Evans Love

July 1, 2011
Biology Lab 4

Bacterial Transformation Lab: Guidance from the [Red], White

and Blue
The ability to manipulate cell DNA artificially isolating, splicing, and
recombining DNA segments through processes that transfer data from one
organism to another underpins modern research in molecular genetics. Studies
using simple prokaryotic species such as E. coli bacteria help clarify the complex
interactions associated with the storage, encoding, replication, and modification
of genomic information, as proteins essential for survival are created from DNA.
Unlike eukaryotic organisms, which procreate via sexual reproduction, many
prokaryotic species reproduce through a form of asexual replication called binary
fission. This cloning method, although faster than finding a mate, greatly reduces
genetic variability. To overcome this disadvantage, prokaryotes have evolved
three different mechanisms for lateral gene transfer: transformation, conjugation,
and transduction.
The current experiment was designed to explore prokaryote transformation
a genetic exchange mechanism through which genomes assimilate DNA
molecules (such as plasmids) from their environment, potentially altering cellular
processes. Plasmids are small in size, allowing them to enter cells with relative
ease; once inside, they both self-replicate rapidly and are distributed by binary
fission. As vectors in cell manipulation, plasmids have been cleverly modified by
genetic engineering to serve as useful tools in molecular biology. The pGAL
plasmid, for example, contains 6751 base pairs, including the E. coli genes LacZ
(which codes for -galactosidase) and ampr (which codes for -lactamase),

although pGAL is replicated without integrating into the E. coli chromosome. The
goal of this experiment was to exhibit how insertion of this plasmid into cells can
affect both cell energy production (by interrupting lactose sugar break down) and
cell protection from antibiotics.

Approach
The experiment was divided into two sections: cell preparation (Day 1) and
observation (Day 2). Cell preparation was split into three separate parts:
competence, transformation, and plating. Because E. coli lack the specialized cell
membrane proteins required for transformation, the competence protocol
stressed actively growing E. coli cells through immersion in an ice-cold calcium
chloride solution to promote membrane permeability to plasmid DNA. During
transformation, the E. coli culture (300 L) was placed into two microcentrifuge
tubes: a CB tube containing the control buffer (25 L) and a pGAL tube
containing the pGAL plasmid solution (25 L @ 0.001 g/L). Both tubes were
then heated in a water bath for 90 sec at 42 C before adding 750 L of recovery
broth to each tube. In the subsequent plating step, 250 L of the CB mixture was
transferred onto a Luria agar plate with X-Gal (Control 1), then 250 L of the CB
mixture was transferred to a plate with X-Gal and ampicillin (Control 2), and
finally 250 L of the pGAL mixture was transferred to an agar plate with X-Gal
and ampicillin (pGAL). After the mixtures were carefully spread on the three
plates, they were allowed to settle for one hour before being placed in a 37 C
incubator for 24 hours. Day 2 observations documented both the qualitative and
quantitative aspects of the resultant cell colonies.

Results and Discussion

Observation data displayed in Figures 1-3 reflects the impact of the pGAL
plasmid on E. coli. As seen in Figure 1, the Control 1 gel medium (Luria agar with
E. coli bacteria and X-Gal) promoted the growth of approximately twenty clear

white E. coli colonies. Given the absence of the pGAL DNA plasmid in this trial, E.
coli were unable to produce the -galactosidase needed to cleave the X-Gal and
form a colored product. Figure 2 data from the Control 2 gel medium (Luria agar
with E. coli bacteria, X-Gal, and ampicillin) demonstrates a toxic environment in
which no E. coli cell growth was allowed. Absence of the pGAL DNA plasmid
inhibited the bacterias ability to produce and secrete the -lactamase needed
inactivate ampicillin. As reflected in Figure 3 results, the pGAL gel medium (Luria
agar with E. coli bacteria, X-Gal, ampicillin, and the pGAL DNA plasmid) promoted
the growth of one clear white and four blue E. coli colonies. In this trial, the blue
colonies most likely took up the pGAL plasmid and began production of both lactamase (needed to inactivate ampicillin) and -galactosidase (required to
cleave the X-Gal and form a colored product). The clear white colony, on the
other hand, most likely assimilated the pGAL plasmid and began -lactamase
production, disrupting the ampicillin; however, a LacZ gene mutation inhibited
production of the -galactosidase needed to cleave the X-Gal and form a colored
product. It should be noted that there was a small chance the white colony may
have genetically mutated in such a way that it did not, in fact, take up the
plasmid but rather otherwise evolved to produce -lactamase or some other
compound needed inactivate the ampicillin.
On completion of experiment observations, transformation efficiencies
were calculated for the pGAL DNA plasmid. Keeping in mind that each colony
grew from one transformed cell, the transformation efficiency was defined as the
number of transformations obtained per microgram of DNA. Transformation
efficiency was calculated using the following formula:

Number of
Colonies
Volume Added
Final Prepared Volume
the Plate

Transformation Efficiency=( Amount of DNA (g)) x ( )

Class data transformation efficiencies ranged from 516 to 3784 transformations
obtained per microgram of DNA with a transformation efficiency of 688 for the
current experiment.

Conclusion
The Bacterial Transformation Lab not only demonstrated the genetic
exchange mechanism of bacterial transformation; it also provided key lessons on
the essential role of accurate controls and the critical importance of
chromatographic aids for DNA recombination and genetic engineering testing. As
shown in the mixed results of trial 3, not all colonies assimilate plasmids equally.
Good controls, coupled with blue/white color screening, can help demarcate
results more fully.
A second take-away and one particularly useful in future experiments
was the recognition that microscopic organisms must be handled meticulously,
precisely, and carefully, especially when using a fertile growth media like Luria
agar. As shown in this experiment, taking extreme precautions to prevent crosscontamination prevented foreign particles from landing on the sticky agar plate
and forming new colonies. This ensured that experimental results were accurate
and useful. Similarly, leaving pipette tips and microcentrifuge tubes exposed to
the air could have allowed microscopic organisms to contaminate the trials.

Bibliography
EDVOTEK. (2005). EDVO-Kit #221: Transformation of E. coli with pGAL
(blue colony). EDVOTEK.

Fig. 1: Control 1. A Luria agar medium containing E. coli bacteria and

X-Gal. Clear white E. coli bacteria colonies formed.

Fig. 2: Control 2. A Luria agar medium containing E. coli bacteria, XGal, and ampicillin. No E. coli bacteria colonies formed.

Fig. 3: pGAL. A Luria agar medium containing E. coli bacteria, X-Gal,

ampicillin, and the pGAL DNA plasmid. One clear white, and four blue
E. coli bacteria colonies formed.