Protein Production Using Transformed Escherichia coli
John T. Johnson
Georgia Gwinnett College
Dates Performed:
Partner:
Instructor:
27-Mar-2013
03-Apr-2013
05-Apr-2013
10-Apr-2013
Sanjin Tankovic
Dr. Cindy Achat-Mendes
Introduction
The first sentence of Avery, MacLeod and McCarty’s seminal
article from 1944 still holds true today:
would engineer an organism to produce a product in large
quantities. The techniques employed included transforming
a bacteria with genes to produce the product (GFP) and genes
for the selection of transformed organisms (bla). A gene
was included (araC) to demonstrate selective expression of
the target protein. The plasmid used (See Figure 1) was an
off-the-shelf plasmid that included the necessary genes. The
protein product was further purified to obtain a quality product free of cellular debris and other contaminants (Bio-Rad,
2002).
Biologists have long attempted by chemical
means to induce in higher organisms predictable
and specific changes which thereafter could be
transmitted in series as hereditary characters
(Avery, MacLead, & McCarty, 1944).
This technique of passing properties of one organism to another by chemical means was first described by Griffith in
1928. In the article, Griffith describes a technique by which
properties of a virulent strain of the bacterium Streptococcus pneumoniae may be transferred to attenuated, non-lethal
cells (Griffith, 1928).
Avery, et al. further narrowed the substance responsible for
transference of function when they isolated fibrous strands
of “active material”. Further testing suggested the material
was desoxyribonucleate (Avery et al., 1944), which we now
know as DNA.
Figure 1. The plasmid transformed into E. coli in this experiment. Decscriptions: ori - origin of replication, araC - arabinose operon, bla - β-lactamase gene for penicillin resistance,
GFP - green fluorescent protein gene.
Materials & Methods
Green fluorescent protein (GFP) was first isolated from the
luminous organs harvested from thousands of jellyfish (Aequorea aequorea) caught over a period of years by Shimomura (Shimomura, 2008). Escherichia coli was first transformed to produce GFP by Chalfie in 1994, and the process
was further refined by Roger Tsien. In 2008 the three shared
the Nobel Prize.
Materials
Bacterial DNA is stored as a large circular molecule of chromosomal DNA. In addition, some bacteria such as E. coli
also have plasmids – small rings of DNA that add functionality to the bacterium. By using restriction enzymes and DNA
ligase, plasmids can be created with DNA of interest to researchers (Campbell & Reece, 2009).
Aseptic techniques were utilized throughout, including, but
not limited to: using sterile disposable pipets, autoclaved
pipette tips and disposable inoculation loops.
The impetus for this experiment was to conduct a small-scale
simulation of the steps by which a biotechnology company
Author contact: jjohnso6@ggc.edu
Materials required for all phases of the experiment are listed
in Table 1 on the next page.
Methods
Transformation. Microcentrifuge tubes were labeled
+pGLO and -pGLO, then 250 µl of transformation solution
(CaCl2) was added to each tube using a sterile pipet. Tubes
were placed on ice. Three large, mucoid, isolated colonies
were transferred from the E. coli starter plate to both of
the +pGLO and -pGLO tubes, then 10 µl of plasmid was
added to the +pGLO tube. Both tubes were incubated on
ice for 10 min. Meanwhile, plates were labeled as shown in
Figure 2 on the following page. Tubes were heat shocked at
2
JOHN T. JOHNSON
Table 1
Materials required.
Quantity
1
2
1
500 µl
500 µl
10 µl
600 mg
30 mg
3.5 ml
50 µl
250 µl
2 ml
250 µl
750 µl
1
1
1
1
2
1
using UV light.
Item
Luria Broth (LB) plate
LB/ampicillin (LB/amp) plates
LB/amp/arabinose (LB/amp/ara) plate
Transformation solution
LB nutrient broth
pGLO plasmid
Arabinose
Ampicillin
Tris, Ethylenediaminetetraacetic acid (TE)
buffer
lysozyme
Binding buffer
Equilibration buffer
Wash buffer
TE (elution buffer)
LB broth capsule
E. coli starter plate
Hydrophobic interaction chromatography
(HIC) column
Column end cap
Culture tubes
Waste beaker
Inoculation loops
Disposable pipets
Microcentrifuge tube holder
Crushed ice in small container
Marking pen
Microcentrifuge tubes
Water bath 42 ◦C
P20 Pipetter and tips
Incubator at 37 ◦C
Incubating shaker
Aluminum foil
Centrifuge
Long wavelength ultraviolet (UV) light
source
42 ◦C for 50 s, then immediately transferred to an ice bath
for 2 min. Tubes were removed from the ice bath to room
temperature, then 250 µl of LB nutrient broth was added
to each tube. Tubes were incubated at room temperature
for 10 min. Tubes were flicked several times and visualized
using UV light.
Selection. A 100 µl aliquot was transferred from the
+pGLO tube to each of the +pGLO plates, and from the
-pGLO tube to each of the -pGLO plates. The cultures were
streaked on each plate. The plates were incubated upside
down (agar side up) at 37 ◦C for 2 d. Plates were visualized
Figure 2. Plating scheme.
Production. Arabinose and ampicillin were reconstituted
by adding 3 ml TE buffer to each vial and swirling to dissolve
the pellet.
Growth medium was prepared by microwaving 50 ml DH2O
to boiling. One LB broth capsule was added and allowed
to dissolve for 20 min. Mixture was swirled, microwaved
to boiling and allowed to dissolve until cool to the touch ≈
50 ◦C. Afterward, 0.5 ml ampicillin solution and 0.5 ml arabinose solution were added and swirled to mix.
Culture tubes were labeled Ara+ and Ara- and prepared with
2 ml growth medium each. The Ara+ tube was inoculated
with one large, mucoid colony from the LB/amp/ara plate
and the Ara- tube was inoculated in similar fashion from the
LB/amp plate. Plates were wrapped in foil and stored at 4 ◦C.
Tubes were shaken at 32 ◦C and 250 rpm for 24 h, then moved
to storage at 4 ◦C. Tubes were visualized using UV light.
Tube Ara- was discarded.
Purification. Tube Ara+ was thawed then centrifuged for
5 min at maximum rpm. The supernatant was discarded.
The pellet was observed under UV light. The pellet was resuspended by adding 250 µl TE buffer and pipetting up and
down several times. One drop of lysozyme was added using a
new pipet. The tube was flicked to mix and frozen at −20 ◦C
for 5 d.
Concentration. Tube Ara+ was thawed and centrifuged
for 10 min at maximum rpm. Meanwhile, the hydrophobic
interaction chromatography (HIC) column was prepared by
shaking it to re-suspend the matrix, then shaking down to
settle the matrix. The top cap was removed, and the bottom
cap was snapped off. The column was allowed to drain for
5 min, then 1 ml equilibration buffer was loaded on the column and allowed to drain until the meniscus was just above
the bed. This procedure was repeated once more, then the
top and bottom of the column were capped. The Ara+ tube
was observed under UV light.
An aliquot of 250 µl of supernatant was transferred from the
Ara+ tube to a new microcentrifuge tube labeled Impure,
then 250 µl of binding buffer was added to the Impure tube.
Three collection tubes were labeled Product1, Product2 and
Product3. The column was drained until the meniscus
reached the top of the matrix bed. The column was placed on
collection tube Product1 and 250 µl of supernatant in binding
buffer was layered on the column. The column was observed
3
PROTEIN PRODUCTION USING TRANSFORMED ESCHERICHIA COLI
under UV light. The entire volume of liquid was allowed to
drain through. Tube Product1 was checked for fluorescence
under UV light.
The column was moved to collection tube Product2. 250 µl
wash buffer was added and allowed to flow through. The
column and tube Product2 were observed under UV light.
of the HIC column was observed to fluoresce under UV light
when the supernatant was loaded. Tube 1 was not observed
to fluoresce. Upon washing into tube 2, the column was observed to fluoresce at matrix at the top, and tube 2 was not
observed to fluoresce under UV light. Upon eluting with TE
buffer into tube 3, the column was observed not to fluoresce,
and tube 3 was observed to fluoresce under UV light.
The column was moved to collection tube Product3 and
750 ul of TE buffer was allowed to flow through. The column
and tube Product3 were observed under UV light.
Results
The +pGLO and -pGLO tubes visualized after transformation showed no signs of fluorescence.
Figure 4. False color image of the three microcentrifuge
tubes from the Concentration phase as imaged in the
Chemi-Doc imager using UV stimulation. L-R: Tube 1 exhibited no fluorescence, Tube 2 exhibited no fluorescence,
Tube 3 exhibited fluorescence
Transformation Efficiency
Figure 3. Inoculated plates after incubation. Upper-left:
-pGLO on LB plate, lower-left: -pGLO on LB/amp plate,
upper-right: +pGLO on LB/amp plate, lower-right: +pGLO
on LB/amp/ara plate.
Figure 3 shows the results of the incubated plates. The
-pGLO on LB plate showed a "lawn" growth of colonies.
The -pGLO on LB/amp showed no growth. The +pGLO
on LB/amp plate showed a single colony. The +pGLO on
LB/amp/ara plate showed five colonies. When visualized
under UV light, the colonies on the +pGLO on LB/amp/ara
plate were observed to fluoresce at a green wavelength. No
fluorescence was observed on the other three plates.
Number of colonies = 5
(1)
DNA plated = 10 µg × 0.08 µg/µl = 0.8 µl
(2)
100 µl
Fraction =
= 0.196
(3)
510 µl
Spread DNA = 0.8 µg × 0.196 = 0.157 µg
(4)
5trans
Trans. Eff. =
≈ 32 transformants/µg
0.157 µg
(5)
Figure 5. Calculation of transformation efficiency.
Discussion
Both Ara+ and Ara- culture tubes fluoresced under UV light.
During the Purification phase, the pellet in the Ara+ tube
fluoresced.
During the Concentration phase, the supernatant in the Ara+
tube fluoresced, while the pellet did not. The liquid on top
The -pGLO plated on the LB plate proliferated since LB is
well suited to growing E. coli, as was the incubation environment of 37 ◦C. The -pGLO plated on the LB/amp plate
failed to proliferate. This is expected since ampicillian inhibits cell-wall synthesis in E. coli (Rogers & Mandelstam,
4
JOHN T. JOHNSON
1962). The +pGLO culture survived on the LB/amp plate
due to the ampicillin resistance conferred by the transformation of the pGLO plasmid and it’s accompanying bac gene
into the organism. This demonstrated that ampicillin could
be used for selection of transformed organisms. The +pGLO
culture also grew on the LB/amp/ara plate. Once again, the
bac gene conferred immunity to ampicillin. In addition, the
arabinose in the LB/amp/ara plate enabled transcription of
the GFP gene and the resulting green fluorescent protein.
This is evidenced by the greenish glow of the colonies on
the lower-right plate in Figure3.
The computed transformation efficiency (See Figure 5 on the
preceding page) is quite low (Achat-Mendes, 2013). A possible cause is presented in the Error Analysis section.
Both Ara+ and Ara- tubes fluoresced showing that the E.
coli from both the LB/amp and LB/amp/are plates contained
the pGLO gene, and that the expression of the pGLO gene
was solely due to the presence or absence of arabinose in the
medium. This demonstrated selective expression.
The HIC was successful and resulted in purification of GFP
as shown in Figure 4 on the previous page. This confirmed
the ability to extract and purify the product from the organisimal debris.
Error Analysis
The low transformation efficiency (See Figure 5 on the preceding page) is probably due to the culture running off the
medium and onto the lid of the petri dish when it was inverted
to be placed in the incubator. A spreading technique, rather
than a streaking technique should result in better growth.
The -pGLO transformant could have been plated on an
LB/ara plate (that is, not containing ampicillin) to prove that
GFP production in the presence of arabinose is not indigenous to E. coli. This is well known and would not have contributed to the goal of the experiment, though it would have
been interesting food for thought.
Conclusion
This experiment confirmed that a bacterium can be transformed and made to selectively express a protein of value.
The experiment also confirmed that the protein may be further purified before sale or use.
References
Achat-Mendes, C. (2013, Apr). Bacterial transformation
[Manual]. 1000 University Center Ln, Lawrenceville,
Ga, 30044.
Avery, O., MacLead, C., & McCarty, M. (1944, Nov). Induction of transformation by a desoxyribonucleic acid
fraction isolated from pneumococcus type iii. Journal
of Experimental Medicine, 79(2), 137-158.
Bio-Rad. (2002). Biotechnology explorer: pglo bacterial
transformation kit [Manual].
Campbell, N., & Reece, J. (2009). Biology (8th ed.). Pearson.
Griffith, F. (1928, Jan). The significance of pneumococcal
types. Journal of Hygiene, 27(2), 113-159.
Rogers, H. J., & Mandelstam, J.
(1962).
Inhibition
of
cell-wall-mucopeptide
formation
in escherichia coli by benzylpenicillin and
6-[d(-)-α-aminophenylacetamidolpenicillanic
acid
(ampicillin). Biochemistry Journal, 84, 299.
Shimomura, O. (2008). Discovery of green fluorescent protein, gfp. Nobel lecture.