Protein Production Using Transformed Escherichia coliJohn T. Johnson

Georgia Gwinnett College

Dates Performed: 27-Mar-201303-Apr-201305-Apr-201310-Apr-2013

Partner: Sanjin TankovicInstructor: Dr. Cindy Achat-Mendes

Introduction

The first sentence of Avery, MacLeod and McCarty’s seminalarticle from 1944 still holds true today:

Biologists have long attempted by chemicalmeans to induce in higher organisms predictableand specific changes which thereafter could betransmitted in series as hereditary characters(Avery, MacLead, & McCarty, 1944).

This technique of passing properties of one organism to an-other by chemical means was first described by Griffith in1928. In the article, Griffith describes a technique by whichproperties of a virulent strain of the bacterium Streptococ-cus pneumoniae may be transferred to attenuated, non-lethalcells (Griffith, 1928).

Avery, et al. further narrowed the substance responsible fortransference of function when they isolated fibrous strandsof “active material”. Further testing suggested the materialwas desoxyribonucleate (Avery et al., 1944), which we nowknow as DNA.

Green fluorescent protein (GFP) was first isolated from theluminous organs harvested from thousands of jellyfish (Ae-quorea aequorea) caught over a period of years by Shimo-mura (Shimomura, 2008). Escherichia coli was first trans-formed to produce GFP by Chalfie in 1994, and the processwas further refined by Roger Tsien. In 2008 the three sharedthe Nobel Prize.

Bacterial DNA is stored as a large circular molecule of chro-mosomal DNA. In addition, some bacteria such as E. colialso have plasmids – small rings of DNA that add functional-ity to the bacterium. By using restriction enzymes and DNAligase, plasmids can be created with DNA of interest to re-searchers (Campbell & Reece, 2009).

The impetus for this experiment was to conduct a small-scalesimulation of the steps by which a biotechnology company

Author contact: jjohnso6@ggc.edu

would engineer an organism to produce a product in largequantities. The techniques employed included transforminga bacteria with genes to produce the product (GFP) and genesfor the selection of transformed organisms (bla). A genewas included (araC) to demonstrate selective expression ofthe target protein. The plasmid used (See Figure 1) was anoff-the-shelf plasmid that included the necessary genes. Theprotein product was further purified to obtain a quality prod-uct free of cellular debris and other contaminants (Bio-Rad,2002).

Figure 1. The plasmid transformed into E. coli in this experi-ment. Decscriptions: ori - origin of replication, araC - arabi-nose operon, bla - β-lactamase gene for penicillin resistance,GFP - green fluorescent protein gene.

Materials & Methods

Materials

Materials required for all phases of the experiment are listedin Table 1 on the next page.

Methods

Aseptic techniques were utilized throughout, including, butnot limited to: using sterile disposable pipets, autoclavedpipette tips and disposable inoculation loops.

Transformation. Microcentrifuge tubes were labeled+pGLO and -pGLO, then 250 µl of transformation solution(CaCl2) was added to each tube using a sterile pipet. Tubeswere placed on ice. Three large, mucoid, isolated colonieswere transferred from the E. coli starter plate to both ofthe +pGLO and -pGLO tubes, then 10 µl of plasmid wasadded to the +pGLO tube. Both tubes were incubated onice for 10 min. Meanwhile, plates were labeled as shown inFigure 2 on the following page. Tubes were heat shocked at

2 JOHN T. JOHNSON

Table 1Materials required.

Quantity Item

1 Luria Broth (LB) plate2 LB/ampicillin (LB/amp) plates1 LB/amp/arabinose (LB/amp/ara) plate

500 µl Transformation solution500 µl LB nutrient broth10 µl pGLO plasmid

600 mg Arabinose30 mg Ampicillin3.5 ml Tris, Ethylenediaminetetraacetic acid (TE)

buffer50 µl lysozyme

250 µl Binding buffer2 ml Equilibration buffer

250 µl Wash buffer750 µl TE (elution buffer)

1 LB broth capsule1 E. coli starter plate1 Hydrophobic interaction chromatography

(HIC) column1 Column end cap2 Culture tubes1 Waste beaker

Inoculation loopsDisposable pipetsMicrocentrifuge tube holderCrushed ice in small containerMarking penMicrocentrifuge tubesWater bath 42 ◦CP20 Pipetter and tipsIncubator at 37 ◦CIncubating shakerAluminum foilCentrifugeLong wavelength ultraviolet (UV) lightsource

42 ◦C for 50 s, then immediately transferred to an ice bathfor 2 min. Tubes were removed from the ice bath to roomtemperature, then 250 µl of LB nutrient broth was addedto each tube. Tubes were incubated at room temperaturefor 10 min. Tubes were flicked several times and visualizedusing 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 werestreaked on each plate. The plates were incubated upsidedown (agar side up) at 37 ◦C for 2 d. Plates were visualized

using UV light.

Figure 2. Plating scheme.

Production. Arabinose and ampicillin were reconstitutedby adding 3 ml TE buffer to each vial and swirling to dissolvethe pellet.

Growth medium was prepared by microwaving 50 ml DH2Oto boiling. One LB broth capsule was added and allowedto dissolve for 20 min. Mixture was swirled, microwavedto boiling and allowed to dissolve until cool to the touch ≈50 ◦C. Afterward, 0.5 ml ampicillin solution and 0.5 ml ara-binose solution were added and swirled to mix.

Culture tubes were labeled Ara+ and Ara- and prepared with2 ml growth medium each. The Ara+ tube was inoculatedwith one large, mucoid colony from the LB/amp/ara plateand the Ara- tube was inoculated in similar fashion from theLB/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 movedto storage at 4 ◦C. Tubes were visualized using UV light.Tube Ara- was discarded.

Purification. Tube Ara+ was thawed then centrifuged for5 min at maximum rpm. The supernatant was discarded.The pellet was observed under UV light. The pellet was re-suspended by adding 250 µl TE buffer and pipetting up anddown several times. One drop of lysozyme was added using anew pipet. The tube was flicked to mix and frozen at −20 ◦Cfor 5 d.

Concentration. Tube Ara+ was thawed and centrifugedfor 10 min at maximum rpm. Meanwhile, the hydrophobicinteraction chromatography (HIC) column was prepared byshaking it to re-suspend the matrix, then shaking down tosettle the matrix. The top cap was removed, and the bottomcap was snapped off. The column was allowed to drain for5 min, then 1 ml equilibration buffer was loaded on the col-umn and allowed to drain until the meniscus was just abovethe bed. This procedure was repeated once more, then thetop and bottom of the column were capped. The Ara+ tubewas observed under UV light.

An aliquot of 250 µl of supernatant was transferred from theAra+ 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 andProduct3. The column was drained until the meniscusreached the top of the matrix bed. The column was placed oncollection tube Product1 and 250 µl of supernatant in bindingbuffer was layered on the column. The column was observed

PROTEIN PRODUCTION USING TRANSFORMED ESCHERICHIA COLI 3

under UV light. The entire volume of liquid was allowed todrain through. Tube Product1 was checked for fluorescenceunder UV light.

The column was moved to collection tube Product2. 250 µlwash buffer was added and allowed to flow through. Thecolumn and tube Product2 were observed under UV light.

The column was moved to collection tube Product3 and750 ul of TE buffer was allowed to flow through. The columnand tube Product3 were observed under UV light.

Results

The +pGLO and -pGLO tubes visualized after transforma-tion showed no signs of fluorescence.

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: +pGLOon 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 +pGLOon LB/amp plate showed a single colony. The +pGLO onLB/amp/ara plate showed five colonies. When visualizedunder UV light, the colonies on the +pGLO on LB/amp/araplate were observed to fluoresce at a green wavelength. Nofluorescence was observed on the other three plates.

Both Ara+ and Ara- culture tubes fluoresced under UV light.

During the Purification phase, the pellet in the Ara+ tubefluoresced.

During the Concentration phase, the supernatant in the Ara+

tube fluoresced, while the pellet did not. The liquid on top

of the HIC column was observed to fluoresce under UV lightwhen the supernatant was loaded. Tube 1 was not observedto fluoresce. Upon washing into tube 2, the column was ob-served to fluoresce at matrix at the top, and tube 2 was notobserved to fluoresce under UV light. Upon eluting with TEbuffer into tube 3, the column was observed not to fluoresce,and tube 3 was observed to fluoresce under UV light.

Figure 4. False color image of the three microcentrifugetubes from the Concentration phase as imaged in theChemi-Doc imager using UV stimulation. L-R: Tube 1 ex-hibited no fluorescence, Tube 2 exhibited no fluorescence,Tube 3 exhibited fluorescence

Transformation Efficiency

Number of colonies = 5 (1)DNA plated = 10 µg × 0.08 µg/µl = 0.8 µl (2)

Fraction =100 µl510 µl

= 0.196 (3)

Spread DNA = 0.8 µg × 0.196 = 0.157 µg (4)

Trans. Eff. =5trans

0.157 µg≈ 32 transformants/µg

(5)

Figure 5. Calculation of transformation efficiency.

Discussion

The -pGLO plated on the LB plate proliferated since LB iswell suited to growing E. coli, as was the incubation envi-ronment of 37 ◦C. The -pGLO plated on the LB/amp platefailed to proliferate. This is expected since ampicillian in-hibits cell-wall synthesis in E. coli (Rogers & Mandelstam,

4 JOHN T. JOHNSON

1962). The +pGLO culture survived on the LB/amp platedue to the ampicillin resistance conferred by the transforma-tion of the pGLO plasmid and it’s accompanying bac geneinto the organism. This demonstrated that ampicillin couldbe used for selection of transformed organisms. The +pGLOculture also grew on the LB/amp/ara plate. Once again, thebac gene conferred immunity to ampicillin. In addition, thearabinose in the LB/amp/ara plate enabled transcription ofthe GFP gene and the resulting green fluorescent protein.This is evidenced by the greenish glow of the colonies onthe lower-right plate in Figure3.

The computed transformation efficiency (See Figure 5 on thepreceding page) is quite low (Achat-Mendes, 2013). A pos-sible 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 containedthe pGLO gene, and that the expression of the pGLO genewas solely due to the presence or absence of arabinose in themedium. This demonstrated selective expression.

The HIC was successful and resulted in purification of GFPas shown in Figure 4 on the previous page. This confirmedthe ability to extract and purify the product from the organ-isimal debris.

Error Analysis

The low transformation efficiency (See Figure 5 on the pre-ceding page) is probably due to the culture running off themedium and onto the lid of the petri dish when it was invertedto be placed in the incubator. A spreading technique, ratherthan a streaking technique should result in better growth.

The -pGLO transformant could have been plated on anLB/ara plate (that is, not containing ampicillin) to prove that

GFP production in the presence of arabinose is not indige-nous to E. coli. This is well known and would not have con-tributed to the goal of the experiment, though it would havebeen interesting food for thought.

Conclusion

This experiment confirmed that a bacterium can be trans-formed and made to selectively express a protein of value.The experiment also confirmed that the protein may be fur-ther 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). In-duction of transformation by a desoxyribonucleic acidfraction isolated from pneumococcus type iii. Journalof Experimental Medicine, 79(2), 137-158.

Bio-Rad. (2002). Biotechnology explorer: pglo bacterialtransformation kit [Manual].

Campbell, N., & Reece, J. (2009). Biology (8th ed.). Pear-son.

Griffith, F. (1928, Jan). The significance of pneumococcaltypes. Journal of Hygiene, 27(2), 113-159.

Rogers, H. J., & Mandelstam, J. (1962). In-hibition of cell-wall-mucopeptide formationin escherichia coli by benzylpenicillin and6-[d(-)-α-aminophenylacetamidolpenicillanic acid(ampicillin). Biochemistry Journal, 84, 299.

Shimomura, O. (2008). Discovery of green fluorescent pro-tein, gfp. Nobel lecture.