieces of the DNA from one
bacterium to another. Transformation involves the transfer of genetic information into a
cell by directly taking up the DNA. This lab uses transformation to insert a specific gene
into a plasmid so that the cell takes on those characteristics for which the gene codes.

Plasmids are small rings of DNA that do carry genetic information. They can transfer
genes, like genes for antibiotic resistance, which can occur naturally within them, or
plasmids can act as carriers or vectors for introducing foreign DNA from other bacteria,
plasmids, or even eukaryotes into recipient bacterial cells. Restriction endonucleases can
be used to cut and insert pieces of foreign DNA into the plasmid vectors. If these plasmid
vectors also carry genes for antibiotic resistance, transformed cells containing plasmids
that carry the foreign DNA of interest in addition to the antibiotic resistance gene can be
easily selected from other cells that do not carry the gene for antibiotic resistance. They
are usually extrachromosomal. This means they exist separately from the chromosome.
Some plasmids replicate only when the bacterial chromosome replicates, and usually
exist only as single copies within the bacterial cell, but still others replicate on their own,
autonomously. There can be anywhere from ten to two hundred copies within a single
bacterial cell. There are specific plasmids called R plasmids that carry genes for
resistance to antibiotics such as ampicillin, kanamycin, or tetracycline.

The bacterium Escherichia coli, or E. coli, is an ideal organism for the molecular
geneticists to manipulate and has been used extensively in recombinant DNA research. It
is a common inhabitant of the human colon and can easily be grown in suspension culture
in a nutrient medium such as Luria broth, or in a petri dish of Luria broth mixed with
agar, or nutrient agar. The single circular chromosome of E. coli contains about five
million DNA base pairs, only one-six hundredth of the haploid amount of DNA in a
human cell. Also, the E. coli cell may contain small plasmids, discussed earlier. The
plasmids are broken up with calcium chloride, and the wanted gene is inserted and the
bacteria can be grown on the nutrient or with an antibiotic to see if the gene has
transformed the bacteria so that they are resistant to the antibiotics.

Materials:
The materials needed in this lab were two Luria agar plates, two Luria agar plates with
ampicillin, two 15mL tubes, one inoculating loop, one bacterial spreader, several sterile
micropipettes, calcium chloride, Luria broth, pAMP solution, a Bunsen burner, hotplate,
ice, and a water bath.
Methods:
Mark one of the sterile 15mL tubes "+" and the other "-", the plus tube obviously having
the plasmid added to it while the other tube does not receive any. Using a sterile
micropipette, add 250 microliters of ice cold 0.05M CaCl2 to each tube. Transfer a large
3mm colony of E. coli from the starter plate to each of the tubes using a sterile
inoculating loop. Try to get the same amount of bacteria into each tube. Be careful not to
transfer any agar. Vigorously tap the loop against the wall of the tube to dislodge the cell
mass. Mix the suspension by repeatedly drawing in and emptying a sterile micropipette
with the suspension. Add ten microliters of pAMP solution directly into the cell
suspension in the tube labeled with a plus sign. Mix by tapping the tube. This solution
contains the antibiotic resistance plasmid. Keep both of these tubes in ice for about 15
minutes. While the tubes are on ice, obtain two LB agar plates and two LB/Amp agar
plates. Label each plate on the bottom as follows: one LB agar plate "LB+" and the other
"LB-." Label one LB/Amp plate "LB/Amp+" and the other plate "LB-." A brief pulse of
heat facilitates entry of foreign DNA into the E. coli cells. Heat-shock cells in both the +
and – tubes by holding in a water bath of 42 degrees Celsius for ninety seconds. It is
essential that cells be given a sharp and distinct shock; so take the tubes directly from the
ice to the water bath. Immediately return the tubes to the ice after ninety seconds. Use a
sterile micropipette to add 250 microliters of Luria broth to each tube. Mix by tapping the
tube. Any transformed cells are now resistant to ampicillin because they contain the gene.
Place 100 microliters of the + cells on the LB+ plate and on the LB- plate, the other cells
should be placed. Immediately spread the cells using a sterile spreading rod. This can be
accomplished by running the rod through the Bunsen burner and allowing to cool by
touching it to the agar on the part of the dish away from the bacteria. Spread the cells and
once again run the rod through the fire to sterilize the rod. Allow the plates to set for
several minutes, then tape the plates together and incubate inverted overnight.

Data:

Luria agar + Lawn

Luria agar - Lawn

Luria agar with ampicillin + None visible

Luria agar with ampicillin - None visible


Total mass of plasmid used 0.05 microliters

Total volume of suspension 510 microliters

Fraction of cell suspension put on the plate 0.1960784314

Total mass of plasmid in fraction 0.0098039216

Number of colonies/ microliter of plasmid None visible

Questions:
Observe the colonies through the bottom of the culture plate. Do not open the plates.
Count the number of individual colonies; use a permanent marker to mark each
colony as it is counted. If cell growth is too dense to count individual colonies, record
"lawn."

LB+ (positive control) – lawn. LB- (positive control) – lawn.

LB/Amp + (experimental) – none visible. LB/Amp- (negative control) – none visible.

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+ and LB- = These are two controls without the presence of ampicillin in the
nutrient. They both had lawns of bacteria colonies on them because the E. coli grow
naturally without the presence of the antibiotic.

b. LB/Amp- and LB/Amp+ = The LB on the ampicillin agar with the addition of the
gene for transforming the plasmids of the E. coli should be able to survive, maintaining
the characteristics of the gene present in their DNA. The LB should not have survived on
the agar with ampicillin because this is how it would occur in nature, and there is no gene
in the bacterium to help resist the antibiotic.

c. LB/Amp+ and LB+ = These two should both have growth. They both were exposed to
the gene that protects E. coli bacteria against ampicillin and so they both should survive
because it shouldn’t matter if the ampicillin is present or not. There is probably less
growth on the ampicillin plate because not all of the bacteria cells could have
transformed; this would have only occurred under optimal conditions.

Determine the total mass of pAMP used. (Ten microliters were used at a concentration
of .005 microgram/microliter) There were .05 microliters used.

Calculate the total volume of cell suspension prepared. There was a total of 510
microliters used.
Now calculate the fraction of the total cell suspension that was spread on the plate.
The fraction of the total cell suspension that was spread was 0.1960784314.

Determine the mass of pAMP in the cell suspension. The mass of the pAMP in the cell
suspension was 0.0098039216.

Determine the number of colonies per microliter of plasmid. Express the number in
scientific notation. There were none visible.

Error Analysis:
There were many possibilities for error in this lab. The amounts of the different solutions
could have been mixed up of misread. Also, there were many lumps in the agar poured
into the plates. The spreading rod that was used to spread the bacteria onto the agar could
have been too hot when it was used to spread, and killed the bacteria, or while the lid of
the petri dish was off, some other contamination from the room could have infected the
bacteria causing different results. Finally, another place of error could have been in
setting of the bacteria on the plates.

Conclusion:
This lab shows the transformation of E. coli bacteria cells can affect the resistance those
bacteria cells have to the antibiotic ampicillin. The transformation takes place when the
plasmids of the bacteria are opened to taking in foreign DNA by the addition of calcium
chloride, and they were finally accepted with the help of heat shock. The results show
that if the bacteria cells were transformed, the cells could grow on the agar plates with
ampicillin, now having a gene for resistance to that antibiotic.

Lab 6B - DNA Fingerprinting

Introduction:
Restriction enzymes are endonucleases that actually cut the phosphodiester bonds on the
sides of deoxyribonucleic acid. These endonucleases recognize specific DNA sequences
in double-stranded DNA, which is usually a four to six base pair sequence of nucleotides.
The endonucleases then digest the DNA at these sites. The resulting product is usually
fragments of DNA of various lengths. Some restriction enzymes cut cleanly through the
DNA double helix while some produce uneven or sticky ends. By using the same
restriction enzyme to cut DNA from different organisms, the sticky ends produced will be
complementary and the DNA from the two different sources can be recombined. In
humans, no two individuals have the exact same restriction enzyme pattern in the DNA
except for identical twins. In DNA, the antiparallel strands are difficult to deal with
considering the restriction enzymes cut from opposite directions. This is the reason for
the complementary ends. The restriction enzymes are named according to a system of
nomenclature. The first letter represents the genus name of the organism. The next two
letters come from the species name. If there is a fourth letter, it stands for the strain of the
organism. Finally, if there are Roman numerals, it represents whether that particular
enzyme was the first or second etc. isolated in that category.
In the electrophoresis chamber, there is placed an agar gel. This gel has wells in it for the
samples of DNA to go into. The agarose gel is covered in a buffer so that the DNA is in a
neutral pH solution. That way, the DNA moves in the direction its charge forces it. Since
the phosphate groups on the skeleton of DNA are negatively charged, the whole molecule
takes on the negative charge. So, when the DNA is placed inside the gel and the
electricity turned on so that the poles are drawing the DNA toward the positive side, it
will move through the gel and separate according to the size of the fragments.

Hypothesis:
By way of electrophoresis, the fragments of DNA of lambda can be separated by the
traveling of the fragments through agar gel according to fragment size; DNA
fingerprinting has occurred.

Materials:
The materials needed for this lab are the following: an electrophoresis chamber, an
agarose gel, lambda DNA digested with endonucleases, tracking dye, micropipette and
tips, running buffer, and an electrical supply.

Methods:
Prepare the agar gel for the electrophoresis by microwaving it for the suggested amount
of time. When the gel has sufficiently hardened, place it in the chamber, pour the running
buffer over the gel and add the DNA samples into the wells with a micropipette. Next, set
the correct voltage and turn on the electricity. Allow this to run until the DNA is almost to
the end of the gel, but do not let it run all the way out. Next, obtain the stain and a
staining tray and let the gel set in the stain for a while. Next, put the gel into distilled
water so that the stain can be taken out of the gel itself, leaving the DNA stained a royal
blue. Look at and measure the gel over a light box, and put data into the data table.
Data:

Table 6.1

HindIII

Actual base pairing sequence Measured Distance (mm)

23,130 12

9,614 18

6,557 22

4,361 28

2,322 41

2,027 43

Table 6.2

EcoRI

Measured Distance Interpolated base Actual base pair


(mm) pairs sequence sequence

Band 1 12 13,500 21,226

Band 2 14 11,000 5,148 or 5,973

Band 3 26 3,700 4,269

Band 4 28 3,150 3,530

Band 5 43 815 2,207

Band 6 47 580 1,904

Band 7 49 500 1,587

Band 8 58 220 1,375


Questions:
Discuss each of the following factors:

Voltage used. If a higher voltage had been used, the DNA would have moved faster
through the agar gel, and slower if the voltage was low.

Running time. If allowed to run longer, the DNA would have eventually ended up into
the running buffer, and lost to the experiment. If not allowed to run long enough, the
bands could merge and be unclear for reading.

Amount of DNA. If more DNA had been used, the bands would have been darker
because more of the fragments would have traveled the same distance in the gel. The
bands would only have been more distinct and distinguishable.

Reversal of polarity. Had the polarity been reversed, the DNA would have been drawn
the other way through the gel, and ended up in the running buffer.

Two small restriction fragments of nearly the same base-pair size appear as a single
band, even when the sample is run to the very end of the gel. What could be dome to
resolve the fragments? Why would it work? I would take the endonucleases needed to
get the two fragment sizes and run an electrophoresis experiment just using those two
sizes. It would probably work because these two fragments just by themselves can’t or
shouldn’t stay together all the way to the end of the gel.
What is a plasmid? How are plasmids used in genetic engineering? Plasmids are
small rings of DNA. They are used in genetic engineering because it is considerably
easier to manipulate them into taking up preferred genes than it is to change the DNA
sequence of the whole cell.

What are restriction enzymes? How do they work? What are recognition sites?
These enzymes are endonucleases that cut the phosphodiether bonds of the DNA. They
only cut at specific proteins, the recognition site.

What is the source of restriction enzymes? What is their function in nature? They
occur naturally in prokaryotes and are used to cut up invading viral DNA that happens to
get through the cell wall and plasma membrane of the bacteria.

Describe the function of electricity and the agarose gel in electrophoresis. The
electricity is used to pull the DNA in a certain direction so that it will separate. The gel is
helpful because it is like a freeze frame that allows the fingerprinting to be visualized.
This could not be done in liquid or any solid.

If a restriction enzyme digest resulted in DNA fragments of the following sizes: 4000,
2500, 2000, and 400 base pairs, sketch the resulting separation by electrophoresis.
Show starting point, positive and negative electrodes, and the resulting bonds.

What are the functions of the loading dye in electrophoresis? How can DNA be
prepared for visualization? The dye allows the DNA to be more distinct so that accurate
measurements can be made in determining the distance traveled and the amount of bands.

Use the graph prepared from the lab data to predict how far (in mm) a fragment of
8000 base pairs would migrate. A piece of DNA of that size would probably run about
17.5 millimeters.

How can a mutation that alters a recognition site be detected by gel electrophoresis?
If you ran the normal and the mutant at the same time, you could see the change in the
band that would be in a different place because it wouldn’t allow the DNA to be cut in
that place.

Error Analysis:
There were not too many errors that could have occurred in this lab, but some of the few
include the adding DNA to the agar gel. The person transferring had to have a steady
hand and good eyes so that the gel wasn’t poked and the DNA made it into the chamber
without problems. The wrong DNA samples were added to the wells, but the right ones
were identified and later labeled correctly, out of order.

Conclusion:
In conclusion, DNA fingerprinting, or electrophoresis is used to determine the size of the
fragments that are cut by restriction enzymes. Restriction enzymes only cut at their
specific protein recognition sites. This is useful because no two restriction enzymes code
for exactly the same recognition site, allowing for a "fingerprint" like uniqueness that is
only possible with one’s DNA. From the data collected in the electrophoresis experiment,
other sizes of parts can be hypothesized by following the size of the base pair to the line
of best fit drawn on the log sheet. This tells you about how many millimeters the base
pair would probably go if allowed the same circumstances.

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