Transformation of Escherichia coli by pUC18 and

a plasmid containing the Lux operon

ABSTRACT
This experiment aims to transform a sample of Escherichia coli through the pUC18 plasmid and a plasmid
containing the lux operon. Practical methods of transformation are showcased such as the use of calcium

chloride and heat shocking and the results are observed through transformation efficiency calculations. The
marker used to aid in transformation efficiency calculation is the ampicillin resistance level of the batch of cells
on a given agar plate. Lastly, transformation via the lux operon will be observed by way of the transformed cells
ability to bio-luminesce.
INTRODUCTION
Biotechnology involves the manipulation of cellular processes through sophisticated methods for industrial
purpose. A central aspect is the manipulation of genetic material to change the resulting genotype of a host cell.
The most common example put forth in recent decades is genetically modified crops that resist the negative
effects of bacterial infestation and other non-ideal environmental hardship. A central part of biotechnology and
genetic engineering is the technique of transformation- helping a host cell to uptake DNA fragments from an
outside source. This can happen naturally in nature when a cell dies or lyses, however the more common
occurrence is within controlled lab experiments such as this. This typically involves assisting the foreign DNA in
transporting across a hosts membrane or cell wall and then attempting to incorporate itself into the cells
genome by DNA repair enzymes. Besides transformation, other techniques are conjugation- the transfer of
genetic material through direct contact among cells and transfection- injecting the foreign DNA via a
bacteriophage. (Raven, et al.)
About 1% of bacterial species are able to uptake DNA without much assistance, termed natural competence.
Utilizing a special set of machinery, these bacteria can transfer foreign DNA across their membrane. Typically,
the foreign DNA strand may bind to the surface of a DNA receptor and then make use of the protein DNA
translocase to transfer it across and into the cytoplasm. The exact process is dependent on whether the bacterium
is gram negative or positive. In gram positive bacteria, a thick peptidoglycan cell wall approximately 150-500
angstroms wide is present. However, its typically porous texture allows for easy diffusion of metabolites and is
also conducive for foreign DNA uptake. On the other hand, gram negative bacteria membranes are more
complex with two layers atop the cytoplasmic membrane: a thin peptidoglycan layer along with an extra
lipopolysaccharide layer containing endotoxins- molecules able to activate a strong immune response. In gramnegative bacteria, the process of uptake is more complicated than simple diffusion and requires pathways to be
formed by secretins to manipulate homeostasis on the outer membrane and allow uptake. (Mitchell, et al.)

The host DNA in this experiment, Escherichia coli is gram-negative. E. coli has become a hallmark for genetic
engineering and recombination in particular. It is a rod-shaped bacteria commonly found in eukaryote intestines
and the ideal laboratory recombination medium thanks to its relatively small 5 million base pair chromosome
and short growth time. Under ideal conditions, the bacterias exponential phase of growth allows it to double in
size every 2-3 minutes. (Interestingly, laboratory strains of E. coli sold around the world today nearly all have a
common ancestor: E. coli K-12, a sample procured from the feces of a British patient suffering from diphtheria
in 1922.) (Bergmans, et al.)
Of course, laboratory recombination does not depend on natural competence and instead procedures exist to
prepare the cell for successful DNA uptake. Two common methods exist, both of which will be utilized in the
methodology for this experiment. The first is the usage of a cation-rich solution (divalent cation) such as
Calcium Chloride. Since the cell membrane is negatively charged due to a phospholipid layer, a cation-rich
solution can form ionic bonds that in turn shield the also-negatively charged plasmid DNA, allowing
intermembrane access. A heat shock assists this process further by loosening pores and even causing slight
damage to cells walls that further increase accessibility. (Raven, et al.)
The actual foreign DNA that will be introduced to the host is by means of a vector- simply put a vessel through
which the DNA can be transported. More specifically a plasmid, a short piece of circular DNA engineered to
easily self-replicate. Two artificial plasmid vectors: pUC18 and a plasmid containing the lux operon are used in
this experiment. pUC18 is among the most popular strains of cloning vectors used today. It contains an
ampicillin resistance gene (Amp) along with a multiple cloning site (MCS), a short piece of DNA with a series
of restriction sites. An MCS, or polylinker, on an engineered plasmid allows scientists to include any DNA of
interest they would like the plasmid to transport into the host. (Zhixing, et al.) Typically, ampicillin resistance is
used only as a marker to identify host cells that successfully transformed whatever DNA of interest was included
in the MCS. (Bergmans, et al.) The pUC18 used in this experiment has no other gene to transport besides Amp.
Instead, the second plasmid used contains both an ampicillin resistance gene as well as the lux operon. The lux
operon codes for bioluminescence- specifically it catalyzes the oxidation of luciferin. When lucferin decays back
to its ground state the energy it releases is partly in the form of a bluish-green glow. (Dunlap, et al.) Thus, this
experiment seeks to observe the efficacy of transformation of these two plasmids along with an illustration of

ampicillin resistance as a marker for biotechnology work. The expectation is successful uptake with partial
ampicillin resistance from both plasmids along with some degree of bioluminescence by the E. coli sample
transformed by the lux plasmid.
METHODS
First, a vial of CaCl2 solution and a vial containing E. coli were placed in an ice bath. With a sterile pipet, 590 L
of CaCl2 solution was placed into the E. coli vial. The tube was mixed by gentle tapping and then allowed to
incubate on ice for 10 minutes. Next, two Eppendorf tubes were procured and labelled C and lux for the
control and lux plasmids, respectively, and placed in an ice bath. Using a micropipette, 5 L each of the control
plasmid and lux plasmid was placed into their respective tubes while still in the ice bath. Gentle tapping was
once again done to aid mixing. Next, 70 L of the now competent E. coli cells were transferred into each of the
two tubes, tapped and allowed to sit in the ice bath for a further 15 minutes. Meanwhile a third Eppendorf tube
was procured and labelled NP for no plasmid and given 35 L of the competent cells. Lastly, the three test
tubes were then transferred into a hot bath at 37 degrees Celsius and allowed to sit for 5 minutes. Using a sterile
pipet 275 L of nutrient broth was added to the C and lux tubes and 150 L of nutrient broth to the NP
tube. They were then allowed to incubate for 45 minutes.
Once the cells sufficiently incubated in the nutrient broth, six agar plates were procured and labelled as follows:
LBc for the control plasmid without Ampicillin, LB/Amp c for the control plasmid with Ampicillin, LBNP
for no plasmid and without Ampicillin, LBlux for the lux plasmid without Ampicillin, LB/Amplux for the lux
plasmid with Ampicillin and lastly LB/AmpNP for the lux plasmid with Ampicillin. Using a sterile pipet, 130
L of the mixed bacterial suspension was removed from the C and lux tubes and placed in their respective
plates. For effective transfer, a cell spreader was used with the following procedure: The spreader was dipped in
ethanol and passed through a flame and allowed to cool for 30 seconds. It was then used to evenly distribute
cells throughout the plate and then repeated for each plate. For the NP plates not containing plasmids, cells
were transferred directly on the plate without the use of a spreader. Once complete, the lids were replaced and
the samples were allowed to incubate at 37 degrees Celsius in a refrigerator.
Once the cells have been given sufficient time to grow, they were retrieved and growth was observed one by one
on the different plates both under a light and in darkness. Results were recorded. (Alberte, et al.)

RESULTS
The results of the experiment were unexpected and indicate a possible failure (to be discussed later). As seen in
Figure 1, the growths were either lawn or none at all. With the control plasmid there was heavy lawn growth but
when ampicillin was introduced the growth was completely non-existent without a single colony being visible.
Likewise, when the lux plasmid was present on a plate there was healthy lawn growth but when ampicillin was
also present the growth was completely absent. E. coli without a plasmid vector present behaved a similar way:
growth observed a heavy lawn pattern without ampicillin but was completely prevented (and more so, existing
growth was heavily negatively impacted) when ampicillin was present. In terms of bioluminescence, the lux
plasmid plates showed no glowing effect in darkness in any of the plates. Though the data was largely corrupted,
transformation efficiencies (the efficiency at which cells uptake foreign DNA) were recorded in Table 1, with the
methodology listed below

LBc

LB/Ampc

LBNP

LBlux

LB/Amplux

LB/AmpNP

Glowing
No glowing

No glowing

Figure 1: E. coli growth on plates. First Row: pUC18 (control) plasmid. Second Row: Lux plasmid.

Transform Efficiency*
Control (pUC18) plasmid

0 transformants/g

Lux plasmid

0 transformants/g

* g plasmid DNA spread per plate = [0.05 (g/L) x 10 (L)] [130 (L) 358 9 (L)] = 0.092 g
Transform Efficiency = (# of colonies) 0.092 g
Table 1: Transformation Efficiency of plates receiving a plasmid vector

DISCUSSION

The experiment was largely a failure in terms of the incongruence between established expectations and the
results that were procured. In a nutshell, there was full lawn growth on plates without ampicillin, irrespective of
whether they contained the pUC18 control plasmid, the lux plasmid or no plasmid. On plates with ampicillin, no
growth occurred and any existing growth was wiped out. Likewise, there was no bioluminescence on any other
plates containing the lux plasmid. This indicates that there was close to zero uptake in the foreign ampicillinresistant DNA provided by either of the two types of plasmids. It was as if no transformation attempt had been
made at all.
On the other hand, what should have occurred according to established expectations and the parameters of the
engineered plasmids and E. coli strain used was lawn growth whenever ampicillin was absent (observed), no
growth when an ampicillin was introduced without a plasmid (observed) but with colonial or dispersed growth in
the two plates where plasmid-treated E. coli was with ampicillin. The expectation being that transformation
efficiency would not be at one hundred percent, some cells would successfully uptake the ampicillin-resistant
genes where others would not result in present but sparse growth. Likewise, bioluminescence was expected in
the agar plate containing ampicillin with lux plasmid treated E. coli but was not present. It was not expected (and
not observed) in the plate containing the lux plasmid without ampicillin however because E. coli, and all cells for
that matter, have basic control of gene expression that allows genes to be turned off when they are not
necessary for homeostasis or survival in general. (Raven, et al.)
The failure of the experiment to see expected results can either be attributed to the genotype of the cells or the
methodology. In terms of the cell genotype, mutations in this sample of E.coli cells could have made them less
susceptible to uptake. Studies have shown some laboratory E. coli strains to have remarkable better
transformation capability due to a different composition of their lipopolysaccharide layer. (Zhixing, et al.)
However the second possibility, poor methodology, is more likely. Increasing transformation efficiency is a
pressing matter due to its necessity for advances in Biotechnology. Ever since the discovery of the positive
effects of Calcium cations on DNA transfer (e.g. calcium chloride), transformation methodology has become
more precise and pathways more closely studied. A common deficiency experienced by researchers is the loss of
competency of E. coli cells stored in cool temperatures for too long which is a possible reason for this
experiments failure. Some researchers have found significantly better retaining of competence with liquid

nitrogen freezing instead. (Inoue, et al.) Other techniques involve taking further advantage of the ionic
interactions of CaCl2 and adding magnesium ions to the solution to speed up ionic bonding between the
positively charged molecules and the negatively charged phospholipid layer thus better preventing repulsion
between it and foreign DNA particles attempting entry through the membrane. (Bergmans, et al.) A fourth
technique to increase competence and DNA uptake that was not utilized and may have helped is electroporation,
introducing an electrical shock to the cells to increase permeability. In some cases, the effectiveness of
electroporation over chemical transformation is remarkable- resulting in transformation efficiency as much as
ten times higher. (Raven, et al.) And yet another technique less commonly used but known to be highly effective
involves pre-treating the vector plasmids with DNA gyrase which introduces negative supercoils that increase
transformation efficiency given successful membrane passage. (Zhixing, et al.)
Though the exact cause of the failure of the E. coli to uptake foreign DNA in this experiment remains unclear, it
provides discussion of uptake factors vital to the continuing advancement of recombination techniques and
biotechnology in general.

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