Life Science April 2001

April 2001 Feature Article

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 A TECHNICAL APPLICATION NEWSLETTER VOL 2   ISSUE 2    April 2001

 Molecular Biology Feature Article


S-Gal™: A Superior Dye to X-gal for Clonal Selection

by Ken Heuermann and Jennifer Cosgrove
Sigma-Aldrich Corporation, St. Louis, MO USA

Introduction

"Blue-white" color screening is well established as a means for identifying a ligation product or indicating the presence of a DNA insert. The method is based upon the ability of ß-galactosidase to hydrolyze 5-bromo-4-chloro-3-indolyl ß-D-galactopyranoside (X-gal)1, resulting in the characteristic blue staining of a colony or phage plaque. The multiple cloning region of numerous plasmid and phage vectors is imbedded within the amino-terminal fragment of an isopropyl ß-D-thiogalactoside (IPTG)-induced ß-galactosidase gene. Successful ligation of a DNA fragment into the multiple cloning site disrupts ß-galactosidase expression; without insertion, expression is uninterrupted. Thus, when the E. coli host, expressing the carboxyterminal portion of ß-galactosidase, is transformed with a plasmid vector or infected with a phage without a DNA insert, a "blue" colony or plaque will result.2 Transformation or infection of a vector with DNA fragment insertion results in a "white" or clear colony or plaque.

Color selection was initially introduced as a means to reduce the labor-intensive process of isolating a desired ligation product, or to distinguish vectors with or without cloned DNA inserts during library construction and screening. With the advent of increasingly efficient cloning systems, this methodology has become less critical for routine cloning — although still providing an additional level of certainty. However, the construction of the genomic sequence for various species and the extensive use of libraries for numerous purposes, including drug discovery, and elucidation pathways associated with development and disease, continue to depend extensively on color selection as a vital component of the high-throughput process. Moreover, automated colony or plaque analyses (counting and "picking") requires a timely discernment of recombinants from non-recombinants, and distinguishable positive signal against the background of the medium.

Despite its utility, the use of X-gal has many drawbacks. Blue and white colonies are often difficult to distinguish, particularly during early colony formation. Slow color development and ambiguous staining often make it necessary to chill plates at 4 °C before colonies or plaques can be picked, adding an additional waiting period before clones can be further analyzed. X-gal is light sensitive, requiring that stock solutions and stored plates be protected from light. Routinely, X-gal is added to agar medium or "top agar" after autoclaving — an inconvenience. Because X-gal is not water-soluble, solutions must be prepared with N, N-dimethylformamide (DMF) a very toxic and possibly carcinogenic reagent. X-gal can be applied to previously poured plates, but color development may be uneven.

S-Gal™ (3,4-cyclohexenoesculetin-ß-D-galactopyranoside), a newly developed ß-galactosidase substrate, directly addresses these concerns (Figure 1). S-Gal™ is autoclavable or microwavable when dry-blended with IPTG in a LB agar medium. The hydrolyzed aglycone (non-sugar portion) reacts with the Fe3+ of added ferric ammonium citrate to produce an intense black stain. This stain provides enhanced contrast and earlier color discernment. S-Gal™ is more soluble in water than X-gal, and is not light sensitive. Functionally, S-Gal™ readily substitutes for X-gal for color selection: darkly stained colonies or plaques indicate the absence of a cloned DNA fragment, while the unstained colony or plaque denotes the presence of a cloned insert. The following report illustrates the advantages of S-Gal™ over the X-gal dye, emphasizing its potential utility for enhancing automated bacterial colony and plaque analyses.

Materials and Methods

All materials were supplied by Sigma-Aldrich Corporation (St. Louis, MO) unless otherwise stated.

Medium Preparation

The following components are added to LB (Miller's formulation) agar medium to prepare S-Gal™/LB agar blend used in this study:

S-Gal™

300

µg/ml

IPTG

30

µg/ml

Ferric Ammonium Citrate

500

µg/ml

S-Gal™/LB agar blend was mixed with deionized water and autoclaved for 20 minutes, at 122 °C and 15 pounds per square inch, using a Getinge autoclave, model GE6915-AR1with PACS 2000 controller (Getinge Castle, Rochester, NY). Twenty minutes represents the time of sustained chamber temperature and pressure of 122 °C and 15 pounds per square inch, respectively. For preparation of microwaved medium, the mix was heated until boiling was observed, followed with swirling to allow the remaining undissolved agar component to go into solution. Ampicillin was added post-autoclaving at 100 µg/ml. Kanamycin is also compatible with S-Gal™ (data not shown). For comparison with standard color selection, X-gal and IPTG were added to LB agar following autoclaving at final concentrations of 267 µg/ml and 67µg/ml, respectively.3

For chemical analysis of S-Gal™ temperature stability, S-Gal™, IPTG, and ferric ammonium citrate were added to water or LB medium at the concentrations previously stated for the LB agar blend, and autoclaved or microwaved in 50-ml aliquots. S-Gal™ dye was initially dissolved in N, N-dimethylformamide at a concentration of 200 mg/ml, and added to water or medium for analysis at the above concentration before autoclaving or microwaving. For this part of the evaluation, a stock solution of S-Gal™ in DMF was used for convenience and consistency. S-Gal™, determined empirically to be approximately 70 percent more soluble than X-gal at room temperature (data not shown), is suitable for the blended medium product but requires dissolving in DMF for preparing more concentrated solutions, such as the stock solution described here. IPTG was added to water or medium from an aqueous stock of 100 mg/ml. Ferric ammonium citrate was prepared as an aqueous stock solution of 200 mg/ml. For microwaving, medium was heated until boiling was first observed, followed with three separate 30-second intervals of further microwaving. The additional intervals of microwaving correspond to additional heating often required to dissolve the agar component when preparing plate medium. Autoclaved samples were heated as previously described for agar medium. Before drawing samples for analysis, microwaved and autoclaved media were brought to their original volume with deionized water.

Application Testing of S-GalTM Temperature Stability

Fifty microliters of subcloning efficiency DH5aTM E. coli (Life Technologies, Gaithersburg, MD) were transformed with 500 ng of pUC18 DNA (Product Code: D4154) or 500 ng of pFLAG-CMV-1-BAP (a component of the Mammalian Transient Expression Kit, Product Code: FLMA). Aliquots of both transformation reactions were spread on test plates containing S-Gal™ or X-gal. When evaluating contrast between stained and unstained colonies, and plate background, plates were spread with different quantities of these two transformation reactions resulting in correspondingly different colony densities. This was done to reflect varying degrees of ligation efficiency or the representation of recombinants versus non-recombinants in a library.

Chemical Analysis of S-Gal™ Temperature Stability

Samples of autoclaved or microwaved media or aqueous solutions containing S-Gal™, and unheated controls, were analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Samples were analyzed on a HP1100 (Hewlett Packard, Palo Alto, CA) using a Discovery C18 column (4.6 mm I.D., 25 cm; Supelco, Bellefonte, PA) at ambient temperature. Sample components were separated using mobile phases A (0.1% trifluoroacetic acid in water) and B (0.08% trifluoroacetic acid in acetonitrile) in a linear gradient from 5 to 80% B. Samples were detected at 220 nanometers. The peak corresponding to S-Gal™ was integrated and tabulated for the study.

Color Development/Contrast Analyses

Colony intensity (light detection, measured as "counts") and color development were determined and recorded using the Gel Doc™ 2000 and Quantity One™ 4.0.3 software from BioRad Laboratories (Hercules, CA). Plate images were viewed by transmitted light from the BioRad UV Transilluminator 2000 with wavelength shifting filter. The relative intensities of ten "positive" (unstained) or negative (dark stained) colonies, or points of background per plate were measured (n=10). A ratio of averaged absolute differences of intensity, between the positive and background measurements, was determined for the media formulations and several plating conditions. A similar comparison was made for the difference of intensity between negative and "positive" colonies.

Results

Autoclavability/Microwavability

Fifty-milliliter aliquots of LB broth or water, with added S-Gal™, IPTG, and ferric ammonium citrate (300 µg/ml, 30 µg/ml, and 500 µg/ml, respectively), were autoclaved or microwaved as described in Materials and Methods. S-Gal™ in samples for each condition, including unheated controls, was analyzed using RP-HPLC, as described in the Materials and Methods. Neither heating regimen resulted in any significant breakdown of S-Gal™ (Figure 2). The data for each condition shown is an average of three injections from the same sample. These samples were tested on two separate occasions (n=2). The standard deviation of these data for S-Gal™ in LB broth ranged from 0.29 — 17.90%, whereas in water, the range was 0.22 — 0.62%. The largest discrepancy (17.9%) was seen for the microwaved LB broth sample, while the standard deviation of the unheated LB control was 0.63%, and 0.29% for the autoclaved LB broth sample.

Repeated Microwaving of Medium

A two-hundred milliliter suspension of S-Gal™/LB agar blend in deionized water was initially autoclaved as described in the Materials and Methods, and subsequently allowed to congeal at 4 °C. The autoclaved medium was then subjected to six 3.5-minute cycles of microwaving, with intervals of congealing at 4 °C. Application testing for black/white selection of medium plated immediately after autoclaving and for each microwave cycle indicated no observable effect on color development, contrast, or bacterial growth.

Color Development

Competent DH5aTM was transformed with a pUC18 or pFLAG-CMV-1-BAP, as described in Materials and Methods. Aliquots of both transformation reactions were spread on LB plates containing S-Gal™, IPTG, and ferric ammonium citrate, or X-gal and IPTG.3 The rate of color development observed was similar for both formulations (Figure 3), with distinction between dark and light colonies observed visually at 16-18 hours post-plating. However, the black S-Gal™ colonies began to appear as early as 14 hours. This enhancement, due to the black staining versus blue, was evident throughout colony development.

Color Contrast

Comparative color intensity of colonies and plate background was analyzed, contrasting S-Gal™/LB agar and X-gal/LB agar media at 23 hours post-plating. Four different conditions, i.e., plates with differing numbers of b-galactosidase-expressing (stained) and non-expressing colonies (unstained), were tested on medium containing S-Gal™ or X-gal. The results in the Table show that across the four densities of colonies plated, discernment of "positive" from "negative" colonies using S-Gal™-containing medium was equivalent to the medium containing X-gal. However, unstained "positive" colonies were consistently more visible over background for medium containing S-Gal™ on the average, 25 percent more visible.

Stability of Prepared Medium

Plated S-Gal™/LB agar medium, containing ampicillin added post-autoclaving or microwaving, was stored at 4 °C for two weeks. Results indicate that such storage has no affect on contrast or color development (data not shown).

Plated microwaved S-Gal™/LB agar medium, with and without added ampicillin, was incubated at 37 °C for 60 hours with no indication of microbial growth, that might occur because of bacterial or fungal spore germination. In addition, microwaved S-Gal™/LB agar plated medium (with added ampicillin) stored for two weeks at 4 °C, showed no sign of contamination when subsequently incubated at 37 °C for 24 hours (data not shown). Ambient light exposure of S-Gal™/LB agar plated medium stored at 4 °C for up to two weeks showed no significant affect on color development and contrast (data not shown).

Discussion

S-Gal™ offers several significant advantages over X-gal. An obvious convenience of S-Gal™ is its autoclavability and microwavability. Autoclaving and microwaving have no significant effect on the compound (Figure 2). This allows the dye to be dry-blended directly into the medium, eliminating the need for preparation and handling of stock solutions in toxic solvents. Prepared medium is stable at 4 °C and, unlike X-gal, S-Gal™ is not light sensitive.

The higher standard deviation observed for the microwaved S-Gal™/LB medium (Figure 2) was most likely due to inconsistent injection of the sample. Triplicate injections of each sample were analyzed. For this particular sample, the area count for the third injection was significantly lower (~28% lower) than for the average of the first two injections (the variation in the area count of the first two injections was less than 5.3%). The S-Gal™ peak area was not the only peak observed to be lower. Consistently, all peak areas recorded from the third injection of sample were approximately 30% lower than those observed from the other two injections, indicating a possible instrument error.

The primary advantages of S-Gal™ over X-gal are found with contrast and color development. Automated analyses, such as counting or screening and recovery of recombinant clones by colony or plaque "picking," require a definitive contrast between positive and negative signal, and the background of the medium. Both contrast and early discernment between positive and negative signal is highly desirable for rapid analyses. The rate of color development on plates containing S-Gal™ or X-gal is similar (Figure 3). In both cases, ß-galactosidase is observed to hydrolyze the O-linkage between the galactoside and respective sidegroup of both molecules with apparently similar efficiency. For X-gal, the result is blue color development. In the case of S-Gal™, two cleaved cyclohexenoesculetin sidegroups (Figure 4) chelate Fe+3 (provided from added ferric ammonium citrate) to produce a black stain, which is more easily detected at a consistently earlier time point.

The density of the bacterial colonies or plaques on plated medium will affect the efficiency of staining by either S-Gal™ or X-gal. A colony or M13 phage plaque will draw dye from the medium in its immediate vicinity. The greater the density of colonies, the less substrate available per colony and, therefore, the less intense the staining of ß-galactosidase-expressing colonies. For this reason, four plating conditions were used for assessing differences in contrast on S-Gal™- and X-gal-containing media. The difference in intensity between positive and negative signal at 23 hours post-plating is approximately equivalent for S-Gal™ and X-gal (Table). However, unstained "positives" on S-Gal™-containing plates are consistently more discernable than X-gal "positives" over the medium background. The combination of enhanced contrast and earlier detection, in addition to stability and greater convenience, signify S-Gal™ as the more suitable dye for automated analyses.

Conclusions

  • S-Gal™/LB agar is stable during autoclaving and microwaving.
  • Autoclaved S-Gal™/LB agar can be stored at 4 °C and re-microwaved (repeatedly) without impairing performance.
  • S-Gal™/LB agar is not light sensitive.
  • S-Gal™-stained colonies are detectable earlier than X-gal-stained colonies.
  • Non-stained colonies (positives) are more easily discerned against the medium (background).

Acknowledgments

We acknowledge our colleagues of the Sigma-Aldrich R&D and Marketing Groups (St. Louis, MO), especially Keming Song, and Tom Hassell, for their help in this work.

References and Note

1. Horwitz, J.P., et al., Substrates for cytochemical demonstration of enzyme activity. I. Some substituted 3-indoyl-ß-glycopyranosides. J. Med. Chem. 7, 574 (1964).

2. Ullman, A., et al., Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the ß-galactosidase structural gene of Escherichia coli. J. Mol. Biol. 24, 339 - 343 (1967).

3. Greenstein, D. and Besmond, C. Preparing and Using M13-Derived Vectors, in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (Eds.), pp. 1.15.1 - 1.15.8., (John Wiley and Sons, New York, 1994).

† Heuermann, K. and Cosgrove, J., S-Gal™: An autoclavable dye for color selection of cloned DNA inserts. BioTechniques, 30(5), 1142-1147 (2001).

About the Authors

Ken Heuermann, M.S., is a senior scientist in Recombinant Protein Expression R&D and Jennifer Cosgrove, B.S., is an associate scientist in Bio-Organic R&D at Sigma-Aldrich, St. Louis, MO.

ORDERING INFORMATION

Product Code

Product Name

Unit

C4478

S-Gal7™/LB Agar Blend

500 ml

 

 

6 x 500 ml

For more information, see Frequently Asked Questions

   
 
   
 

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