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Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis
利用lacZ报告基因对形成杂环的蓝藻多变鱼腥藻中的基因表达进行荧光原位定位   

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Abstract

One of the most successful fluorescent proteins, used as a reporter of gene expression in many bacterial, plant and animals, is green fluorescent protein and its modified forms, which also function well in cyanobacteria. However, these fluorescent proteins do not allow rapid and economical quantitation of the reporter gene product, as does the popular reporter gene lacZ, encoding the enzyme β-galactosidase. We provide here a protocol for the in situ localization of β-galactosidase activity in cyanobacterial cells. This allows the same strain to be used for both a simple, quantitative, colorimetric assay with the substrate ortho-nitrophenyl-β-galactoside (ONPG) and for sensitive, fluorescence-based, cell-type localization of gene expression using 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG).

Keywords: β-galactosidase(β-半乳糖苷酶), in situ localization(原位定位), Heterocysts(异形细胞), Cyanobacteria(蓝藻), lacZ reporter(lacZ报告基因)

Background

Anabaena variabilis is a filamentous cyanobacterium that differentiates specialized cells called heterocysts that function specifically for nitrogen fixation (Kumar et al., 2010; Maldener and Muro-Pastor, 2010). We use the lacZ gene of Escherichia coli as a transcriptional reporter of cyanobacterial gene expression because of the ease of a quantitative, enzymatic, colorimetric, β-galactosidase assay in 96-well plates (Griffith and Wolf, 2002) and the ability to use the same strain for in situ localization of gene expression using the fluorescent substrate 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG) (Thiel et al., 1995; Ma et al., 2016). One of the earliest reports of lacZ as a reporter was the fusion of malF, encoding the maltose transporter, to lacZ, which resulted in localization of β-galactosidase activity to the cytoplasmic membrane in E. coli (Silhavy et al., 1976). Since then lacZ has been used as a reporter in bacterial, plant and animal systems; e.g., the stable transfection of mouse tumor cells with lacZ allowed single cell histochemical staining using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (Arlt et al., 2012). In fact, most cellular localization of expression of lacZ has used X-gal, which is relatively inexpensive, easy to use and provides an easy visual screen. Our initial attempts to use X-gal and other chromogenic substrates in Anabaena were unsuccessful because the colored products were toxic to cyanobacteria and often resulted in cell lysis. In addition, the cyanobacterial pigments, including chlorophyll, phycocyanin, and carotenoids, made color detection difficult. We also attempted to use the fluorescent substrate, 4-methylumbelliferone β-D-galactopyranoside, whose product, 4-methylumbelliferone, emits in the blue range; however, we were not able to detect fluorescence over the background fluorescence of the cells. Finally we tried fluorescein β-D-galactopyranoside (FDG), a very sensitive fluorogenic substrate for β-galactosidase. FDG, which is not fluorescent, is hydrolyzed in two steps by β-galactosidase, first to fluorescein monogalactoside and then to fluorescein. We modified the method developed to visualize lacZ expression during sporulation in Bacillus subtilis (Bylund et al., 1994; Chung et al., 1995). That protocol specified 5-octanolyaminofluorescein di-β-D-galactopyranoside (C8-FDG); however we had poor results with C8-FDG, so we tried the more lipophilic 5-dodecanolyaminofluorescein di-β-D-galactopyranoside (C12-FDG) (Miao et al., 1993; Plovins et al., 1994; Zhang et al., 1991), which has 12 carbons added to the fluorescein in FDG. C12-FDG proved to function well in cyanobacteria. Using C12-FDG we have been able to easily visualize heterocyst-specific expression of genes, such as cnfR1, the activator of the nitrogenase genes in heterocysts (Pratte and Thiel, 2016), fused to lacZ (Figure 1).

Materials and Reagents

  1. 1.7 ml Advant microtubes (MIDSCI, catalog number: AVSS1700 )
  2. Aluminum foil
  3. 0.22 µm filter (Thermo Fisher Scientific, Fisher Scientific, catalog number: 09-720-004 )
  4. Microscope cover glass (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-545A )
  5. Microscope slides (Thermo Fisher Scientific, Fisher Scientific, catalog number: 12-550-A3 )
  6. BP830, an A. variabilis ATCC 29413 derivative, containing a pcnfR1:lacZ fusion (Pratte and Thiel, 2016)
  7. Ammonium chloride (NH4Cl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: A661-500 )
  8. TES buffer (AG Scientific, catalog number: T-1050 )
  9. DMSO (Dimethyl sulfoxide) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP231-1 )
  10. Millipore water
  11. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: M63-500 )
  12. Calcium chloride dihydrate (CaCl2·2H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP510-500 )
  13. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: S271-1 )
  14. Potassium phosphate dibasic anhydrous (K2HPO4) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P288-500 )
  15. Manganese chloride tetrahydrate (MnCl2·4H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: M87-100 )
  16. Sodium molybdate dihydrate (Na2MoO4·2H2O) (Sigma-Aldrich, catalog number: M1003 )
  17. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: Z76-500 )
  18. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP346-500 )
  19. Boric acid (H3BO3) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP168-500 )
  20. Cobaltous chloride hexahydrate (CoCl2·6H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: C371-100 )
  21. Potassium hydroxide (KOH) (Thermo Fisher Scientific, Fisher Scientific, catalog number: P250-500 )
  22. Ethylenediaminetetraacetic acid (Na2EDTA·2H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP120-1 )
  23. Ferrous sulfate heptahydrate (FeSO4·7H2O) (Thermo Fisher Scientific, Fisher Scientific, catalog number: I146-500 )
  24. 25% glutaraldehyde solution (Sigma-Aldrich, catalog number: G5882 )
  25. ImaGene GreenTM C12FDG lacZ Gene Expression Kit (Thermo Fisher Scientific, catalog number: I2904 )
  26. p-Phenylenediamine (Sigma-Aldrich, catalog number: P-6001 )
  27. Glycerol (Thermo Fisher Scientific, catalog number: G33-1 )
  28. Sodium bicarbonate (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP328-500 )
  29. Allen and Arnon (AA) medium (Allen and Arnon, 1955): (AA/8 = 8-fold dilution of AA) (see Recipes)
    1. AA/8 media
    2. AA Phosphate stock solution
    3. K2HPO4 stock solution
    4. Microelements stock solution
    5. AA FeEDTA solution
  30. 0.04% glutaraldehyde solution (see Recipes)
  31. 100 µM 5-dodecanoylaminefluorescein di-β-d galactopyranoside (C12-FDG) in 25% DMSO (see Recipes)
  32. 0.5 M carbonate buffer (see Recipes)
  33. Antifade solution (see Recipes)

Equipment

  1. 125-ml glass flasks (Thermo Fisher Scientific, Fisher Scientific, catalog number: 10-040D )
  2. Plugs for 125-ml flasks (Thermo Fisher Scientific, Fisher Scientific, catalog number: 1412740C )
  3. Shaker (set at 170 rpm) (Eppendorf, New BrunswickTM, model: Innova® 2100 )
  4. Centrifuge (Eppendorf, model: 5415D )*
  5. Incubator (waterbath) (set at 37 °C) (Polyscience, model: 2LS-M )*
  6. Environmental chamber set at 30 °C with 70% humidity and light
  7. Spectrophotometer (Bibby Scientific, JENWAY, model: 7300 )
  8. Zeiss Confocal LSM700 using a Plan-Apochromat 63x/1.4 Oil DIC M27 objective (Carl Zeiss, model: LSM700)

*Note: These products have been discontinued.

Procedure

  1. Culture growth
    1. Starting from colonies on agar plates, inoculate strains of A. variabilis or Anabaena sp. PCC 7120 containing lacZ fusions (constructed as described in Pratte and Thiel, 2016) in AA/8 containing 5 mM NH4Cl and 10 mM TES, pH 7.2 and antibiotics, when necessary. Shake cultures at 170 rpm at 30 °C in 100-120 μE/m2 s light and allow to grow for about 10 generations (3-4 days).
    2. Two days prior to nitrogen-stepdown, dilute cultures 1:100 in AA/8 containing 5 mM NH4Cl and 10 mM TES, pH 7.2 and allow them to continue growing at 30 °C with shaking and light to an OD720 of 0.1-0.2. Cyanobacteria should be actively growing for several generations so that they differentiate heterocysts well after the removal of fixed nitrogen. Consistency in growth conditions improves the reproducibility of β-galactosidase production in the cultures.
    3. Wash actively growing (step A2) cyanobacterial cultures 3 x in AA/8 to remove nitrogen. Resuspend cultures to an OD720 of < 0.1 in 125-ml flask containing 50 ml AA/8 with (+N) or without (-N) 5 mM NH4Cl and 10 mM TES, pH 7.2 and grow for 24 h with light and shaking. Check cultures for heterocysts prior to starting in situ localization assays.

  2. In situ localization
    1. Spin down 2-5 ml culture in 1.7 ml Eppendorf tube for 5 min at 16,000 x g in microfuge.
    2. Wash cells twice with 500 µl water to remove growth medium.
    3. Fix cells in 500 µl 0.04% glutaraldehyde at 25 °C for 15 min.
    4. Centrifuge cells for 1 min at 16,000 x g in microfuge and remove glutaraldehyde solution.
    5. Wash pellet twice with 500 µl water to remove residual glutaraldehyde.
    6. Resuspend pellet in 30 µl of substrate - 100 µM 5-dodecanoylaminefluorescein di-β-d galactopyranosine (C12-FDG) in 25% DMSO and incubate in the dark at 37 °C for 30 min.
    7. Centrifuge cells for 1 min at 16,000 x g in microfuge and remove substrate.
    8. Wash pellet twice with 500 µl water to remove residual substrate.
      Note: This step is important to achieve low background fluorescence.
    9. Resuspend pellet in 20 µl of antifade solution to keep the fluorescence stable. Keep cells in the dark until visualizing on microscope. Proceed immediately to imaging.
    10. Add ~2 µl of prepared cells to microscope slide with cover slip and visualize on a Zeiss Confocal LSM700 using a plan Apochromat 63x/1.4 Oil DIC M27 objective. Expression of lacZ in cells was visualized using excitation (488 nm) and emission (400-557 nm) wavelengths (from an argon ion laser) specific to detect fluorescein fluorescence, while cyanobacterial autofluorescence was visualized using excitation (561 nm) and emission (565-700 nm) wavelengths to detect cyanobacterial phycobiliprotein fluorescence. Typically we observe hundreds of filaments and then choose fields with sufficient representative filaments for imaging.

Data analysis

Long, planar cyanobacterial filaments on a glass microscope slide under a cover slip are identified in the samples using bright-field microscopy with a plan Apochromat 63x/1.4 Oil DIC M27 objective. Bright- field images are obtained using the transmitted light channel during a 30 cycle, time-series acquisition. Images of phycobiliprotein autofluorescence of the same filaments are acquired by excitation using 561 nm irradiation from an argon ion laser and visualization at 565-700 nm using a 30 cycle, time-series acquisition. The gain is set at 600 and the focus is adjusted slightly to optimize phycobiliprotein autofluorescence emitted from the cells during a live, continuous fast scan. Typically, vegetative cells show higher levels of phycobiliprotein autofluorescence than heterocysts. Fluorescein, measuring lacZ expression, is excited using 488 nm irradiation from an argon laser and visualized at 400-557 nm using a 30 cycle, time-series acquisition. The gain to detect fluorescein is typically set at 800, but can be adjusted to detect lower levels of fluorescence. Cells emitting lower levels of fluorescence need a higher gain to visualize the fluorescence, whereas higher levels of fluorescence can be seen at lower gains. Images are saved and then converted to TIFF files for final analysis of the level and cell-type specific expression of lacZ (fluorescein) in the experimental versus the control strains. Examples of typical images are shown in Figure 1.
 

Figure 1. In situ localization of lacZ expression in an A. variabilis FD strain containing a cnfR1:lacZ reporter (BP830) (Pratte and Thiel, 2016) grown aerobically for 24 h after nitrogen depletion (-N) or with fixed nitrogen (+N). Arrows indicate representative heterocysts. Left panels: light micrographs; Middle panels: red fluorescence from photosynthetic pigments in cyanobacteria; Right panels: fluorescein fluorescence from cleavage of 5-dodecanoyl-fluorescein-β-D-galacto-pyranoside by β-galactosidase.

Notes

  1. Wash the cells treated with C12-FDG thoroughly to remove as much of the substrate as possible because it can lead to high background fluorescence.
  2. Protect cells from the light after they have been treated with C12-FDG, including when they are on the microscope slide.
  3. Use an anti-fade solution to prevent bleaching of the fluorescein by the excitation light.
  4. If C12-FDG does not work well in your cells, try unmodified FDG or other FDG derivatives: 5-pentafluorobenzoylamino-fluorescein, C8-FDG, or 5-chloromethylfluorescein (http://www.mobitec.de/probes/docs/sections/1002.pdf).
  5. If you prefer red fluorescence you can try resorufin or C8-resorufin labeled β-d-galactopyranosides (http://www.mobitec.de/probes/docs/sections/1002.pdf).

Recipes

  1. Allen and Arnon medium (AA) (diluted 8-fold [AA/8])
    Note: Anabaena variabilis is grown in an eight-fold dilution of Allen and Arnon medium (Allen and Arnon, 1955).
    1. AA/8 media
      3.1 ml AA-Phosphate stock solution
      0.8 ml K2HPO4 stock solution
      996.0 ml Millipore water
      Aliquot 50 ml in 125-ml flasks with plugs and aluminum foil covering, and then autoclave for 30 min at 121 °C
    2. AA Phosphate stock solution
      500 ml 4% MgSO4·7H2O (final concentration 1%)
      500 ml 1.2% CaCl2·2H2O (final concentration 0.3%)
      500 ml 3.8% NaCl (final concentration 0.95%)
      500 ml microelements stock solution
    3. K2HPO4 stock solution
      28.0 g K2HPO4
      500.0 ml Millipore water
    4. Microelements stock solution
      160.0 ml AA Fe-EDTA solution
      360 mg MnCl2·4H2O
      35 mg Na2MoO4·2H2O
      44.0 mg ZnSO4·7H2O
      15.8 mg CuSO4·5H2O
      572.0 mg H3BO3
      8.0 mg CoCl2·6H2O
      1,090.0 ml Millipore water
    5. AA FeEDTA solution
  2. 0.04% glutaraldehyde solution
    40 µl 1% glutaraldehyde solution
    960 µl Millipore water
    (1% glutaraldehyde solution: 40 µl [25% glutaradehyde solution] added 960 µl [Millipore water])
  3. 100 µM 5-dodecanoylaminefluorescein di-β-d galactopyranoside (C12-FDG) in 25% DMSO
    5 µl 20 mM C12-FDG (supplied in ImaGene GreenTM C12FDG lacZ Gene Expression Kit)
    250 µl 100% DMSO
    745 µl sterile Millipore water
  4. 0.5 M carbonate buffer pH 8.0
    5.42  sodium bicarbonate
    9 ml Millipore water
    Adjust pH to 8.0 and bring volume to 10 ml with Millipore water
  5. Antifade solution
    Dissolve 30 mg p-Phenylenediamine in 4 ml of sterile Millipore water
    Add 6.0 ml glycerol
    Add 1.0 ml 0.5 M carbonate buffer (pH 8.0)
    Filter through a 0.22-µm filter to remove any undissolved chemical and store in 0.5 ml aliquots at -80 °C in the dark

Acknowledgments

This protocol was based on an earlier protocol for Bacillus subtilis (Bylund et al., 1994) that was modified for use in cyanobacteria. Support for this research was provided by National Science Foundation grant MCB-1052241.

References

  1. Allen, M. B. and Arnon, D. I. (1955). Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol 30(4): 366-372.
  2. Arlt, M. J., Born, W. and Fuchs, B. (2012). Improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and X-Gal staining of lacZ-tagged tumor cells. J Vis Exp (66): e4162.
  3. Bylund, J. E., Zhang, L., Haines, M. A., Higgins, M. L. and Piggot, P. J. (1994). Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis. J Bacteriol 176(10): 2898-2905.
  4. Chung, J. D., Conner, S. and Stephanopoulos, G. (1995). Flow cytometric study of differentiating cultures of Bacillus subtilis. Cytometry 20(4): 324-333.
  5. Currier, T. C. and Wolk, C. P. (1979). Characteristics of Anabaena variabilis influencing plaque formation by cyanophage N-1. J Bacteriol 139(1): 88-92.
  6. Griffith, K. L. and Wolf, R. E. (2002). Measuring β-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays. Biochem Biophy Res Co 290: 397-402.
  7. Kumar, K., Mella-Herrera, R. A. and Golden, J. W. (2010). Cyanobacterial heterocysts. Cold Spring Harb Perspect Biol 2(4): a000315.
  8. Ma, P., Mori, T., Zhao, C., Thiel, T. and Johnson, C. H. (2016). Evolution of KaiC-dependent timekeepers: A proto-circadian timing mechanism confers adaptive fitness in the purple bacterium Rhodopseudomonas palustris. PLoS Genet 12(3): e1005922.
  9. Maldener, I. and Muro-Pastor, A. M. (2010). Cyanobacterial Heterocysts. eLS. John Wiley & Sons Ltd, Chichester.
  10. Miao, F., Todd, P. and Kompala, D. S. (1993). A single-cell assay of β-galactosidase in recombinant Escherichia coli using flow cytometry. Biotechnol Bioeng 42: 708-715.
  11. Plovins, A., Alvarez, A. M., Ibañez, M., Molina, M. and Nombela, C. (1994). Use of fluorescein-di-β-D-galactopyranoside (FDG) and C12-FDG as substrates for β-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells. Appl Environ Microbiol 60: 4638-4641.
  12. Pratte, B. S. and Thiel, T. (2016). Homologous regulators, CnfR1 and CnfR2, activate expression of two distinct nitrogenase gene clusters in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. Mol Microbiol 100(6): 1096-1109.
  13. Silhavy, T. J., Casadaban, M. J., Shuman, H. A. and Beckwith, J. R. (1976). Conversion of β-galactosidase to a membrane-bound state by gene fusion. Proc Natl Acad Sci U S A 73(10): 3423-3427.
  14. Thiel, T., Lyons, E. M., Erker, J. C. and Ernst, A. (1995). A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium. Proc Natl Acad Sci U S A 92(20): 9358-9362.
  15. Zhang, Y. Z., Naleway, J. J., Larison, K. D., Huang, Z. J. and Haugland, R. P. (1991). Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic β-galactosidase substrates. FASEB J 5(15): 3108-3113.

简介

在许多细菌,植物和动物中用作基因表达的报告基因的最成功的荧光蛋白之一是绿色荧光蛋白及其修饰形式,其在蓝细菌中也起良好作用。然而,与编码β-半乳糖苷酶的流行的报道基因lacZ一样,这些荧光蛋白不允许报道基因产物的快速和经济的定量。我们在这里提供了在蓝细菌细胞中原位β-半乳糖苷酶活性定位的方案。这允许将相同的菌株用于具有底物邻硝基苯基-β-半乳糖苷(ONPG)的简单,定量,比色测定,并且用于灵敏的,基于荧光的细胞型定位使用5-十二烷酰氨基荧光素二-β-D-吡喃半乳糖苷(C12-FDG)的基因表达。

背景 鱼腥藻变种是一种丝状蓝细菌,其区分称为异养细胞的特异性细胞,其特异性用于固氮(Kumar等人,2010; Maldener and Muro Pastor,2010)。由于在96孔中易于定量,酶,比色,β-半乳糖苷酶测定,我们使用大肠埃希氏菌的 lacZ 基因作为蓝细菌基因表达的转录报告基因(Griffith和Wolf,2002)和使用相同的菌株用于使用荧光底物5-十二烷基聚氨基荧光素二-β-D-吡喃半乳糖苷(C12-FDG)的原位定位基因表达的能力( Thiel等人,1995; Ma等人,2016)。作为报道者的最早的报告之一是将编码麦芽糖转运蛋白的malF融合到lacZ,导致β-半乳糖苷酶活性定位于细胞质膜中的膜。大肠杆菌(Silhavy等人,1976)。此后,lacZ 已被用作细菌,植物和动物系统的记者;例如,用lacZ 稳定转染小鼠肿瘤细胞允许使用显色底物5-溴-4-氯-3-吲哚基-β-吡喃半乳糖苷(X-Gal)(Arlt等人,2012)。事实上,大多数细胞定位的表达的lacZ 已经使用了相对便宜的X-gal,易于使用,并提供了一个简单的视觉屏幕。我们初步尝试使用Xaram和其他显色底物在鱼腥藻中不成功,因为有色产品对蓝细菌有毒,并且经常导致细胞裂解。此外,蓝藻色素,包括叶绿素,藻蓝蛋白和类胡萝卜素,使得颜色检测困难。我们还尝试使用其产物4-甲基伞形酮在蓝色范围内发射的荧光底物4-甲基伞形酮β-D-吡喃半乳糖苷。然而,我们无法在细胞的背景荧光中检测荧光。最后,我们尝试了荧光素β-D-吡喃半乳糖苷(FDG),一种非常敏感的β-半乳糖苷酶的荧光底物。不荧光的FDG通过β-半乳糖苷酶分两步进行水解,首先用荧光素单糖苷,然后进行荧光素水解。我们修改了在枯草芽孢杆菌(Bilund等人,1994; Chung等人)中孢子形成过程中开发的可视化表达lacZ 的方法, /em>。,1995)。该方案规定5-辛氨基荧光素二-β-D-吡喃半乳糖苷(C8-FDG);然而,我们对C8-FDG的结果差,所以我们尝试了更亲脂的5-十二烷基聚氨基荧光素二-β-D-吡喃半乳糖苷(C12-FDG)(Miao等人,1993; Plovins等人,1994; Zhang等人,1991),其中FDG中的荧光素中添加了12个碳。 C12-FDG在蓝细菌中表现良好。使用C12-FDG,我们已经能够容易地观察基因的异型细胞特异性表达,例如,与异源囊中的氮酶基因的活化剂(Pratte和Thiel,2016)融合的基因,例如cnfR1 lacZ (图1)。

关键字:β-半乳糖苷酶, 原位定位, 异形细胞, 蓝藻, lacZ报告基因

材料和试剂

  1. 1.7ml Advant微管(MIDSCI,目录号:AVSS1700)
  2. 铝箔
  3. 0.22μm过滤器(Thermo Fisher Scientific,Fisher Scientific,目录号:09-720-004)
  4. 显微镜盖玻璃(Thermo Fisher Scientific,Fisher Scientific,目录号:12-545A)
  5. 显微镜载玻片(Thermo Fisher Scientific,Fisher Scientific,目录号:12-550-A3)
  6. BP830,a。变体ATCC 29413衍生物,其含有pcnfR1:lacZ 融合物(Pratte和Thiel,2016)
  7. 氯化铵(NH 4 Cl)(Thermo Fisher Scientific,Fisher Scientific,目录号:A661-500)
  8. TES缓冲器(AG Scientific,目录号:T-1050)
  9. DMSO(二甲基亚砜)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP231-1)
  10. Millipore水
  11. 硫酸镁七水合物(MgSO 4·7H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:M63-500)
  12. 氯化钙二水合物(CaCl 2·2H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP510-500)
  13. 氯化钠(NaCl)(Thermo Fisher Scientific,Fisher Scientific,目录号:S271-1)
  14. 无水磷酸氢钾(K 2/2 HPO 4)(Thermo Fisher Scientific,Fisher Scientific,目录号:P288-500)
  15. 四氯化锰(MnCl 2·4H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:M87-100)
  16. 钼酸钠二水合物(Na 2 MoO 4·2H 2 O)(Sigma-Aldrich,目录号:M1003)
  17. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:Z76-500)
  18. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP346-500)
  19. 硼酸(H 3 3 BO 3)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP168-500)
  20. 六氯化钴(CoCl 2·6H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:C371-100)
  21. 氢氧化钾(KOH)(Thermo Fisher Scientific,Fisher Scientific,目录号:P250-500)
  22. 乙二胺四乙酸(Na 2 EDTA·2H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP120-1)
  23. 硫酸亚铁七水合物(FeSO 4·7H 2 O)(Thermo Fisher Scientific,Fisher Scientific,目录号:I146-500)
  24. 25%戊二醛溶液(Sigma-Aldrich,目录号:G5882)
  25. 基因表达试剂盒(Thermo Fisher Scientific,目录号:I2904)
    <><
  26. 对苯二胺(Sigma-Aldrich,目录号:P-6001)
  27. 甘油(Thermo Fisher Scientific,目录号:G33-1)
  28. 碳酸氢钠(Thermo Fisher Scientific,Fisher Scientific,目录号:BP328-500)
  29. Allen和Arnon(AA)培养基(Allen和Arnon,1955):(AA/8 = AA的8倍稀释度)(参见食谱)
    1. AA/8媒体
    2. AA磷酸盐储备溶液
    3. K 2> HPO 4 储备溶液
    4. 微量元素库存解决方案
    5. AA FeEDTA解决方案
  30. 0.04%戊二醛溶液(参见食谱)
  31. 在25%DMSO中的100μM5-十二酰胺荧光素二-β-d吡喃半乳糖苷(C12-FDG)(参见食谱)
  32. 0.5 M碳酸盐缓冲液(见配方)
  33. 防腐解决方案(见配方)

设备

  1. 125毫升玻璃烧瓶(Thermo Fisher Scientific,Fisher Scientific,目录号:10-040D)
  2. 用于125ml烧瓶的塞子(Thermo Fisher Scientific,Fisher Scientific,目录号:1412740C)
  3. 振荡器(设定在170rpm)(Eppendorf,New Brunswick TM ,型号:Innova ® 2100)
  4. 离心机(Eppendorf,型号:5415D)*
  5. 孵化器(水浴)(37°C)(Polyscience,型号:2LS-M)*
  6. 环境室设置在30°C,70%湿度和浅色
  7. 分光光度计(Bibby Scientific,JENWAY,型号:7300)
  8. Zeiss Confocal LSM700使用Plan-Apochromat 63x/1.4 Oil DIC M27物镜(Carl Zeiss,型号:LSM700)

注意:这些产品已经停产。

程序

  1. 文化成长
    1. 从琼脂平板上的菌落开始,接种A株菌株。变种或鱼腥藻含有含有5mM NH 4 Cl和10mM TES,pH 7.2的AA/8中的含有lacZ融合体(Pratte和Thiel,2016所述构建)的PCC 7120和抗生素,必要时。在30°C,以100-120μE/m 2的光照下以170rpm振荡培养物,并允许生长约10代(3-4天)。
    2. 氮降解前两天,在含有5mM NH 4 Cl和10mM TES(pH7.2)的AA/8中稀释培养物1:100,并允许它们在30℃下摇动继续生长,光到OD 为0.1-0.2。蓝细菌应积极生长数代,以便在去除固定氮后能很好地分离异养细胞。生长条件的一致性提高了培养物中β-半乳糖苷酶生产的重现性
    3. 在AA/8中洗涤积极生长(步骤A2)3×的蓝藻培养物以除去氮。将培养物重悬于720nm的OD < 0.1在含有(+ N)或不含(-N)5mM NH 4 Cl和50mM TES,pH 7.2的50ml AA/8的125ml烧瓶中,并用光和生长24小时,摇动在开始原位定位测定之前检查杂种的培养物。

  2. 原位本地化
    1. 在微量离心机中以16,000 x g的速度将2-5ml培养物在1.7ml Eppendorf管中旋转5分钟。
    2. 用500μl水洗涤细胞两次以除去生长培养基
    3. 将细胞在25℃下在500μl0.04%戊二醛中固定15分钟
    4. 在微量离心机中以16,000 x g离心细胞1分钟,并除去戊二醛溶液。
    5. 用500μl水洗涤沉淀两次以除去残留的戊二醛
    6. 将沉淀重悬于30μl底物 - 100μM5-十二烷酰胺荧光素二-β-d吡喃半乳糖(C12-FDG)的25%DMSO中,并在37℃下暗处孵育30分钟。
    7. 在微量离心机中以16,000 x g离心细胞1分钟,并除去底物
    8. 用500μl水洗涤沉淀两次以除去残留的底物 注意:这一步对于实现低背景荧光很重要。
    9. 将沉淀物重悬于20μl抗真菌液中以保持荧光稳定。将细胞保持在黑暗中,直到显微镜观察。立即进行成像。
    10. 添加〜2μl准备的细胞到具有盖滑动的显微镜载玻片上并且使用计划的Apochromat 63x/1.4油DIC M27目标在Zeiss Confocal LSM700上显现。使用激发(488nm)和发射(400-557nm)波长(来自氩离子激光)来特异性检测荧光素荧光的细胞中的lacZ 的表达,而蓝细菌自发荧光用激发( 561nm)和发射(565-700nm)波长以检测蓝藻藻胆蛋白荧光。通常我们观察数百根长丝,然后选择具有足够代表性丝线的场用于成像。

数据分析

使用具有平面Apochromat 63x/1.4油DIC M27物镜的明视野显微镜在样品中鉴定了在盖玻片下的玻璃显微镜载玻片上的长平面蓝细菌细丝。在30周期,时间序列采集期间,使用透射光通道获得亮场图像。通过使用来自氩离子激光的561nm照射的激发获得相同细丝的藻胆蛋白自发荧光的图像,并使用30周期时间序列采集在565-700nm的可视化。增益设置为600,并且稍微调整焦点,以在活体连续快速扫描期间优化从细胞发出的藻胆蛋白自发荧光。通常,营养细胞比异囊细胞显示更高水平的藻胆蛋白自身荧光。使用来自氩激光器的488nm照射激发荧光素,测量lacZ 表达,并使用30周期时间序列采集在400-557nm显现。检测荧光素的增益通常设定在800,但可以调整以检测较低水平的荧光。发射较低荧光水平的细胞需要更高的增益来显现荧光,而较低的增益可以看到更高的荧光水平。图像被保存,然后转换成TIFF文件,以便最终分析实验对照菌株中的lacZ(荧光素)的水平和细胞类型特异性表达。典型图像的例子如图1所示  

图1. 原位 本地化 lacZ 表达式 A。含有cnfR1:lacZ 报告者(BP830)(Pratte和Thiel,2016)的变应性 氮耗竭(-N)或固定氮(+ N)。箭头表示代表性的异型囊。左图:光照片;中间板:蓝藻中光合色素的红色荧光;右图:β-半乳糖苷酶切割5-十二酰基荧光素-β-D-半乳糖吡喃糖苷的荧光素荧光。

笔记

  1. 用C12-FDG彻底洗涤细胞以除去尽可能多的底物,因为它可以导致高背景荧光。
  2. 使用C12-FDG处理细胞后,保护细胞,包括当它们在显微镜载玻片上时。
  3. 使用防褪色溶液来防止荧光素被激发光漂白。
  4. 如果C12-FDG在细胞中不能正常工作,请尝试未修饰的FDG或其他FDG衍生物:5-五氟苯甲酰氨基荧光素,C8-FDG或5-氯甲基荧光素( http://www.mobitec.de/probes/docs/sections/1002.pdf )。
  5. 如果您喜欢红色荧光,您可以尝试使用resorufin或C8-resorufin标记的β-D-吡喃半乳糖苷( http://www.mobitec.de/probes/docs/sections/1002.pdf )。

食谱

  1. Allen和Arnon培养基(AA)(稀释8倍[AA/8])
    注意:鱼腥藻以八倍稀释的Allen和Arnon培养基(Allen和Arnon,1955)生长。
    1. AA/8媒体
      3.1毫升AA-磷酸盐储备溶液
      0.8ml K 2 HPO 4储备溶液
      996.0毫升Millipore水
      将等分试样在125毫升带有塞子和铝箔覆盖的烧瓶中,然后在121℃下高压灭菌30分钟。
    2. AA磷酸盐储备溶液
      500ml 4%MgSO 4·7H 2 O(终浓度1%)
      500ml 1.2%CaCl 2·2H 2 O(终浓度0.3%)
      500 ml 3.8%NaCl(最终浓度为0.95%)
      500毫升微量元素储备溶液
    3. K 2> HPO 4 储备溶液
      28.0g K 2 HPO 4
      500.0毫升Millipore水
    4. 微量元素库存解决方案
      160.0 ml AA Fe-EDTA溶液
      360mg MnCl 2·4H 2 O
      35mg Na 2 MoO 4·2H 2 O 44.0mg ZnSO 4·7H 2 O
      15.8mg CuSO 4·5H 2 O
      572.0mg H 3 BO 3
      8.0mg CoCl 2·6H 2 O
      1,090.0毫升Millipore水
    5. AA FeEDTA溶液
  2. 0.04%戊二醛溶液
    40μl1%戊二醛溶液
    960微升Millipore水
    (1%戊二醛溶液:40μl[25%戊二醛溶液])加入960μl[Millipore水])
  3. 在25%DMSO中的100μM5-十二烷酰胺荧光素二-β-d半乳糖吡喃糖苷(C12-FDG)
    5μl20mM C12-FDG(以ImaGene Green提供)基因表达试剂盒
    250μl100%DMSO
    745微升无菌微孔水
  4. 0.5M碳酸盐缓冲液pH 8.0
    5.42 碳酸氢钠
    9毫升Millipore水
    将pH调至8.0,并用Millipore水将体积调至10ml
  5. 防风解决方案
    将30毫克对苯二胺溶于4毫升无菌微孔水中 加入6.0毫升甘油 加入1.0 ml 0.5 M碳酸盐缓冲液(pH 8.0) 过滤0.22μm过滤器以除去任何未溶解的化学物质,并在-80℃黑暗中储存在0.5ml等分试样中。

致谢

该方案基于经修饰用于蓝细菌的枯草芽孢杆菌(Bylund等人,1994)的早期方案。国家科学基金资助项目MCB-1052241提供了本次研究的支持。

参考文献

  1. Allen,MB和Arnon,DI(1955)。  研究在固氮蓝绿藻上。 I. Anabaena cylindrica Lemm的生长和固氮 植物生理30 /(3):366-372。
  2. Arlt,MJ,Born,W. and Fuchs,B。(2012)。  通过组合肺组织原位灌注和λ-lacZ标记的肿瘤细胞的X-Gal染色改善小鼠单细胞分辨率肺转移的可视化。 (66):e4162。
  3. Bylund,JE,Zhang,L.,Haines,MA,Higgins,ML和Piggot,PJ(1994)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih。 gov/pubmed/8188591"target ="_ blank">通过荧光显微镜分析在枯草芽孢杆菌的孢子形成过程中细胞间特异性基因表达的发展。细菌> 176(10):2898-2905。
  4. Chung,JD,Conner,S.和Stephanopoulos,G。(1995)。枯草芽孢杆菌分化培养物的流式细胞术研究。细胞计数 20(4):324-333。
  5. Currier,TC and Wolk,CP(1979)。  特征通过蓝藻N-1影响斑块形成的不同变异体。 139细菌杆菌139(1):88-92。
  6. Griffith,KL and Wolf,RE(2002)。  测量细菌中的β-半乳糖苷酶活性:96孔阵列中的细胞生长,透化和酶测定。生物化学Biophy Res Co 290:397-402。
  7. Kumar,K.,Mella-Herrera,RA和Golden,JW(2010)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/20452939"目标="_ blank">蓝藻细菌。冷泉Harb Perspect Biol 2(4):a000315。
  8. Ma,P.,Mori,T.,Zhao,C.,Thiel,T.and Johnson,CH(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm开眼科依赖计时员的演变:原始昼夜节律计时机制赋予紫色细菌红假单胞菌中的适应性适应度。 > PLoS Genet 12(3):e1005922。
  9. Maldener,I。和Muro-Pastor,AM(2010)。< a class ="ke-insertfile"href ="http://onlinelibrary.wiley.com/doi/10.1002/9780470015902.a0000306.pub2/abstract"目标="_ blank">蓝藻异种囊。 John Wiley& Sons有限公司,奇切斯特。
  10. Miao,F.,Todd,P.和Kompala,DS(1993)。  使用流式细胞术进行重组大肠杆菌中的β-半乳糖苷酶的单细胞测定。生物技术Bioeng 42:708-715。 />
  11. Plovins,A.,Alvarez,AM,Ibañez,M.,Molina,M。和Nombela,C。(1994)。使用荧光素二-β-D-吡喃半乳糖苷(FDG)和C12-FDG作为动物,细菌和酵母细胞中流式细胞术检测β-半乳糖苷酶的底物。 Appl Environ Microbiol 60:4638-4641。
  12. Pratte,BS和Thiel,T.(2016)。同源调节剂CnfR1和CnfR2激活丝状蓝细菌斑马鱼ATCC 29413中两种不同的氮酶基因簇的表达.Mol Microbiol 100(6):1096 -1109。
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  15. Zhang,YZ,Naleway,JJ,Larison,KD,Huang,ZJ和Haugland,RP(1991)。使用新的亲脂性荧光β-半乳糖苷酶底物检测活细胞中的lacZ 基因表达。 FASEB J 15):3108-3113。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pratte, B. S. and Thiel, T. (2017). Fluorescence in situ Localization of Gene Expression Using a lacZ Reporter in the Heterocyst-forming Cyanobacterium Anabaena variabilis. Bio-protocol 7(1): e2084. DOI: 10.21769/BioProtoc.2084.
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