New and Improved: The Art of Bacterial Transformation

Introduction

Purpose: The objectives of the New and Improved: Bacterial Culture

Transformation Lab are: to observe standard bacterial growth under various
conditions including the transformation of bacteria; to understand how the process
of transformation occurs, as well as the biological results and consequences that
come of transformation; and to understand the importance of transformation in
prokaryotic and eukaryotic life cycles.

Background: Transformation is the “process by which the genetic material

carried by an individual cell is altered by the incorporation of foreign
(exogenous) DNA into its genome” (MedicineNet.com, “Definition of Genetic
transformation”). Transformation in bacterial cells occurs when the cell
incorporates naked DNA into its genetic material; in a laboratory setting, this is
encouraged by placing the mixtures of transformation solution and plasmid DNA (in
+pGLO tube only) on ice, then rapidly transferring them to a hot water bath for
about fifty seconds, and then placing them back on ice again – this procedure is
called heat shock and “increases the permeability of the cell membrane to DNA”
(lab directions). The agent which the new genetic material is incorporated into
is the bacterial plasmid.
A plasmid is a circular deoxyribonucleic acid (DNA) molecule that replicates
independently of the bacterial chromosome and often is the avenue for which a
bacteria gains resistance to an antibiotic. Recombinant plasmids are those which
have DNA from two or more sources incorporated into a single plasmid. To make
recombinant plasmids, two different plasmids are cut with the same restriction
enzyme: this restriction enzyme only cuts at particular restriction sites, so the
type of cut it makes in one plasmid will be the same type of cut in another
plasmid. The cut must produce “sticky ends” so that the plasmid DNA can bind to
any other plasmid DNA with complementary base pairs. Once cut, the two plasmids
are mixed and the complementary sticky ends for each plasmid are sealed by DNA
Ligase.
The pGLO plasmid – which contains the genes for GFP, or Green Fluorescent Protein,
and the enzyme ß-lactamase, which provides resistance to the antibiotic ampicillin
– will be incorporated into the genome of the E. Coli bacteria used in the lab.
pGLO is originally “from the bioluminescent jellyfish, Aequorea Victoria which
allows the jellyfish to fluoresce and glow in the dark. E. coli can be
transformed to” make the GFP protein and “express this gene” which will “cause the
E. coli to glow green when exposed to UV light” (The Mystery Behind pGlo and GFP).
The aforementioned antibiotic ampicillin is used in this lab to demonstrate
the effect of recombinant plasmids and transformation. By applying ampicillin to
a Petri dish with and without pGLO, we can see the success of an antibiotic
against bacteria as well as the success of recombinant bacteria against an
antibiotic. Since pGLO harbors ß-lactamase – a restriction enzyme which cuts the
DNA of an antibiotic to render it ineffective – along with GFP, any bacteria with
the pGLO recombinant plasmid can better resist the antibiotic ampicillin.
In our experiment, the control treatment is the dish with bacteria, but
lacking pGLO, ampicillin and agarose. On this dish, we can see how the bacteria
grow unimpeded or aided by any other substance. The experimental groups are the
plate with bacteria, ampicilline and pGLO, the plate with only bacteria and
ampicillin, and the plate with ampicillin, agarose and pGLO.

Hypothesis:
What plates will have growth and why?: The plates with pGLO, because they are
resistant to antibiotics.
What plates will not have growth and why?: The plate with ampicillin and no pGLO,
because the bacteria are compromised by an antibiotic and do not have a
recombinant plasmid.
What plates will glow under a UV light and why?: Plates with pGLO because the
plasmid contains the gene for glowing.

Results (Data & Analysis)

Discussion/Conclusion

To conduct this lab, we first took two solutions of transformation solution and
placed a starter colony of bacteria in each one. Then, to one solution, we added
a pGLO plasmid DNA solution. We then incubated both solutions, treated them with
a heat shock, added LB nutrient broth to both and incubated them once more. Then,
on two Petri dishes we spread the solution with pGLO, while on two more we spread
the solution lacking pGLO. On one pGLO Petri dish we spread ampicillin; on the
other, we spread ampicillin and agarose. On one Petri dish without pGLO we spread
ampicillin; on the other, we spread nothing. Finally, we incubated the four Petri
dishes four approximately 24 hours and let the bacteria grow. After the final
incubation period, the dish with only the initial solution and no pGLO developed
into a lawn of bacteria, coverin about 85% of the dish. The plate with
ampicillin, but no pGLO, produced no bacteria. The dish with both pGLO and
ampcilin produced about 548 colonies of bacteria. The dish with ampicillin,
agarose and pGLO produced about 810 colonies of bacteria.
The lack of growth on the plate with only ampicillin and the bacterial
solution is due to the fact that ampicillin is an antibiotic which inhibits
production of bacterial cell walls. Thus, no bacteria can be produced unless
recombination occurs. The plate with only bacteria and no pGLO produced an entire
lawn because the bacteria were able to grow unchecked by an antibiotic. The plate
with pGLO and ampicillin produced bacteria regardless of the antibiotics presence
because the bacteria possess a plasmid that has undergone recombination and
therefore become resistant to the antibiotic. The plate with pGLO, agarose and
ampicillin grew nearly one-and-a-half times the number of colonies as the plate
with only pGLO and ampicillin because the sugar agarose is a nutrient for the
bacteria which helps them divide at a quicker rate. The only plate to glow was
the plate with ampicillin, agarose and pGLO; a possible explanation for why this
plate glowed when exposed to UV light but the plate with only pGLO and ampicillin
did not is that the presence of agarose causes the operon that is responsible for
glowing to become activated in order to break down agarose and make it usable by
the bacterial cell.
We know transformation occurred because there would be no bacterial colonies
on the plate with both pGLO and ampicillin if transformation had not occurred.
The plasmids on this plate had to be recombinant in order to survive in the
presence of an antibiotic. The transformation efficiency of 5162.52 indicates
that approximately 5163 cells were transformed for every one microgram of DNA.
Possible sources of error in our experiment include inaccurately measuring
the number of bacterial colonies that grew on each plate, insufficiently mixing
the solutions of DNA after adding restriction enzymes or not returning the
mixtures to ice quickly enough after during the heat shock.

Literature Cited

"Definition of Genetic Transformation." MedicineNet. 07 June 1999. 12 Apr. 2008

<http://www.medterms.com/script/main/art.asp?articlekey=9803>.

"The Mystery Behind PGlo and GFP." Chemistry and Biochemistry At MC. 2002. The
Monmouth College Biochemistry Staff. 12 Apr. 2008
<http://department.monm.edu/chemistry/chemistry330/fall2002/nauclair/>.