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 amp
r
(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:
Tiansfoimation Eficiency
Numbei of Blue Colonies
Amount of BNA g
x
Final Piepaieu volume
volume Auueu to the Plate

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 cross-contamination 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, X-Gal, 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.