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FEMS Microbiology Letters 100 (1992) 87-90 87 © 1992 Federation of European Microbiological Societies 0378-1097/92/$05.00 Published by Elsevier FEMSLE 80028 Experimental conditions may affect reproducibility of the/3-galactosidase assay Alessio G i a c o m i n i , Viviana Corich, F r a n c i s c o J. O l l e r o 1, A n d r e a S q u a r t i n i a n d M a r c
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  FEMS Microbiology Letters 100 (1992) 87-90 © 1992 Federation of European Microbiological Societies 0378-1097/92/ 05.00 Published by Elsevier 87 FEMSLE 80028 Experimental conditions may affect reproducibility of the/3 galactosidase assay Alessio Giacomini, Viviana Corich, Francisco J. Ollero 1, Andrea Squartini and Marco P. Nuti Dipartimento di Biotecnologie Agrarie, UniL,ersith degli Studi di Padol;a, PadoL a, Italy Received 15 June 1992 Accepted 19 June 1992 Key words: Escherichia coli;/3 Galactosidase; lacZ Gene; Miller s assay 1. SUMMARY Several experimental conditions and parame- ters contributing to the determination of /3- galactosidase activity, as proposed in Miller s as- say, were studied. Use of the absorbance correc- tion factor and the nature and concentration of permeabilizing agents were taken into account as different experimental conditions. Reaction time, culture volume, and growth stage were investi- gated as equation parameters. From a quantita- tive point of view the results, in terms of Miller units, are markedly affected by variation in these conditions. Therefore, to ensure reproducibility it is advisable to use constant values for all the parameters. Correspondence to: M.P. Nuti, Dipartimento di Biotecnologie Agrarie, Universit5 degli Studi di Padova, via Gradenigo 6, 35131 Padova, Italy. 1 Present address: Departamento de Microbiologia, Facultad de Biologia, Universidad de Sevilla, Apdo 1095, 41080 Sevilla, Spain. 2. INTRODUCTION The activity of /3-galactosidase is widely used as a marker phenotype in several biological sys- tems [1]. The availability of substrates other than lactose whose cleavage releases compounds which can be easily detected allows assay of enzyme activity on both a qualitative and quantitative basis. Several expression vectors rely on fusions with the lacZ gene from Escherichia coli. Moni- toring the level of lacZ expression in strains containing such constructs can report on the ef- fect of the cloned fragments. Among the different methods commonly used to measure /3-galac- tosidase activity, the assay proposed by Miller [2] is one of the most popular, due to its simple procedure based on bacterial cell permeabiliza- tion followed by spectrophotometrical measure- ment of o-nitrophenol released from its galacto- side conjugate (o-nitrophenyl-/3-D-galactoside, ONPG). While studying the strength of bacterial pro- moters we experienced a problem of reproducibil- ity of the results when using this assay. Therefore  88 we carried out a detailed analysis of the different parameters appearing in Miller s procedure in order to evaluate whether these can affect the overall results. We considered two E. coli strains, one carrying a complete lactose operon on the chromosome, the other, a Lac mutant, contain- ing the lacZ gene on a plasmid. This gene, driven by a synthetic promoter, is constitutively ex- pressed at high levels. 3. MATERIALS AND METHODS 3.1. Bacterial strains, plasmids and growth condi- tions Escherichia coli strain DH1 [3] and JM109 lacZAM15) [4] were used. The tetracycline-re- sistant plasmid pMPT15 is a derivative of the incP vector pMP220 [5], containing a synthetic promoter in front of the E. coli lacZ gene (A. Giacomini, F.J. Ollero, A. Squartini and M.P. Nuti, unpublished). Cells were grown at 37°C with agitation in 1 x A medium-glucose [6] supplemented with thi- amine. Tetracycline (10/zg/ml) was added when required; isopropyl-l-thio-/3-D-galactoside (IPTG, 1 mM) was added to all samples to ensure com- parability. 3.2. [3-Galactosidase assay The method of Miller [2] was followed. Either toluene or chloroform-SDS in varying amounts were used to permeabilize cells. The correction coefficient of the absorbance value proposed by the author was adopted; in some cases, bacteria were centrifuged at 14000 rpm for 2 min. 4. RESULTS 4.1. General considerations /3-Galactosidase activities are expressed in Miller units according to the following equation: OD42 o X (1.75 X ODs5o) Units = 1000 × OD6o o X time x volume The expression of the lacZ gene in strain DH1 is regulated by the inducer IPTG. In the case of Table 1 /3-Galactosidase activity (Miller units) A: Determined by using the correction factor (1.75 x ODss0). B: Determined after centrifugation of the samples DHl JM109 JM109 pMPT15 A 1878 23 20499 B 2580 356 21170 strains JM109 pMPT15, IPTG has no effect due to the absence of the lac operator. The activity of strain DH1 ranges between 1000 and 3000 units when induced, while for strain JM109 pMPT15 the activity is around 20000. The variability of these data was observed upon changing the pa- rameters of the equation. Each value shown below is the mean of three independent measurements. 4.2. Effect of the correction factor This factor (1.75 x ODs50) is used to compen- sate for that portion of 0042 o due to light scatter- ing by bacterial cells. As an alternative, the srci- nal protocol suggests removing cells by centrifu- gation before reading the absorbance at 420 nm. Table 1 shows a comparison of the two proce- dures. These methods are not alternative since we constantly obtained higher values when cells were centrifuged. By using the latter procedure we also obtained more homogeneous measure- ments from replicates of the same sample. More- over, when dealing with very low activities, i.e. low OD420, the spectrophotometric reading at 550 nm can strongly affect the result, since the sub- tracted value, ODss0X 1.75, is proportionally high, and can approach or, due to experimental error, be greater than the 0042o leading to nega- tive activity values. Therefore, centrifugation is a superior procedure since a potential source of error, the second spectrophotometric reading, is no longer required. For such reasons centrifuga- tion was adopted as the standard choice. 4.3. Effect of the permeabilizing agent Two alternative treatments are proposed by the protocol. The first makes use of a drop of toluene followed by a 40-min incubation at 37°C; the second is based on the addition of a drop of  Table 2 /3-Galactosidase activity Miller units) A: Using toluene as permeabilizing agent B: Using SDS-chloroform as permeabilizing agent DH1 JM109 JM109 pMPT15 A 2 322 317 20 072 B 2580 356 21 170 0.1 SDS plus two drops of chloroform. In Table 2 we show a comparison of the two treatments. Values obtained by SDS-chloroform permeabi- lization were constantly higher. The SDS-chloro- form treatment was chosen for its speed and convenience. 4.4. Effect of SDS chloroform concentration The srcinal protocol indicates the amounts of chloroform and SDS in 'drops'. The actual vol- ume of liquid contained in a drop released from a Pasteur pipette may vary from a few microliters up to more than 50 depending on length, shape, total volume of liquid in the pipette, use of rub- ber bulb or gravity to release drops, etc. For this reason we investigated the effect of different quantities of the two reagents, maintaining the 1 : 2 ratio proposed by Miller (Table 3). Sub-opti- mal amounts (5 ~1 SDS, 10/zl chloroform) giving inefficient permeabilization, fail to reveal full ac- tivity, leading to considerable underestimates. On the other hand, volumes exceeding 15 /xl SDS and 30 /xl chloroform progressively affect the reaction in a negative fashion, presumably be- cause of an inhibiting effect on the enzyme. Table 4 /3-Galactosidase activities Miller units) as tion time 89 a function of reac- Time DHI Time JM109 pMPT15 rain) s) 1 28O9 2O 22675 2 2386 40 19563 5 1942 60 17956 15 1850 120 16222 30 1745 180 15086 45 1581 240 12388 Therefore it is advisable to use a measured amount of the two reagents and we routinely dispensed 10/~1 SDS and 20 /xl chloroform into our reaction tubes. 4.5. Activity as a function of reaction time /3-Galactosidase activity should be indepen- dent from the duration of the reaction since longer reaction times, giving higher OD420 values, should yield the same result in terms of Miller units. However, the results (Table 4) indicate that there is a marked decrease in apparent activity at increasing reaction times. 4.6. Activity as a function of added culture volume Increasing the volume leads to a considerable decrease in the apparent activity (Table 5). 4. 7. Activity as a function of culture growth stage The srcinal protocol recommends the use of cultures within a range of optical density at 600 nm between 0.28 and 0.70, in which the activity Table 3 /3-Galactosidase activity Miller units) as affected by the use of different amounts of SDS-chloroform to permeabilize the cells SDS Chloroform DH1 JM109 pMPT15 /~1) ~1) 5 10 1326 5199 10 20 1443 15532 15 30 1452 15757 25 50 1379 15189 40 80 1306 12272 Table 5 /3-Galactosidase activities Miller units) as a funtion of added culture volume Culture volume DH1 JM109 pMPT15 150 2417 16433 300 2246 13 552 450 1999 11463 600 1776 9 635 750 1492 8 229 900 1446 6 421  90 Table 6 /3-Galactosidase activities (Miller units) as a function of growth stage OD6o o DH1 OD6o o JM109 pMPT15 0.104 2243 0.150 13155 o. 160 2 041 0.245 13 809 0.250 2 044 0.361 12 991 0.332 1626 0.528 11817 0.487 1797 0.647 10 865 0.644 1414 should remain constant. However, the trend ap- pearing from data in Table 6 is a reduction of the units, which one can obtain by using older cul- tures. Our experiment deals exclusively with lac genes of E coli controlled by IPTG inducible (DH1) or constitutive (JM109 pMPT15) promot- ers. In many instances researchers would make use of gene fusions where lac genes may be driven by different exogenous promoters. As a consequence, the /3-galactosidase activity re- ported as a function of culture age will depend on the expression of the specific promoter at a given stage of the growth phase. 4 8 Effect of ONPG concentration To ensure linearity of the assay, the ONPG concentration must be in excess. A concentration of 4 mg/ml in the stock solution is reported as saturating. Results shown in Table 7 show that a moderate increase in activity can be achieved when using higher concentrations. Table 7 /3-Galactosidase activities (Miller units) as a function of ONPG concentration ONPG stock DH1 JM109 pMPT15 concentration (mg/ml) 4 1463 16393 10 1552 17166 20 1671 17574 5. DISCUSSION The assay proposed by Miller is one of the most widely used procedures for qualitative and quantitative determination of/3-galactosidase ac- tivity. Our data indicate that, in its essential for- mulation, the test is not particularly reproducible, at least in E coli In order to maximize repro- ducibility it appears to be essential to standardize the different parameters. Therefore data appear- ing in the literature cannot be accurately com- pared unless the exact experimental conditions are stated. However, from a qualitative, or not strictly quantitative point of view, the test retains all its validity, chiefly due to its simple and rapid procedure. From a preliminary examination of our data it also appears that there could be a correlation between the reduction of activity and some exper- imental conditions. Further studies may lead to the discovery of possible correction coefficients for the parameters of the equation. ACKNOWLEDGEMENTS This work was supported by EEC Biotechnol- ogy Action Program, contract No. 0359-I REFERENCES [1] Sambrook, J., Fritsch, E.F. and Maniatis T. (1989) In: Molecular Cloning: A Laboratory Manual, 2nd Edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [2] Miller, J.H. (1972) Experiments in Molecular Genetics, pp. 352-355. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [3] Hanahan, D. (1983) J. Mol. Biol. 166, 557-580. [4] Yanisch-Perron, C., Vieira, J. and Messing J. (1985) Gene 33, 103-119. [5] Spaink, H.P., Okker, R.J.H., Wijffelman, C.A., Pees, E. and Lugtenberg, B.J.J. (1987) Plant Mol. Biol. 9, 27-39. [6] Lech, K. and Brent R. (1987) In: Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D., Seidman, J.G., Smith J.A., Struhl, K., Eds.), John Wiley Sons, New York.

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