Cost effectiveness of colony lysis and colony PCR methods for screening of recombinant Escherichia coli colonies ⎯ A comparative study
P Azhahianambi1, S Ghosh*, C Ashok Kumar2 & V V S Suryanarayana2
Entomology Laboratory, Division of Parasitology, Indian Veterinary Research Institute, Izatnagar 243122, India Received16 November 2007; revised 18 August 2008
Economizing the research protocols by using low cost technologies is the need of laboratories of developing world.
Screening of recombinant E. coli colonies is the crucial step in gene cloning and expression studies. In the present study, the cost effectiveness of colony lysis method and colony PCR method in the screening of recombinant E. coli colonies was compared. The colony lysis method was 20 two times more cost effective and less time consuming and can be used to screen the recombinant E. coli colonies in large scale instead of colony PCR method.
Keywords: Colony lysis, Colony PCR, Cost effectiveness, Recombinant E. coli
Due to number of political and social commitments of the governments of developing countries including India, research support in the form of public funding to biomedical research is not proportionately increasing. Therefore, there is always an added enthusiasm among the researchers working in different universities, research institutes, pharmaceutical establishments and in diagnostic laboratories, in the search for cheaper and quicker methodologies in biological research. Among the different programme running in the research establishments, expression of targeted protein(s) for development of vaccines and diagnostics is highly prioritized. For the generation of recombinant proteins, prokaryotic expression systems are extensively used and the methodologies for the expression of proteins have been standardized1,2. Gene cloning followed by screening of the recombinant Escherichia coli colonies for the presence of target gene is usually done by colony PCR method3. Colony lysis is a simple method to screen the recombinant bacterial clones4,5. Commercial kits based on this principle are available in market (CLONE
CHECKERTM system, Lifetechnologies and Ready- LyseTM, Epicentre biotechnologies). The colony lysis method employed in the present communication is the modified method of Sekar 4. An attempt has been made to standardize the colony lysis method for screening E.
coli colonies and compared with colony PCR method.
Materials and Methods
Materials used ⎯ Tris, SDS, acrylamide, N, N, methylene bisacrylamide, glacial acetic acid, TEMED, ammonium per sulphate, Ammonium sulphate, sodium chloride, 5,5'dithiobis-2- nitrobenzoic acid (DTNB), EDTA, calcium chloride, ampicilin, kenamycin, RNA isolation kit, pQE30, Oligodt primer, RT kit, E. coli M15 cells, RNase A from Qiagen, USA, RNA storage buffer and Super Taq were obtained from Ambion, LB media, LB agar, nuclease free water and Urea from Amresco, MLV- Reverse Transcriptase from Invitrogen, Taq DNA polymerase, Hot start Taq DNA polymerase, dNTPs, Gene rulerTM100 DNA ladder plus, Gene rulerTM1 kb DNA ladder, Restriction enzymes BamHI, XbaI, from MBI-Fermentas were used in this study, Oligonuleotide primers (GCC, IDT), and agarose molecular biology grade were also used in the study.
Total RNA isolation⎯ The total RNA was isolated from the eggs of Hyalomma anatolicum anatolicum Izatnagar isolate using the commercial RNA isolation kit (RNeasy, Qiagen).
RT-PCR amplification of the Bm86 homologue of H. a. anatolicum Izatnagar isolate⎯A two step reverse transcription–polymerase chain reaction was
_____________
Present addresses:
1Centre for Biosystem Research, University of Maryland Biotechnology Institute, Rockville, MS, USA
2 Molecular Virology Laboratory,
Indian Veterinary Research Institute, Bangalore 560 012 India
*Correspondent author Telephone: 91 581 2302368 Fax : 91 581 2302368 Email: sghoshp@yahoo.co.in
INDIAN J EXP BIOL, OCTOBER 2008
732
performed to amplify the Bm86 homologue of H. a.
anatolicum. The first strand cDNA was synthesized in a reverse transcription reaction using oligo dT primer.
Subsequently, the Bm86 homologue of H. a.
anatolicum was amplified by polymerase chain reaction using a primer pair designed based on the published sequence (AF347079) with BamHI and XbaI restriction enzyme sites in the forward and reverse primer, respectively6.
Cloning of the Bm86 homologue of H. a.
anatolicum ⎯ The PCR amplified product and the prokaryotic expression vector pQE30 were separately digested with BamHI and XbaI restriction enzymes and subsequently the gel purified digested PCR product and pQE30 were ligated together by a standard ligation reaction. The ligated product was used to transform the competent E. coli M15 cells which was treated with 0.1M CaCl23. The recombinant M15 colonies were selected based on ampicillin and kanamycin resistance.
Screening of the selected E. coli colonies for the presence of insert (Bm86 homologue gene of H. a.
anatolicum)
Colony lysis method: Pin head size E. coli colonies were taken from the respective bacterial streaks from the LB agar plates. The bacterial colonies were suspended separately in 25 μl colony lysis buffer [100 mM Tris, pH 8.0, 10 mM EDTA, pH 8.0, 0.1%
lysozyme, 30 µl RNase A (stock solution 10 mg/ml)]
and incubated at 37oC for 1 hr. After 1 hr of incubation, equal volume of tris (pH 8.0) saturated phenol: chloroform (1:1) was added to the lysed cell suspension and the mixture was vortexed for 1 min.
Then the sample was spun at 10000 rpm for 10 min.
The whole aqueous supernatant was loaded on the 0.8% agarose gel and resolved completely. In the same way an E.coli M15 colony with empty pQE30 was processed and resolved in the same gel as a control. The gel was visualized under UV light to compare the level of migration of recombinant and non-recombinant colonies.
Colony PCR: One in ten dilution of the recombinant E. coli culture or the recombinant colony diluted in nuclease free water was boiled for 10 min and spun at 8000 rpm for 10 min. Supernatant (1 μl) was taken as template for colony PCR. A 25 μl PCR reaction was set up using 2.5 µl of 10X PCR buffer (MBI-Fermentas), 2.5 mM MgCl2 (MBI-Fermentas), 200 µM of each dNTP (MBI-Fermentas), 25 pM of each of forward and reverse primers (IDT, GCC) and
1 unit of Taq DNA polymerase (MBI-Fermentas).
This mixture was incubated in a thermocycler (PTC- 200, MJ Research) with the following cycling conditions: initial denaturation at 95oC for 5 min and further 30 cycles at 94oC for 1 min, 57 oC for 1 min and 72oC for 2 min and a final extension at 72oC for 10 min. At the end, 10 µl of colony PCR mixture of each corresponding colonies were loaded in 1 % agarose gel and resolved.
Calculation of total cost involved in screening of single E. coli colony by colony lysis method and colony PCR method ⎯ All the chemicals and enzymes used in this study were purchased from Genetix Biotech Asia Pvt. Ltd. The quantity of chemicals and enzymes used to process an E. coli colony by colony lysis and colony PCR methods and the corresponding costs of the chemicals and enzymes in Indian rupee was calculated. For this calculation, the price list 2005-2006 of Genetix Biotech Asia Pvt.
Ltd was taken as a reference.
Calculation of cost effectiveness ⎯ The cost effectiveness was calculated as per the following formula:
method lysis colony sigle screening in involved cost Total
PCR colony by colony single screening in involved cost Total ess effectiven
Cost =
Results
RT-PCR amplification of Bm86 homologue of H. a.
anatolicum ⎯ The PCR product was resolved in the 1% agarose gel (with ethidium bromide) electrophoresis and the 1990 bp amplicon was visualized under UV light (Fig. 1).
Colony lysis to confirm the E.coli-pQEHA86F colony ⎯ Migration of the empty plasmid, pQE30 of non-recombinant E. coli colonies ahead of the recombinant plasmid constructs of recombinant E. coli colonies was visualized and the presence of insert was confirmed (Fig. 2).
Colony PCR ⎯ Colony PCR was done from the individual recombinant E. coli colonies or cultures to confirm the presence of recombinant plasmid constructs inside E. coli. The amplification of 1990 bp PCR product confirmed the presence of recombinant plasmid construct consisting of the gene (Fig. 3).
Cost involved in the screening of single E. coli colonies ⎯ The calculation of the total cost involved in the screening of single E. coli colony by colony lysis method and colony PCR method is presented in Tables 1 and 2. The total cost involved in the
screening of single E. coli colony by colony lysis method and colony PCR method was Rs. 0.93 and Rs.
49.72, respectively. The colony lysis method was highly cost effective method for the screening of
recombinant E. coli colonies as compared to colony PCR method. The colony lysis method was 53 times cheaper than the colony PCR method, in Indian condition.
Calculation of time duration to perform colony lysis method and colony PCR method ⎯ Colony lysis method required 30 min for the incubation to lyse the cells followed by 10 min centrifugation and 30 min agarose gel electrophoresis. The total time required to perform the single colony lysis was 1 hr 10 min. In Colony PCR method initial 10 min boiling to lyse the cells followed by 2.30 hr for PCR reaction and 30 min for agarose gel electrophoresis. Total, 3 hr 10 min time was spent to perform single colony PCR. Thus, the colony lysis method was 2.7 fold faster than the colony PCR method.
Discussion
The entomology laboratory of the Division of Parasitology, IVRI is engaged with the development of recombinant vaccine against Ixodid tick, H .a.
anatolicum, vector of Theileria annulata which is the causative agent of Tropical Bovine Theileriosis.
Bm86 is a protective midgut antigen identified and isolated from cattle tick, Boophilus microplus7. TickGARD and Gavac are Bm86 based commercial recombinant vaccines against B. microplus available in Australia and Cuba and in Latin American
Fig. 1 ⎯ RT-PCR amplification of Bm86 homologue gene (Haa86) of H. a. anatolicum Izatnagar isolate [Lane 1- Bm86 homologue gene (Haa86) of H. a. anatolicum Izatnagar isolate (1990 bp), M- Gene rulerTM100 DNA ladder plus (MBI- Fermentas) 100 bp - 3000 bp].
Fig. 2 ⎯ 1% agarose gel showing the migration of plasmids extracted by colony lysis method from a recombinant (E. coli- pQEHA86) and non-recombinant (E. coli-pQE30) colonies [Lane 1- Migration pattern of recombinant plasmid construct, pQEHA86, Lane 2- Migration pattern of empty plasmid, pQE30, which migrates ahead of the recombinant plasmid construct, pQEHA86].
Fig. 3 ⎯ Colony PCR amplification of the 1990 bp Bm86 homologue gene of H. a. anatolicum Izatnagar isolate from different E. coli-pQEHA86 colonies [Lane 1 to 10- 1990 bp Bm86 homologue of H. a. anatolicum Izatnagar isolate amplified from five different E. coli-pQEHA86 colonies, N- Negative control. E.
coli DH5α colonies with out plasmid construct, M- Gene rulerTM100 DNA ladder plus (MBI-Fermentas) 100 bp - 3000 bp].
INDIAN J EXP BIOL, OCTOBER 2008
734
countries, respectively8-10. In our laboratory, the Bm86 homologue gene of H. a. anatolicum Izatnagar isolate was cloned, expressed and characterized. In an attempt to express the Bm86 homologue protein in prokaryotic system, the gene was cloned in the prokaryotic cloning vector pQE30/ E. coli M15. This opportunity was utilized to compare the colony lysis method and colony PCR method, especially the cost- effectiveness in the screening of recombinant E. coli M15 colonies.
Though the recombinant E. coli colonies were initially selected based on the antibiotic resistance, there is always likelihood for false positive selection due to recircularization of the linearized plasmid vectors that have lost some bases at their ends due to digestion with contaminating exonucleases11. Therefore, confirmation of the presence of target gene or recombinant plasmid in the recombinant E. coli colonies, which are selected, based on the antibiotic resistance is imperative.
Principally, colony lysis method is the combination of alkaline lysis method of plasmid isolation followed by phenol-chloroform extraction of the plasmid DNA3,11. The recombinant plasmid containing the target gene could be identified by resolving the recombinant plasmid with control empty plasmid in agarose gel. The super coiled empty plasmid from control E. coli colony migrates ahead of super coiled recombinant plasmid because of the size difference.
In the present experiment, colony PCR and colony lysis methods were employed to screen the recombinant E. coli colonies which were initially selected based on antibiotic resistance. Both the techniques were equally efficient in screening the E. coli colonies however, the colony lysis method was found to be cheaper and faster than the colony PCR method. The cost effective comparison was entirely based on the cost of reagents used for both the techniques. The cost of instruments, annual
Table 1 ⎯ Quantity and cost of reagents required to screen single E. coli colony by colony lysis method Sl. No. Chemicals/Enzymes Quantity Price in Indian rupees Cost of reagents to
prepare 10 ml colony lysis solution (Indian rupees)
1 Tris base (Amresco) 1 kg 4890.00 0.59
2 0.5 M EDTA pH 8.0 (Amresco) 100 ml 1323.00 2.65
3 Lysozyme (Amresco) 10 g 5275.00 5.28
4 Ribonuclease A (MBI-Fermentas) 10 mg 2141.00 64.23
5 Nuclease free water (Amresco) 500 ml 1968.00 39.63
Total cost of reagents required to prepare 10 ml colony lysis solution 112.11 Cost of 25 μl of colony lysis buffer required to screen single colony 0.28
6 Saturated phenol : Chloroform (1:1) 100 ml 2596.00 0.65/25 μl
Total cost involved in screening single colony by colony lysis method 0.93 Table 2 ⎯ Quantity and cost of reagents required to screen single E. coli colony by colony PCR method
Sl. No. Chemicals/Enzymes Quantity Price in
Indian rupees Quantity and cost (Rs.) of reagents required to screen single colony
1 Taq buffer 10X (MBI, Fermentas) 5 ml 898.00 2.5 μl 0.45
2 MgCl2 (25mM) (MBI, Fermentas) 5 ml 898.00 1.0 μl 0.18
3 dNTP (2mM each) (MBI, Fermentas) 1 ml 1105.00 2.5 μl 2.76
4 Forward primer (IDT, GCC) 35 bp (Rs. 27/bp) 25 nM 945.00 25 pM 0.95
5 Reverse primer (IDT, GCC) 35 bp (Rs. 27/bp) 25 nM 945.00 25 pM 0.95
6 Taq DNA Polymerase (recombinant) (MBI, Fermentas)
100 units 1037.00 1 unit 10.37
7 Nuclease free water (Amresco) 500 ml 1968.00 (66 μl)
16 μl + 50 μl*
0.26 8 Gene ruler DNA ladder, 100 bp plus
(MBI, Fermentas)
100 applications 3384.00 One application 33.80
Total cost involved in screening single colony by colony PCR 49.72
* To make one in ten dilution of the E. coli culture and subsequent boiling
maintenance cost of the instruments, labour cost and the laboratory infrastructure facility were not included. Thermocycler (MJ research) and incubator (37oC) (Scientronic, New Delhi) were the specific instruments used in colony PCR and colony lysis method, respectively. The cost of thermocycler used in colony PCR method is higher than the incubator used in the colony lysis method and it indicates the higher running cost of thermocycler/hour when compared to the running cost of incubator. Moreover, for performing colony PCR and the handling of the thermocycler requires a person with technical knowhow who supposed to be paid more. The involvement of man hour is less in the colony lysis method as it is 2.7 fold quicker than colony PCR method. Colony lysis method permits screening of approximately double the number of E. coli colonies in the same time required to perform colony PCR. In the laboratories with limited resources, by employing colony lysis method to screen the recombinant E. coli colonies, the thermocycler can be made available for other’s work at the same time to improve the working efficiency of the laboratory.
It is concluded that the colony lysis method to screen the recombinant E. coli colonies is cheaper, quicker and as efficient as colony PCR method.
Moreover, colony lysis method may improve the working efficiency of biomedical laboratories and save lot of money in the large scale screening of recombinant E. coli colonies in the protein expression studies.
Acknowledgement
The financial assistance to the senior author in the form of senior research fellowship from IVRI and the
financial support in the form of research project from Department of Biotechnology, Government of India, are acknowledged.
References
1 Bera B C, Chaudhuri P, Bhattacharya D, Bera, A K &
Das S K, Cloning and Sequencing and expression of cDNA of bovine neutrophile β-defensin from water buffalo (Bubalus bubalis), Int J Immunogen, 34 (2007) 173.
2 Mallick A I, Singha H, Chaudhuri P, Nadeem A, Khan S A, Dar K A, Owais M, Liposomised recombinant ribosomal L7/L12 protein protects BALB/c mice against Brucella abortus 544 infection, Vaccine, 25 (2007) 3692.
3 Sambrook J & Russel D W, Molecular cloning: A laboratory manual, Third edition (Cold Spring Harbor Laboratory Press, New York) 2001.
4 Sekar V, A rapid screening procedure for the identification of recombinant bacterial clones, Biotechniques, 5 (1987) 11.
5 Sekar V, Thompson D V, Maroney M J, Bookland R G &
Adang M J, Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var tenebrionis, PNAS, USA, 84 (1987) 7036.
6 de Vos S, Zeinstra L, Taoufic O, Willadsen P & Jongejan F, Evidence for the utility from Boophilus microplus in vaccination against other tick species, Exp Appl Acarol, 25 (2001) 245.
7 Willadsen P, McKenna R V & Riding G A, Isolation from the cattle tick Boophilus microplus, of antigenic material capable of eliciting a protective immunological response in the bovine host, Int J Parasitol, 18 (1988) 183.
8 Willadsen P, Bird P, Cobon G S & Hungerford J, Commercialization of a recombinant vaccine against Boophilus microplus, Parasitology, 110 (1995) S 43.
9 Canales M, Enriquez A, Ramos E, Cabrera D, Dandie H, Soto A, Falcon V, Rodriguez M & de la Fuente J, Large scale production in Pichia pastoris of the recombinant vaccine GavacTM against cattle tick, Vaccine, 15 (1997) 414.
10 Willadsen P & Kemp D H, Challenges and opportunities in the integrated control of parasites: The example of ticks and tick borne diseases J Parasitol, 89 (2003) S245.
11 Malo M S & Husain Z, Positive selection vectors for high fidelity PCR cloning, Biotechniques, 34 (2003)1250.
