jou rna l h om epage: www.elsev ier .com/ locate /be j
egular article
he effects of protein solubility on the RNA Integrity Number (RIN)or recombinant Escherichia coli
ary Alice Salazara,1, Lawrence P. Fernandoa,1,2, Faraz Baigb, Sarah W. Harcumb,∗
Department of Chemistry, Clemson University, United StatesDepartment of Bioengineering, Clemson University, United States
r t i c l e i n f o
rticle history:eceived 23 January 2013eceived in revised form 9 July 2013ccepted 28 July 2013vailable online 8 August 2013
eywords:ggregation
a b s t r a c t
High quality, intact messenger RNA (mRNA) is required for DNA microarray and reverse transcriptasepolymerase chain reaction analysis and is generally obtained from total RNA isolations. The most widelyrecognized measure of RNA integrity is the RNA Integrity Number (RIN) obtained from the Agilent Bioana-lyzer, as it provides sizing, quantification, and quality control measures. This work describes comparisonsof the RIN values obtained for recombinant Escherichia coli. Uninduced recombinant E. coli cultures wereexamined, as well as induced cultures that produced either a soluble or insoluble recombinant protein.The uninduced cultures and the induced cultures producing soluble protein had higher RIN values than
roteaseurificationecombinant DNANA
nclusion bodies
the induced cultures producing insoluble protein. These lower RIN values for E. coli producing the insol-uble protein indicate that cellular degradation of the ribosomal RNA species is the likely cause of thelower RIN values. As the use of DNA microarrays and other gene expression tools increase in usage in theindustrial recombinant protein production community, these results suggest the need for further studiesto determine acceptable RIN ranges for gene expression analysis and effects of various culture conditions
inant
on RIN values for recomb
. Introduction
Escherichia coli is used to produce a wide range of recom-inant proteins, many of which are used as therapeutic agents1,2]. E. coli has many advantages over the other host organismsor the large-scale production of recombinant proteins includingenome simplicity, well understood genetics and metabolism, andast growth rates on inexpensive growth medium [3,4]. One majorisadvantage of recombinant protein production in E. coli is its ten-ency to produce insoluble inclusion bodies of the desired targetecombinant protein [4–14]. Many efforts to control inclusion bodyormation include overexpression of chaperones [15–20], codonptimization [21,22], and decreased culture temperatures [23–25].espite these advances to control inclusion bodies, it is still notossible to a priori predict the solubility state of a new recombinant
rotein [16,26–30].
To gain a better understanding of inclusion body formation,NA microarrays have been used [27,31]. In order to conduct DNA
∗ Corresponding author at: Department of Bioengineering, 301 Rhodes, Clemsonniversity, Clemson, SC 29634-0905, United States. Tel.: +1 864 656 6865.
E-mail address: [email protected] (S.W. Harcum).1 These authors contributed equally.2 Present address: Department of Biomedical Engineering, University of Florida,nited States.
microarray or any gene expression analysis, high quality messengerRNA (mRNA) is required [32–34]. Most RNA purification techniquesfor prokaryotes isolate and amplify the total RNA, which includesmRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA) species,since prokaryotic mRNA lacks a stable poly(A) tail [35]. There areseveral methods that are routinely used to evaluate RNA quan-tity and quality, the most common being RNA absorbance [36].Absorbance methods indicate purity and concentration, but can-not distinguish the RNA species or intactness [36]. Electrophoresismethods allow visualization of the ribosomal RNA species, but arelimited in quantification precision [36–38]. Due to the limitationof the absorbance and traditional electrophoresis methods, DNAmicroarray manufacturers (i.e., Affymetrix, Illumina, Aglient, andRoche Nimblegen) highly recommend analysis of RNA quality usingthe Agilent Bioanalyzer.
The Agilent Bioanalyzer 2100 is a microfluidics-based platformthat generates an electropherogram and simulated gel imagethat provides sizing, RNA quantification, and quality control,which is reported as the RNA Integrity Number (RIN) [37,39].The RIN value is calculated from the proportion of expected RNAfragment sizes and is independent of variance due to sampleconcentration [37,39]. Low RIN values are usually attributed to
ribosome degradation during the isolation and purification steps;however, these detailed characterization studies were largelyfocused on eukaryotic RNA [37,39–44]. Two recent investigationsexamined RIN values in prokaryotes; wild-type E. coli in meat
amples [45] and several bacterial species found in human stoolamples [46]. To illustrate the lack of knowledge for RIN values forecombinant E. coli, the Agilent RNA Integrity Database (RINdb)ontains only one E. coli electropherogram out of the 646 entrieshttp://www.chem.agilent.com/rin/ rinDetail.aspx?rID=4156,ccessed May 30, 2013); and this electropherogram is for anormal” wild-type E. coli culture, and has a RIN value of only 1.0.
In this study, RIN values were evaluated for recombinant E. coliultures in preparation for DNA microarray analysis. Both inducednd uninduced cultures were evaluated. Additionally, the effectsf soluble and insoluble protein productions were compared. Allultures were synchronized with respect to growth phase and cellensities at induction. Samples were harvested in parallel from theoluble and insoluble protein producing cultures. The total RNAas isolated and purified in parallel using standard RNA isolation
nd purification techniques. The isolated total RNA was evaluatedy standard absorbance techniques. And, the Agilent Bioanalyzer100 with the Prokaryotic Total RNA Nano software was used tobtain RIN values for all samples. A statistical comparison of theIN values obtained for the total RNA samples was conducted.
. Materials and methods
.1. Bacterial strains and plasmids
E. coli MG1655 obtained from American Type Culture Collec-ion (ATCC) were transformed with either pTVP1GFP or pGFPCATlasmids. Both plasmids are isopropyl ß-D thiogalactopyranosideIPTG) inducible via a trc promoter and encode ampicillin resis-ance. The pTVP1GFP plasmid (donated by A. Villaverde) encodesor a fusion protein (VP1GFP) which contains the VP1 capsid pro-ein from the Foot and Mouth disease virus [47] fused to a greenuorescent protein (mGFP) [48]. The pGFPCAT plasmid encodes ahloramphenicol acetyltransferase (CAT) and mGFP fusion and wasonstructed by replacing the GFPUV in the pTrcHis-GFPUV/CAT plas-id (donated by W.E. Bentley [49]) with the mGFP from pTVP1GFP.
he primers used for the mGFP substitution were: Forward: 5′ GTC CAT ATG AGC AAA GGA GAA GAA CTT TTC 3′ and Reverse: 5′ GTC CAT ATG TGT AGA GCT CAT CCA TGC CAT GTG TAA TCC 3′.
.2. Culture conditions
Cells were cultured in a minimal medium as described pre-iously [50,51]. One milliliter of E. coli from frozen stocks wasdded to minimal medium in the presence of ampicillin (40 �g/mL,yclone) [36] and grown overnight at 37 ◦C and 250 rpm (Newrunswick Scientific, C24 incubator shaker) to an optical densityOD600) of 2.5 OD, where 1 OD is equivalent to 0.50 g dry celleight per L. From the overnight cultures, E. coli pGFPCAT andTVP1GFP were added separately to 500 mL shake flasks (120 mLorking volume) at 37 ◦C in a water bath shaker at 200 rpm (Newrunswick Scientific, C76 incubator shaker) for an initial cell den-ity of 0.05 OD. Cell growth was monitored by optical densitysing a spectrophotometer (Spectronic 20 Genesys), where sam-les were taken without stopping agitation or removing the flasksrom the water bath. Samples for cell densities were diluted witheionized water to obtain absorbance readings in the linear range0–0.25 OD units). Cultures were induced (1 mM IPTG) in the mid-xponential phase (OD600 = 0.5). For the RNA isolations, samplesere collected prior to induction (time 0-min) and 5-, 20-, 40-, and
0-min post-induction for the induced cultures and at 60-min for
arallel uninduced cultures. The uninduced samples were obtainednly for times 0- and 60-min. The entire culture broth sampleas immediately stabilized in RNAProtectTM Bacteria Reagent (Qia-
en) and processed as per manual instructions. These protected
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samples were centrifuged (14,500 × g, 10 min, Hermle LabnetZ383K centrifuge), and growth media and RNAProtectTM BacteriaReagent were removed. Cell pellets were stored at −80 ◦C untilused for RNA isolation. Additionally, parallel samples were har-vested and processed for protein characterization [52]. All cultureconditions were conducted in biological triplicates.
2.3. Protein expression analysis
The CAT activity of the fusion GFPCAT protein was measuredusing the kinetic assay described by Rodriguez and Tait (1983)and adapted to a 96-well plate format [52,53]. Additionally, CATbiological activity was confirmed by the ability of induced E. colipGFPCAT to grow on high-levels of chloramphenicol (0.61 mM)containing LB plates. VP1GFP protein production was confirmedby obtaining fluorescence emission spectra for whole cells fixedwith 2% formaldehyde and immediately assayed [54–56]. Fluo-rescence measurements were taken using a BD Influx Cell Sorter(formerly Cytopeia) with a 488-nm Argon excitation laser and a530/540 emission filter. The mean signal acquisition from 50,000cells was used to characterize sample intensity. VP1GFP inclusionbodies were confirmed by fluorescence microscopy (Nikon Ti, 60XTIRF oil) [57].
2.4. RNA isolation and characterization
Total RNA was isolated using the RNAeasy® Bacteria Kits (Qia-gen, #74524 and #75144) were used depending on the cellnumbers to be processed). RNA was quantified by a Nanodropspectrophotometer (ND 1000, Thermo Scientific). The Agilent Bio-analyzer 2100 and Agilent RNA 6000 Nanochip kits were used toassess the total RNA quality as per manual instruction (Agilent).The Agilent 2100 Expert software (Version B.02.07.SI532) was usedwith the Prokaryote total RNA series II assay settings. No significantdifferences in RIN values were observed between different dilutionsof the same sample.
2.5. Gene expression analysis
Total RNA was used to synthesize the first strand cDNA usingthe Superscript First-Strand Synthesis System for RT-PCR (Invi-trogen, Inc.) as per the Nimblegen instruction manual (Version3.2). The RNA 6000 Nanochip Kit was also used to quantify mRNA(using the mRNA protocol) after the second strand synthesis. Cus-tom E. coli DNA microarrays (12 arrays per slide × 135 K probesper array) were prepared by Roche NimbleGen with 4281 E. coligenes, mGFP, TVP1, ampicillin resistance gene (Ampr), and CATprobes (45-60mer, 10 probes per target, 3 copies of each probeon array). The DNA microarrays were processed at Florida StateUniversity’s NimbleGen Certified Microarray Facility in Tallahas-see, Florida. NimbleGen’s NimbleScan software normalizes thegene expression levels with a quantile normalization methodin order to reduce obscuring variation between samples. Thesoftware uses a Robust Multichip Average (RMA) algorithm to gen-erate Calls files ( RMA.calls) that contain normalized average geneexpression values [58–60]. The probe sequences and raw geneexpression data have been deposited in the National Center forBiotechnology Information’s Gene Expression Omnibus (Accessionnumber: GSE47732). The DNA microarray data was imported intoArrayStarTM from the RMA.calls files. Technical replicate expression
levels were scaled using the “global averaging” data transformation.An ANOVA test (p ≤ 0.10) was conducted on the gene expres-sion values for the biological triplicates to screen for differentiallyexpressed genes (ArrayStarTM).
Fig. 1. Growth profiles and protein levels for synchronized E. coli cultures. (A) E. colipTVP1GFP cultures uninduced ( ) and induced ( ); (B) E. coli pGFPCAT culturesuninduced ( ) and induced ( ). The solid and dashed lines represent the expo-nential growth rate model fits for the induced and uninduced cultures, respectively,where all cultures had growth rates equal to approximately 0.57 h−1 (C) CAT activ-
M.A. Salazar et al. / Biochemical E
.6. Statistical analysis
Statistical analysis was conducted using the generalized linearodel (GLM) method (p ≤ 0.05) with the JMP 10 software (SAS Insti-
ute Inc.). For the RIN values and the growth rates, the effectorsere protein carried on the plasmid (VP1GFP and GFPCAT) and the
nduction state (uninduced and induced) of the cultures. Addition-lly, for the RIN values, interaction effects were examined for therotein and induction conditions, as well as any time dependencyffects. For the RIN values, post hoc testing was conducted usinghe LSMean Tukey’s HSD test.
. Results and discussion
.1. Growth profiles and protein expression
The overall objective of these studies was to characterize theynamics of gene expression variability in E. coli due to insolu-le and soluble protein production and specifically to examinehe RIN values. The pTVP1GFP and pGFPCAT plasmids and theP1GFP and GFPCAT proteins were selected to minimize the differ-nces between the culture conditions, except for protein solubility.pecifically, the reasons for the selection of these two systemsre: (1) VP1GFP is a well-characterized inclusion body-prone pro-ein with fluorescence in both insoluble and soluble conformations15,48]; (2) CAT and GFP are both well-characterized soluble pro-eins [53,55,61–64]; (3) the two fusion proteins (GFPCAT andP1GFP) have similar molecular weights; and (4) both plasmidsre very similar with a common lineage (e.g., ampicillin resistance,BR322 origin, lacI expression, and trc promoter) [15,65]. Thus, anyifferences observed in the RNA electropherogram profiles and RINalues can be attributed to the protein solubility.
In order to evaluate RIN values for the different cultureonditions, total RNA was isolated from synchronized culturesxpressing either the soluble protein GFPCAT or the inclusion body-rone protein VP1GFP. The growth profiles for these cultures arehown in Fig. 1 for triplicate cultures. Each culture condition wasonducted in triplicate and the culture times have been alignedo the induction time, which corresponds to a cell density ofpproximately 0.5 OD and mid-exponential growth. The samplessed for the total RNA isolation were taken just prior to induc-ion (time 0-min) and 5-, 20-, 40-, and 60-min post-induction.he time 0-min represents uninduced cultures only. Additionally,he uninduced cultures were sampled at 60-min past the synchro-ization cell density. The pre-induction and post-induction growthates were analyzed using the GLM method. It was determinedhat the growth rates were not different for the cultures due tohe protein produced (VP1GFP and GFPCAT) or the induction stateuninduced and induced) (p > 0.05). The exponential growth rate
odel fit for all cultures prior to and up to 1-h post-induction were.568 ± 0.013 h−1 (standard error), and is highlighted in Fig. 1 byhe overlapping exponential model line fits, one for each of the fourulture conditions examined (uninduced GFPCAT, induced GFPCAT,ninduced VP1GFP, and induced VP1GFP). Additionally, inductionith 1 mM IPTG did not significantly alter the growth rate of the cul-
ures (p > 0.05). In minimal medium, it is commonly observed thatnduction does not alter the culture growth rate [50,53]. In contrast,n richer medium decreased growth rates have been observed at theime of induction [66]. In this study, the growth rates and cell densi-ies were not different for the cultures producing either the GFPCATsoluble) or VP1GFP (insoluble) protein for any of the analyzed time
oints.
The CAT activity of the GFPCAT protein was quantified (Fig. 1)nd shows an approximately 8-fold increase in protein enzymectivity by 60-min post-induction and an approximately 20-fold
ity for GFPCAT (lighter bars - red) and fluorescence for VP1GFP (darker bars - blue).Error bars represent 95% confidence intervals. (For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of the article.)
increase at 4-h post-induction. Additionally, induced GFPCATcells grew on chloramphenicol (0.61 mM) LB plates, whereasno growth was observed for uninduced cells (data not shown).
The VP1GFP protein production was quantified by fluorescenceintensity per cell using flow cytometry (Fig. 1). Flow cytometryanalysis of the induced VP1GFP cultures showed the fluorescentintensity increased approximately 8-fold at 60-min post-induction
132 M.A. Salazar et al. / Biochemical Engine
Fig. 2. Representative electropherograms for total RNA samples using the AgilentBioanalyzer. (A) E. coli pTVP1GFP uninduced at time 0-min and (B) E. coli pTVP1GFP5ri
amflbits
3
tctaiOfi2b([Batfp7a
opt(af
-min post-induction. The electropherogram peaks include the dye front (dye), 16SRNA (16S), and 23S rRNA (23S). The RIN values and RNA sample concentrations arendicated for each sample. Peak intensities are shown as fluorescence units (FU).
nd approximately 30-fold at 3.5-h post-induction. Fluorescenceicroscopy showed the VP1GFP protein was localized in the
agella end of the cells, which was also reported and characterizedy Dr. Villaverde’s research group as inclusion bodies [57]. The
nduction levels for both GFPCAT and VP1GFP were similar underhe experimental conditions, which was expected for these veryimilar plasmid constructs.
.2. Total RNA isolation and characterization
The cell pellet samples were harvested from synchronized cul-ures and were processed in parallel. To obtain total RNA from theell pellets, a set of samples representing all the triplicate condi-ions was also processed in parallel. Thus, one set of the uninducednd induced, and GFPCAT and VP1GFP conditions were processedn parallel from the synchronized culture for total RNA analysis.nce the total RNA was obtained, the isolated RNA was quanti-ed using a Nanodrop spectrophotometer. The 260/280 nm and the60/230 nm ratios demonstrated that cellular contaminates hadeen sufficiently removed (≥2.0) and that the purity was sufficient2.0–2.2), respectively, for all samples with no statistical differences67,68]. The isolated total RNA was then examined using the Agilentioanalyzer for the biological triplicates, again grouped to includet least one complete set of the 12 conditions. Representative elec-ropherograms obtained using the Agilent Bioanalyzer are shownor VP1GFP cultures in Fig. 2; uninduced 0-min and induced 5-minost-induction. The uninduced VP1GFP culture had a RIN value of.7, which is considered acceptable [45], while the induced culture
had much lower RIN value of 4.8 [37,39].The RIN algorithms were entirely developed using 1208 eukary-
tic data set, where the 5S, 18S and 28S peaks were used [37]. Forrokaryotes, the RIN values are calculated from the entire elec-
rophoretic trace with weighting given to the (1) total RNA ratio,2) 23S peak height, (3) 23S area ratio, (4) comparison of the 16Snd 23S area to the fast region area, (5) a linear regression of theast region end point, (6) detected fragment amounts in the fast
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region, (7) the presence or absence of the 16S peak, and (8) a com-parison of the overall mean value to the median value [37]. Thefast region is defined as the area between the 5S and 18S peaks;however, for E. coli a 5S peak is not detected, and thus not usedin the calculation; and the 16S peak replaces the 18S peak [45].Therefore, for E. coli, the fast region is between the dye front andthe 16S peaks and includes the elution times between approxi-mately 25 and 40 s. Due to the parallel processing for all samples,including sample harvesting, sample protection, sample storage,and RNA isolation procedures, this anomaly in the RIN values couldnot be attributed to ribosome degradation alone during processes.A statistically analysis was conducted to determine if the lower RINvalues could be attributed to protein solubility.
To evaluate the RIN values statistically, the RIN values for theuninduced time 0- and 60-min samples as well as the induced 5-,20-, 40-, and 60-min samples were used. The average RIN valueswith standard error are shown for the VP1GFP and GFPCAT cul-tures in Fig. 3. A statistical analysis was conducted to determinethe significance of the RIN values using the generalized linear model(GLM) method (p ≤ 0.05) with the JMP 10 software (SAS InstituteInc.). Both the protein and the induction state were determined tobe statistically significant for the RIN values. Post hoc analysis usingthe least square mean differences with Tukey HSD determinedthat the uninduced culture RIN values, regardless of the proteinencoded by the plasmid, were not significant (p > 0.05). The posthoc analysis also determined that the induced GFPCAT culture RINvalues were not significantly different from the uninduced cultures(p > 0.05). However, the VP1GFP induced culture RIN values weredetermined to be statistically different (p ≤ 0.05) from the unin-duced cultures and the induced GFPCAT cultures. Additionally, theobserved decrease in RIN values was at 5-minute post-inductionfor the VP1GFP cultures and had no time dependence up to 60-minpost-induction (p > 0.05). This rapid RIN value decrease followed byno change in RIN values would indicate a counterbalancing cellularresponse allowing for stabilized overall RNA integrity.
Ribosomes are composed of both protein and RNA, and ribosomeabundance has been observed to decrease due to recombinant pro-tein production in E. coli [66,69–73]. Gene expression analyses ofE. coli producing insoluble versus soluble proteins have demon-strated higher levels of the heat shock response genes [27,31]. And,it is well known that the heat-shock response activates proteaseactivity in response to insoluble recombinant protein produc-tion [27,64,74], where recombinant protein production has beenobserved to have elevated protease activity as well [75]. Increasedprotease activity toward the ribosomes could cause the lower RINvalues for the cultures producing the insoluble protein. A counter-acting cellular response that could increase ribosome abundancewould be increased ribosomal subunit gene expression.
In this study, 25 of the 55 ribosomal subunits genes(rplACDEFJLMNQRX, rpmDIJ, and rpsABFGHLMNPU) had statisticallysignificant gene expression levels (p ≤ 0.10). The dynamic behav-ior for all 25 ribosomal subunit genes was coordinated across the12 samples. The average ribosomal subunit gene expression pro-files are shown in Fig. 3 for the uninduced and induced GFPCAT andVP1GFP cultures (Supplemental Data has the profiles for all 25 ribo-somal subunit genes by culture condition). The ribosomal subunitgene expressions levels increased linearly for the GFPCAT culturesdue to the protein induction (p ≤ 0.05), and remained unchanged forboth the uninduced VP1GFP and GFPCAT cultures (p > 0.05). For theinduced VP1GFP cultures, the 25 ribosomal subunit gene expres-sion levels at 60-min post-induction were higher than any othercondition (p ≤ 0.05). As expected, the housekeeping genes (mdoG
and crp) [76] were not significantly affected (p > 0.10) by the con-ditions. The RIN and ribosomal subunit gene expression profilessupport the hypothesis that VP1GFP production causes increasedribosome degradation due to heat shock response proteases, and
M.A. Salazar et al. / Biochemical Engine
Fig. 3. RNA Integrity Number (RIN) values, and ribosomal subunit and housekeep-ing gene expression levels. (A) RIN values; (B) Normalized gene expression forthe average of the 25 ribosomal subunit genes (rplACDEFJLMNQRX, rpmDIJ, andrpsABFGHLMNPU); and (C) Normalized gene expression for two housekeeping genes,mdoG and crp. E. coli pTVP1GFP cultures uninduced ( ) and induced ( ); andE. coli pGFPCAT cultures uninduced ( ) and induced ( ). Error bars represent thestandard error.
ering Journal 79 (2013) 129– 135 133
these negative effects are counterbalanced by increased ribosomalsubunit gene expression levels, resulting in a plateau in the RINvalues.
4. Conclusions
Recombinant E. coli prior to induction of a target protein has RINvalues that indicate acceptable RNA integrity for gene expressionanalysis. Induction of an insoluble protein resulted in lower RINvalues compared to soluble protein induction, as well as to the unin-duced cultures. The observed RIN values for the induced insolubleprotein cultures would normally indicate ribosome degradationduring the isolation and purification steps; however, since paral-lel processing was used for the induced soluble protein and theuninduced cultures, the apparent ribosome degradation most likelyoccurred in the cells in response to the insoluble protein produc-tion. Specifically, increased protease activity due to a heat shockresponse caused a rapid decrease in the RIN value, indicative ofribosome degradation. Interestingly, the RIN values stabilized, sug-gesting a counterbalancing cellular response, which is likely due toincreased ribosomal subunit gene expression levels. Since the DNAmicroarray community uses the RIN values to assess RNA integrityas a critical first step in gene expression analysis, and becausethere is limited data for both wild-type and recombinant E. coliunder various culture conditions, these widely different RIN valuesobserved between the induced insoluble protein cultures and boththe induced soluble protein and the uninduced cultures indicatethat further analysis is needed to determine the effects of variousculture conditions on RIN values for recombinant E. coli.
Acknowledgements
The pTVP1GFP plasmid was generously provided by E. Garcia-Fruitos and A. Villaverde, Universitat Autònoma de Barcelona. ThepGFPCAT plasmid was constructed by M.T. Morris, Clemson Univer-sity. The pTrcHis-GFPUV/CAT plasmid was donated by W.E. Bentley,University of Maryland. The authors also would wish to thank N.Vyavahare, Clemson University, for the use of the Agilent Bioana-lyzer 2100; Dr. Terri Bruce of the Clemson Light Imaging Facilityfor microscopy assistance; and Arthur Nathan Brodsky for review-ing the manuscript. Funding was provided by the National ScienceFoundation under NSF Award CBET 0738162 and by an InstitutionalDevelopment Award (IDeA) from the National Institute of GeneralMedical Sciences of the National Institutes of Health under grantnumber P20GM103444.
Appendix A. Supplementary data
Supplementary material related to this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bej.2013.07.011.
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