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Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria
Cryobiology 53 (2006) 276–278 www.elsevier.com/locate/ycryo
Brief communication
Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria 夽 Elias Anastassopoulos TEI Larissas, School of Agricultural Technology, Department of Plant Production, Perifereiaki Odos Trikalon Karditsas, 41335 Larissa, Greece Received 4 April 2006; accepted 30 May 2006 Available online 18 July 2006
Abstract An agar plate freezing assay is described based on the incorporation of Xuorescein dye in agar medium. Upon addition of Xuorescein the medium becomes transparent. This facilitates the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. In comparison with known assays for the screening of transformants, the proposed assay is very accurate and reproducible. It may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes. © 2006 Elsevier Inc. All rights reserved. Keywords: Ice nucleation; In situ; Selection; Freezing; Agar; Fluorescence; Assay
Biological ice nucleation is a phenomenon in which ice nucleating proteins trigger the liquid to solid phase transition of water, resulting in the formation of ice. Genes encoding ice nucleation proteins have been cloned primarily from bacteria, though there are other organisms, with proven ice nucleating activity, such as fungi and lichens (for a review see [1,7]). Moreover, ice nucleation genes have been used as reporter molecules in a variety of studies (for a review see [4]). Once initiated by ice nucleation, freezing is very rapid, therefore it is very diYcult to detect the bac夽
This work was supported by the General Secretary of Research and Technology, Hellas. The assay described in this paper is a subject of a patent pending. E-mail address: [email protected]. 0011-2240/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2006.05.006
terium that initiated the event in a mixture of bacteria (e.g. in environmental samples or transformants) grown in liquid or in solid media. For liquid cultures, a droplet freezing test can be applied, which involves successive dilutions, splitting the culture into small droplets and measuring the ice nucleation activity of each droplet (for a review see [6]). For bacteria grown in agar plates, it is very diYcult to detect which colony initiated freezing, due to the opacity of the medium, both before and after the freezing event. For colony screening, a technique analogous to the classical replica plating (replica freezing) has been used which involves printing the colonies on aluminum foil and spraying the foil with a Wne mist, before subjecting the colonies at appropriate subzero temperatures [3].
E. Anastassopoulos / Cryobiology 53 (2006) 276–278
The application of solutions of Xuorescein dye in biological ice nucleation studies is not new, since it has been used for spraying aluminum foils in droplet freezing assays [2]. In this report, Xuorescein dye was incorporated in the agar medium, to make it transparent and enable the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. The inaZ gene from Pseudomonas syrignae, was cloned under the control of Plac promoter into pBSK(-) (Stratagene, La Jolla, CA, USA), and the derived plasmid, pBSK(-)/Zani, was used to transform Escherichia coli strain JM83, following standard cloning procedures [5]. No IPTG induction was used. The transparent medium was prepared by adding 0.05 g/ mL of Xuorescein (Sigma, St. Louis, MO, USA) and 100 g/mL ampicillin (Cooper SA, Greece) to LB medium. Agar plates were left to solidify, prior to
277
plating the bacteria. After an incubation period at 23 °C for 48 h the colonies formed were pierced with a needle, so that some bacteria to be inserted in the medium. After piercing, the plates were subjected to freezing by Xoating them on the surface of an alcohol bath, (Lauda RE120, Messgeraete Werk Lauda, West Germany), maintained at ¡7 °C. The inaZ gene was expressed in transformed E. coli cells, resulting in detectable ice nucleation activity, at ¡7 °C (Fig. 1A–F). Colonies transformed with the plasmid pBSK(-), negative controls, did not show any ice nucleation activity at ¡7 °C (data not shown). In comparison with known techniques for the screening of transformants, the proposed technique is very accurate and reproducible. The agar plates can be repeatedly frozen 2–3 times for veriWcation of results, though the texture of the medium changes during repeated freezing. The technique is not destructive to
Fig. 1. (A–I). Sequential images of a freezing event initiated by a colony of ice nucleating JM83 E. coli cells harboring the plasmid pBSK(-)/Zani. Images were captured using a digital camera (Olympus Camedia C-350 Zoom) mounted on top of a magniWer lens (Original Hanau FLUOTEST). Note a second freezing event (E–I), that is partly masked by the initial freezing event. The arrow indicates the beginning of a second freezing event.
278
E. Anastassopoulos / Cryobiology 53 (2006) 276–278
bacteria, since they can be recovered after the plates have been frozen. A limiting factor for the use of inaZ carrying plasmids as cloning vehicles is the rather large size of the bacteria ice nucleation genes, about 3–4 kb. The freezing bath can be substituted by any other apparatus with a surface that can be maintained at such temperatures, e.g. a peltier cold plate. Also, it is necessary to pierce the solid medium with bacteria, so that some cells can be inserted in the medium. The technique may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes. Acknowledgments I thank Professor Nick Panopoulos for critically reading the manuscript, Mr. Petros Kyrkoudis for technical assistance and Mr. Eleftherios Kardamakis for his contribution to the art work.
References [1] E.N. Ashworth, T.L. Kieft, Ice nucleation activity associated with plants and fungi, in: R.E. Lee, G.J. Warren, L.V. Gusta (Eds.), Biological Ice Nucleation and Its Applications, APS Press, St. Paul, MN, 1995, pp. 137–162. [2] G. Gotz, E. Meszaros, G. Vali, Atmospheric Particles and Nuclei, Akademiai Kiado, Budapest, 1991. [3] C.S. Orser, B.J. Staskawicz, N.J. Panopoulos, D. Dahlbeck, S.E. Lindow, Cloning and expression of bacterial ice nucleation genes in Escherichia coli, J. Bacteriol. 41 (1985) 359–366. [4] N.J. Panopoulos, Ice nucleation genes as reporters, in: R.E. Lee, G.J. Warren, L.V. Gusta (Eds.), Biological Ice Nucleation and Its Applications, APS Press, St. Paul, MN, 1995, pp. 271–281. [5] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989. [6] G. Vali, Principles of ice nucleation, in: R.E. Lee, G.J. Warren, L.V. Gusta (Eds.), Biological Ice Nucleation and Its Applications, APS Press, St. Paul, MN, 1995, pp. 1–28. [7] G.J. Warren, IdentiWcation and analysis of ina genes and proteins, in: R.E. Lee, G.J. Warren, L.V. Gusta (Eds.), Biological Ice Nucleation and Its Applications, APS Press, St. Paul, MN, 1995, pp. 85–99.
Brief communication
Agar plate freezing assay for the in situ selection of transformed ice nucleating bacteria 夽 Elias Anastassopoulos TEI Larissas, School of Agricultural Technology, Department of Plant Production, Perifereiaki Odos Trikalon Karditsas, 41335 Larissa, Greece Received 4 April 2006; accepted 30 May 2006 Available online 18 July 2006
Abstract An agar plate freezing assay is described based on the incorporation of Xuorescein dye in agar medium. Upon addition of Xuorescein the medium becomes transparent. This facilitates the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. In comparison with known assays for the screening of transformants, the proposed assay is very accurate and reproducible. It may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes. © 2006 Elsevier Inc. All rights reserved. Keywords: Ice nucleation; In situ; Selection; Freezing; Agar; Fluorescence; Assay
Biological ice nucleation is a phenomenon in which ice nucleating proteins trigger the liquid to solid phase transition of water, resulting in the formation of ice. Genes encoding ice nucleation proteins have been cloned primarily from bacteria, though there are other organisms, with proven ice nucleating activity, such as fungi and lichens (for a review see [1,7]). Moreover, ice nucleation genes have been used as reporter molecules in a variety of studies (for a review see [4]). Once initiated by ice nucleation, freezing is very rapid, therefore it is very diYcult to detect the bac夽
This work was supported by the General Secretary of Research and Technology, Hellas. The assay described in this paper is a subject of a patent pending. E-mail address: [email protected]. 0011-2240/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2006.05.006
terium that initiated the event in a mixture of bacteria (e.g. in environmental samples or transformants) grown in liquid or in solid media. For liquid cultures, a droplet freezing test can be applied, which involves successive dilutions, splitting the culture into small droplets and measuring the ice nucleation activity of each droplet (for a review see [6]). For bacteria grown in agar plates, it is very diYcult to detect which colony initiated freezing, due to the opacity of the medium, both before and after the freezing event. For colony screening, a technique analogous to the classical replica plating (replica freezing) has been used which involves printing the colonies on aluminum foil and spraying the foil with a Wne mist, before subjecting the colonies at appropriate subzero temperatures [3].
E. Anastassopoulos / Cryobiology 53 (2006) 276–278
The application of solutions of Xuorescein dye in biological ice nucleation studies is not new, since it has been used for spraying aluminum foils in droplet freezing assays [2]. In this report, Xuorescein dye was incorporated in the agar medium, to make it transparent and enable the monitoring of the ice nucleation event in vivo and the subsequent in situ selection of transformed ice nucleating bacteria. The inaZ gene from Pseudomonas syrignae, was cloned under the control of Plac promoter into pBSK(-) (Stratagene, La Jolla, CA, USA), and the derived plasmid, pBSK(-)/Zani, was used to transform Escherichia coli strain JM83, following standard cloning procedures [5]. No IPTG induction was used. The transparent medium was prepared by adding 0.05 g/ mL of Xuorescein (Sigma, St. Louis, MO, USA) and 100 g/mL ampicillin (Cooper SA, Greece) to LB medium. Agar plates were left to solidify, prior to
277
plating the bacteria. After an incubation period at 23 °C for 48 h the colonies formed were pierced with a needle, so that some bacteria to be inserted in the medium. After piercing, the plates were subjected to freezing by Xoating them on the surface of an alcohol bath, (Lauda RE120, Messgeraete Werk Lauda, West Germany), maintained at ¡7 °C. The inaZ gene was expressed in transformed E. coli cells, resulting in detectable ice nucleation activity, at ¡7 °C (Fig. 1A–F). Colonies transformed with the plasmid pBSK(-), negative controls, did not show any ice nucleation activity at ¡7 °C (data not shown). In comparison with known techniques for the screening of transformants, the proposed technique is very accurate and reproducible. The agar plates can be repeatedly frozen 2–3 times for veriWcation of results, though the texture of the medium changes during repeated freezing. The technique is not destructive to
Fig. 1. (A–I). Sequential images of a freezing event initiated by a colony of ice nucleating JM83 E. coli cells harboring the plasmid pBSK(-)/Zani. Images were captured using a digital camera (Olympus Camedia C-350 Zoom) mounted on top of a magniWer lens (Original Hanau FLUOTEST). Note a second freezing event (E–I), that is partly masked by the initial freezing event. The arrow indicates the beginning of a second freezing event.
278
E. Anastassopoulos / Cryobiology 53 (2006) 276–278
bacteria, since they can be recovered after the plates have been frozen. A limiting factor for the use of inaZ carrying plasmids as cloning vehicles is the rather large size of the bacteria ice nucleation genes, about 3–4 kb. The freezing bath can be substituted by any other apparatus with a surface that can be maintained at such temperatures, e.g. a peltier cold plate. Also, it is necessary to pierce the solid medium with bacteria, so that some cells can be inserted in the medium. The technique may be applied in environmental samples screening for ice nucleating organisms, or in cDNA or genomic libraries for identifying novel ice nucleation genes. It may also prove useful in comparative studies of the ice nucleation activity, e.g. in directed evolution experiments involving ice nucleation genes. Acknowledgments I thank Professor Nick Panopoulos for critically reading the manuscript, Mr. Petros Kyrkoudis for technical assistance and Mr. Eleftherios Kardamakis for his contribution to the art work.
References [1] E.N. Ashworth, T.L. Kieft, Ice nucleation activity associated with plants and fungi, in: R.E. Lee, G.J. Warren, L.V. Gusta (Eds.), Biological Ice Nucleation and Its Applications, APS Press, St. Paul, MN, 1995, pp. 137–162. [2] G. Gotz, E. Meszaros, G. Vali, Atmospheric Particles and Nuclei, Akademiai Kiado, Budapest, 1991. [3] C.S. Orser, B.J. Staskawicz, N.J. Panopoulos, D. Dahlbeck, S.E. Lindow, Cloning and expression of bacterial ice nucleation genes in Escherichia coli, J. Bacteriol. 41 (1985) 359–366. [4] N.J. Panopoulos, Ice nucleation genes as reporters, in: R.E. Lee, G.J. Warren, L.V. Gusta (Eds.), Biological Ice Nucleation and Its Applications, APS Press, St. Paul, MN, 1995, pp. 271–281. [5] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989. [6] G. Vali, Principles of ice nucleation, in: R.E. Lee, G.J. Warren, L.V. Gusta (Eds.), Biological Ice Nucleation and Its Applications, APS Press, St. Paul, MN, 1995, pp. 1–28. [7] G.J. Warren, IdentiWcation and analysis of ina genes and proteins, in: R.E. Lee, G.J. Warren, L.V. Gusta (Eds.), Biological Ice Nucleation and Its Applications, APS Press, St. Paul, MN, 1995, pp. 85–99.