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In vivo Analysis of Cyclic di-GMP Cyclase and Phosphodiesterase Activity in Escherichia coli Using a Vc2 Riboswitch-based Assay
使用基于Vc2 Riboswitch的测定法体内分析大肠杆菌中环状di-GMP环化酶和磷酸二酯酶活性   

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Ying  LiuYing Liu*Hyunhee  KimHyunhee Kim*Ute   RömlingUte Römling  (*共同第一作者)
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Journal of Bacteriology
Sep 2017

Abstract

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that regulates distinct aspects of bacterial physiology. It is synthesized by diguanylate cyclases (DGCs) and hydrolyzed by phosphodiesterases (PDEs). To date, the activities of DGC and PDE are commonly assessed by phenotypic assays, mass spectrometry analysis of intracellular c-di-GMP concentration, or riboswitch-based fluorescent biosensors. However, some of these methods require cutting-edge equipment, which might not be available in every laboratory. Here, we report a new simple, convenient and cost-effective system to assess the function of DGCs and PDEs in E. coli. This system utilizes the high specificity of a riboswitch to c-di-GMP and its ability to regulate the expression of a downstream β-galactosidase reporter gene in response to c-di-GMP concentrations. In this protocol, we delineate the construction of this system and its use to assess the activity of DGC and PDE enzymes.

Keywords: Cyclic di-guanylate monophosphate (c-di-GMP) (环鸟苷酸单磷酸酯(c-di-GMP)), Diguanylate cyclase (DGC) (Diguanylate环化酶(DGC)), Phosphodiesterase (PDE) (磷酸二酯酶(PDE)), Riboswitch (核糖开关), β-Galactosidase (β-半乳糖苷酶)

Background

Cyclic-di-GMP is an important and ubiquitous second messenger in bacteria, which regulates a variety of processes, such as motility-to-sessility transition, biofilm formation, virulence, and cell cycle progression (Römling et al., 2013). The GG(D/E)EF domain has diguanylate cyclase (DGC) activity and it is responsible for the synthesis of c-di-GMP from two GTPs, which is a two-step reaction with 5’-pppGpG as intermediate and two molecules of pyrophosphate as by-products (Ryjenkov et al., 2005). Phosphodiesterases (PDE) with an EAL or an HD-GYP domain hydrolyze c-di-GMP into linear 5’-pGpG (Schmidt et al., 2005) and GMP (Ryan et al., 2006), respectively.

Several tools have been developed to monitor intracellular cyclic di-nucleotide levels and to identify proteins involved in cyclic di-nucleotide signaling, for example, protein-based fluorescence resonance energy transfer (FRET) biosensor (Christen et al., 2010), riboswitch-based fluorescent biosensor (Kellenberger et al., 2015), and riboswitch-based dual-fluorescence reporter (Zhou et al., 2016). However, these tools monitor altered fluorescence of reporters and require the access to flow cytometry or fluorescence microscopy. Here, we report the development of an alternative assay to monitor the intracellular c-di-GMP concentration, namely by monitoring the alteration in β-galactosidase activity in agar-growing cells. For that, the Vc2 riboswitch (Sudarsan et al., 2008) is fused translationally to lacZY and integrated into the chromosome of E. coli strain TOP10. Vc2 is an ‘off’ riboswitch from Vibrio cholerae and thus down-regulates the expression of β-galactosidase when c-di-GMP is bound (Figure 1). The stable integration into the Tn7 attachment site in the chromosome of E. coli avoids copy number effects and eliminates the need to use an antibiotic resistance marker. Changes in c-di-GMP levels are subsequently translated to the alteration in β-galactosidase expression, which is reflected by the color change of the colony growing on an agar plate containing 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). This assay can be used, for instance, to reveal the function of proteins under physiological condition and to assess the enzymatic activity of proteins that are challenging to be purified and tested in vitro. However, the Vc2-based assay described here is a qualitative assessment of the change in intracellular c-di-GMP concentration. Quantification is not crucial for a screening assay, but would be advantageous in, for example, measuring the activity of enzymes. We have demonstrated that the Vc2-based assay can be exploited to verify the activity of both DGCs and PDEs in vivo (El Mouali et al., 2017).


Figure 1. The principle of a riboswitch-based screening system. The Vc2 riboswitch is located upstream of the β-galactosidase open reading frame to control its expression in response to the variation in c-di-GMP concentration. When c-di-GMP is present in high abundance due to the overexpression of DGCs, the expression of β-galactosidase is down-regulated resulting in a white colony on an X-gal containing plate. In contrast, when PDEs are overexpressed, generating low intracellular c-di-GMP concentration, the colony is blue. In E. coli TOP10 wild type cells, there are residual amounts of c-di-GMP, resulting in a light blue colony (adapted from El Mouali et al., 2017).

Materials and Reagents

  1. PCR tubes
  2. Petri dish 92 x 16 mm (SARSTEDT, catalog number: 82.1472.001 )
  3. Syringe filters, 0.2 μm pore size (SARSTEDT, catalog number: 83.1826.001 )
  4. Microcentrifuge tubes of 1.5 ml (SARSTEDT, catalog number: 72.690.001 )
  5. Pipette tips (1,000 μl: SARSTEDT, catalog number: 70.762.100 ; 1-200 μl: Corning, catalog number: 4804 ; 0.1-10 μl: Gilson, catalog number: F171103 )
  6. Spectrophotometer cuvettes (SARSTEDT, catalog number: 67.742 )
  7. 3-part disposable HSW SOFT-JECT® syringes (5 ml: Henke Sass Wolf, catalog number: 5050.X00V0 ; 10 ml: Henke Sass Wolf, catalog number: 5100.X00V0 )
  8. Aluminum foil
  9. Plasmids used in this study:

  10. Calcium chloride competent E. coli TOP10 cells (Hanahan, 1983)
  11. Primers used in this study (ordered from Sigma-Aldrich):

    aThe restriction sites are underlined.
  12. GenElute Plasmid Miniprep Kit (Sigma-Aldrich, catalog number: PLN350 )
  13. Sterile distilled, deionized water (diH2O)
  14. Phusion High-fidelity DNA polymerase (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: F530S )
  15. dNTP mix (10 mM each) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0192 )
  16. Agarose (Sigma-Aldrich, catalog number: A9539 )
  17. SmartLadder for DNA: 200-10,000 bp (Eurogentec, catalog number: MW-1700-10 )
  18. GelRedTM nucleic acid gel stain (Biotium, catalog number: 41003 )
  19. NotI restriction enzyme (New England Biolabs, catalog number: R0189S )
  20. PacI restriction enzyme (New England Biolabs, catalog number: R0547S )
  21. PCR purification kit (QIAGEN, catalog number: 28106 )
  22. Rapid DNA ligase kit (Roche Diagnostics, catalog number: 10716359001 )
  23. Taq DNA polymerase with standard Taq buffer (New England Biolabs, catalog number: M0273S )
  24. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
  25. Tryptone (BD, BactoTM, catalog number: 211705 )
  26. Yeast extract (BD, BactoTM, catalog number: 212750 )
  27. Agar (BD, DifcoTM, catalog number: 281230 )
  28. Tris base (Sigma-Aldrich, catalog number: 93350 )
  29. Glacial acetic acid (Sigma-Aldrich, catalog number: ARK2183 )
  30. EDTA (Sigma-Aldrich, catalog number: E9884 )
  31. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518 )
  32. Gentamicin sulfate salt (Sigma-Aldrich, catalog number: G3632 )
  33. L-(+)-arabinose (Sigma-Aldrich, catalog number: A3256 )
  34. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, catalog number: I6758 )
  35. Dimethyl sulfoxide (Sigma-Aldrich, catalog number: D8418 )
  36. 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) (Roche Diagnostic, catalog number: 10703729001 )
  37. LB (Luria-Bertani) medium (see Recipes)
  38. LB (Luria-Bertani) agar (see Recipes)
  39. TAE buffer (see Recipes)
  40. 100 mg/ml ampicillin stock (see Recipes)
  41. 30 mg/ml gentamicin stock (see Recipes)
  42. 100 mM IPTG stock (see Recipes)
  43. 20 mg/ml X-gal stock (see Recipes)

Equipment

  1. Erlenmeyer flasks (50 ml/100 ml/250 ml)
  2. Pipettes [e.g., PIPETMAN® P20 (Gilson, catalog number: F123600 ), P200 (Gilson, catalog number: F123601 ), or P1000 (Gilson, catalog number: F123602 )]
  3. SureCycler 8800 thermal cycler (Agilent Technologies, model: SureCycler 8800 , catalog number: G8800A)
  4. HeraeusTM PicoTM 17 Microcentrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM PicoTM 17 , catalog number: 75002410)
  5. Mini-Sub® cell GT Horizontal Electrophoresis System (Bio-Rad Laboratories, catalog number: 1704406 )
  6. 15-well comb (Bio-Rad Laboratories, catalog number: 1704464 )
  7. Sub-Cell GT UV-Transparent Mini-Gel Tray (Bio-Rad Laboratories, catalog number: 1704436 )
  8. Electrophoresis power supply
  9. Heraeus® microbiological incubator (Thermo Fisherer Scientific, Thermo ScientificTM, model: Heraeus® microbiological incubator )
  10. Multitron Standard shaker (INFORS HT)
  11. Gel DocTM XR+ Gel Documentation System (Bio-Rad Laboratories, catalog number: 1708195 )
  12. BioPhotometer (Eppendorf, model: 6131 )
  13. NanoDropTM 2000c Spectrophotometers (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000c , catalog number: ND-2000C)
  14. Thermomixer compact (Eppendorf, model: 5350 )
  15. Digital camera

Procedure

Practically, the workflow can be divided into two parts, as shown in Figure 2. The first part is to construct the reporter strain, which takes approximately 12-15 days (Steps 1-3 of the protocol). The second part is to assess the activity of DGC and PDE by the reporter strain (Steps 4-5). This will take 7-9 days.


Figure 2. The overview of the workflow. The procedure is divided into two parts: 1) construction of the reporter strain and 2) assessment of c-di-GMP cyclase or phosphodiesterase activity by the reporter. It takes approximately 12-15 days to construct the reporter strain, and another 7-9 days to assess the target.

  1. Amplify Vc2 and lacZY by PCR:
    1. The template for the PCR is Vc2-pRS414 (kindly received from Prof. R. R. Breaker, Yale University), in which the fragment containing riboswitch along with its native promoter was cloned in frame to the lacZ reporter gene (Sudarsan et al., 2008).
    2. The primers used are:
      Vc2lacZY_F_PacI: GCGTTAATTAAATAACGCCTATATTTGAAAGCTTG
      Vc2lacZY_R_NotI: GATCAGCGGCCGCTTAAGCGACTTCATTCACCTG
      The restriction sites PacI and NotI are underlined.
    3. Prepare PCR reaction mix:

    4. Thermocycling conditions:

    5. Check the PCR products by gel electrophoresis using 1% TAE agarose gel. The expected size is 4,733 bp.
  2. Clone Vc2lacZY into pGRG25 vector:
    1. Double digest the PCR product Vc2lacZY and the plasmid pGRG25 with PacI and NotI at 37 °C overnight. To reduce star activity, it is advisable to use the fewest units possible to achieve the digestion. After digestion, it is necessary to check the size of products using agarose gel electrophoresis. The enzymes are inactivated at 65 °C for 20 min.
    2. Remove the restriction enzymes using PCR purification kit. The concentration of digested Vc2lacZY and pGRG25 are measured by NanoDrop.
    3. Ligate pGRG25 and Vc2lacZY of molar ratio 1:1 at 12 °C overnight using T4 DNA ligase following the manufacturer’s instruction. To verify that the vector is completely cut, ligate the digested vector alone. If the vector is not cut by the two restriction enzymes efficiently, re-circulation will happen and colonies will appear after transformation.
    4. Transform the ligation products into chemically competent TOP10 cells and select for the recombinant plasmids on LB agar plate containing 100 μg/ml ampicillin. The pGRG25 vector is temperature-sensitive, so the plate should be incubated at 32 °C or even lower temperature.
    5. Verify the recovered colonies by colony PCR. First, prepare the master mix for Taq DNA polymerase with standard Taq buffer (from New England Biolabs) following manufacturer’s instruction. Then 25 µl aliquots are added to the PCR tubes. Sterile pipette tips are used to pick single colonies and mix with reaction buffer. An initial 5 min denaturation at 95 °C is recommended for colony PCR with Taq DNA polymerase. The expected size is 518 bp. The primers used are:
      pGRG25control_F: CACTTATCTGGTTGGTCGACACT
      Vc2lacZY_R: CTGCAAGGCGATTAAGTTGG
      The details for the colony PCR can be found in the protocol by Azevedo et al. (2017).
    6. Confirm the sequence integrity of the riboswitch in the positive clones by DNA sequencing using the same primers for colony PCR.
  3. Integrate Vc2lacZY into the Tn7 attachment site of E. coli chromosome (Note 1):
    1. Streak positive colonies on LB agar plate supplemented with 100 μg/ml ampicillin and incubate the cells at 32 °C.
    2. Inoculate approximately 10 colonies into 3 ml of ampicillin-free LB medium at 32 °C, 200 rpm. Add 0.1% L-arabinose (w/v) to facilitate the incorporation of Vc2lacZY into E. coli Tn7 attachment site. The integration results in a new strain E. coli TOP10 attTn7::Vc2lacZY.
    3. Dilute the overnight culture by a factor of 108 and plate 50 μl of the diluted culture on LB plate to acquire single colonies. Incubate the plate at 42 °C to block the replication of the temperature-sensitive pGRG25 vector.
    4. Streak individual colonies on LB plate and repeat the incubation at 42 °C to make sure that the plasmid is lost.
    5. Confirm the integration of Vc2lacZY into the chromosome by colony PCR using Taq DNA polymerase. The primers used are:
      pGRGcontrol_F: GATGCTGGTGGCGAAGCTGT
      pGRGcontrol_R: GATGACGGTTTGTCACATGGA
      The expected size for positive clone and control is 6,328 bp and 678 bp, respectively.
  4. Assess the activity of diguanylate cyclases by Vc2-based screening: take the established DGC AdrA, YdeH and STM4551 as examples
    1. Prepare calcium chloride competent E. coli TOP10 attTn7::Vc2lacZY cells.
    2. Transform pBAD vectors (Note 2) harboring DGC (AdrA, YdeH and STM4551) and catalytically inactive mutants (STM4551E267A and YdeH-mut) as well as empty vectors into the chemically competent cells by heat shock at 42 °C for 40 sec. Add 1 ml LB medium free of antibiotics into the microcentrifuge tubes and incubate the tubes at 37 °C, 200 rpm for 1 h to let the cells recover.
    3. Plate 200 μl cells on LB agar plates containing 100 μg/ml ampicillin and incubate the plates at 37 °C overnight.
    4. Streak single colonies on ampicillin-containing LB agar plates.
    5. Pick one colony for each strain and culture the colony in 5 ml of LB medium with 100 μg/ml ampicillin at 37 °C, 200 rpm overnight.
    6. Measure the OD600 of overnight culture and adjust the OD600 to 0.1.
    7. Grow cells at 37 °C, 200 rpm. When OD600 reaches 0.6-1.0, spot 3 μl culture on LB plates with the addition of 100 μg/ml ampicillin, 80 μg/ml X-gal, 0.1% (w/v) L-arabinose (Note 3). It is important that cells with DGC, corresponding mutant, and vector control have similar OD600.
    8. Incubate the plates at 28 °C and monitor the color of colonies up to 72 h. A DGC activity will lead to a white colony indicative of high c-di-GMP levels. Take pictures of the plate using a digital camera.
  5. Investigate the activity of PDE: take the established PDE STM3611 as an example
    1. The intracellular concentration of c-di-GMP is low in TOP10 cells, therefore Vc2lacZY-harboring cells have a basal level of β-galactosidase activity resulting in a blue colony, which makes it impossible to distinguish the color further enhanced by phosphodiesterase. The activity of PDE can, however, be reliably investigated by co-expression of a DGC. Therefore, pWJB30 (DGC AdrA cloned into pBAD30) and pSTM3611 (PDE STM3611 cloned into pSRKGm) (Notes 2 and 4) are co-transformed into chemically competent TOP10::Vc2lacZY cells. As controls, empty vectors are co-transformed or in combination with the DGC/PDE. The combinations are listed in Table 1.

      Table 1. The combination of plasmids co-transformed into chemically competent TOP10::Vc2lacZY cells


    2. Plate cells on LB agar plates containing both ampicillin (100 μg/ml) and gentamicin (30 μg/ml). The plates are incubated at 37 °C overnight.
    3. Streak the positive colonies on antibiotics-containing LB agar plates.
    4. Inoculate at least three individual colonies per strain in 5 ml of LB medium containing both ampicillin and gentamicin. Grow cells at 37 °C, 200 rpm overnight.
    5. Measure OD600 of overnight culture and dilute the culture to OD600 of 0.1 with LB medium containing antibiotics. Let the cells grow to exponential phase (OD600 ~0.6). It is critical that all samples have similar OD600 values.
    6. Spot 3 μl of the cultures on LB agar plates containing ampicillin (100 μg/ml), gentamicin (30 μg/ml), IPTG (0.75 mM), L-arabinose (0.003%, w/v) and X-Gal (80 μg/ml) (Note 3).
    7. Incubate the plate at 28 °C and record the color of the colonies up to 72 h. While expression of the DGC alone leads to white colonies, co-expression of an active PDE will lead to blue colonies.
    8. Take pictures of the plate using a digital camera.

Data analysis

  1. Assessment of the activity of DGC by the Vc2-based assay: after spotted on X-gal containing plates, the color of colonies is monitored periodically. Figure 3 shows the color after 24 h incubation at 28 °C. It is explicit that cells expressing DGCs have whiter color due to the riboswitch inhibition of β-galactosidase expression upon high c-di-GMP levels compared with the corresponding mutant and vector control.


    Figure 3. Detection of established diguanylate cyclases by the Vc2-based assay. DGC AdrA, STM4551 and catalytic inactive STM4551E267A were cloned into pBAD30 generating pWJB30, p4551 and p4551E267A, respectively. Another DGC YdeH and its mutant YdeHG206AG207A were cloned into pBAD28 resulting in pBAD28ydeH and pBAD28ydeH-mut. Cells expressing functional DGC (AdrA/STM4551/YdeH) are white, while cells with vector control or catalytically inactive mutant (STM4551E267A/YdeHG206AG207A) are blue. Growth was at 28 °C for 24 h on LB agar plates supplemented with 100 μg/ml ampicillin, 80 μg/ml X-gal and 0.1% arabinose (w/v).

  2. Assessment of the enzymatic activity of a PDE by the Vc2-based assay: Figure 4 shows the color of colonies after incubation at 28 °C for 48 h. Cells expressing the DGC AdrA alone (row 2) or co-expressing AdrA and catalytically inactive PDE STM3611E136A (row 5) are white. However, cells are blue when wild type STM3611 is co-expressed with AdrA (row 4), which illustrates the enzymatic activity of STM3611 and the decrease of the intracellular c-di-GMP concentration upon expression.


    Figure 4. Detection of phosphodiesterase activity of PDE STM3611 by the Vc2-based assay. Phosphodiesterase STM3611 and catalytic inactive STM3611E136A were cloned into pSRKGm generating pSTM3611 and pSTM3611E136A, respectively. Diguanylate cyclase AdrA was cloned into pBAD30 resulting in pWJB30. Cells expressing the DGC AdrA alone have a white color. Upon co-expressing the PDE STM3611 with AdrA, the color turns to blue indicating low c-di-GMP level. However, cells expressing the DGC AdrA with catalytically inactive PDE STM3611E136A are white. Growth was at 28 °C for 48 h on LB agar plate supplemented with 100 μg/ml ampicillin, 30 μg/ml gentamicin, 80 μg/ml X-gal, 0.003% arabinose (wt/vol), and 0.75 mM IPTG (adapted from El Mouali et al., 2017).

Notes

  1. The protocol for the integration of target genes into the E. coli chromosomal Tn7 attachment site is delineated by G. J. McKenzie and N. L. Craig (McKenzie and Craig, 2006).
  2. We cloned diguanylate cyclases into pBAD28/pBAD30 vectors and phosphodiesterases into the pSRKGm vector. Other expression vectors can also be used depending on the availability.
  3. Different L-(+)-arabinose and IPTG concentrations need to be tested to achieve the most discriminating color contrast between diguanylate cyclase, phosphodiesterase and control.
  4. It is important that diguanylate cyclase and phosphodiesterase are cloned into compatible vectors containing different antibiotic resistance markers and compatible origins of replication.
  5. The specificity of the Vc2-based riboswitch towards other cyclic di-nucleotides has not been tested in this work.

Recipes

  1. LB (Luria-Bertani) medium for 400 ml
    4 g NaCl
    4 g tryptone
    2 g yeast extract
    Add dH2O to 400 ml
    Autoclave at 121 °C for 15 min
  2. LB (Luria-Bertani) agar for 400 ml
    4 g NaCl
    4 g tryptone
    2 g yeast extract
    6 g agar
    Add dH2O to 400 ml
    Autoclave at 121 °C for 15 min
    Pour into Petri dishes (add antibiotics, X-gal, L-arabinose, or IPTG if required)
    Let cool completely and store at 4 °C
  3. TAE buffer (50x stock solution)
    Dissolve 242 g Tris base in water
    Add 57.1 ml glacial acetic acid
    Add 100 ml of 0.5 M EDTA (pH 8.0)
    Add water up to 1 L
  4. 100 mg/ml ampicillin stock
    Dissolve 500 mg ampicillin sodium salt in 5 ml deionized water
    Filter the solution with 0.2 µm sterile syringe filter
    Make 1 ml aliquot of solution into sterile microcentrifuge tubes
    Store at -20 °C
  5. 30 mg/ml gentamicin stock
    Dissolve 150 mg gentamicin sulfate salt in 5 ml deionized water
    Filter the solution with 0.2 µm sterile syringe filter
    Make 1 ml aliquot of solution into sterile microcentrifuge tubes
    Store at -20 °C
  6. 100 mM IPTG stock
    Dissolve 1.191 g IPTG in 5 ml deionized water
    Filter the solution with 0.2 µm sterile syringe filter
    Make 1 ml aliquot of solution into sterile microcentrifuge tubes
    Store at -20 °C
  7. 20 mg/ml X-gal stock
    Dissolve 100 mg X-gal in 5 ml DMSO
    Wrap the tube with aluminum foil to protect from light
    Store at -20 °C
    The solution is stable for 6-12 months at -20 °C

Acknowledgments

The authors would like to thank Prof. Dr. Ronald R. Breaker for sharing Vc2-pRS414 riboswitch construct. Prof. Ute Römling conceived this study. Ying Liu developed the assay and assessed DGC activity. Hyunhee Kim developed the assay to assess PDE activity. This work was supported by the Swedish Research Council for Natural Sciences and Engineering (grant 621-2013-4809) and the Karolinska Institutet. This modified protocol is based on a previously published work (El Mouali et al., 2017). The authors declare no conflicts of interest or competing interests.

References

  1. Ahmad, I., Lamprokostopoulou, A., Le Guyon, S., Streck, E., Barthel, M., Peters, V., Hardt, W. D. and Römling, U. (2011). Complex c-di-GMP signaling networks mediate transition between virulence properties and biofilm formation in Salmonella enterica serovar Typhimurium. PLoS One 6(12): e28351.
  2. Azevedo, F., Pereira, H. and Johansson, B. (2017). Colony PCR. Methods Mol Biol 1620: 129-139.
  3. Christen, M., Kulasekara, H. D., Christen, B., Kulasekara, B. R., Hoffman, L. R. and Miller, S. I. (2010). Asymmetrical distribution of the second messenger c-di-GMP upon bacterial cell division. Science 328(5983): 1295-1297.
  4. El Mouali, Y., Kim, H., Ahmad, I., Brauner, A., Liu, Y., Skurnik, M., Galperin, M. Y. and Römling, U. (2017). Stand-alone EAL domain proteins form a distinct subclass of EAL proteins involved in regulation of cell motility and biofilm formation in enterobacteria. J Bacteriol 199(18): pii:e0079-17.
  5. Guzman, L. M., Belin, D., Carson, M. J. and Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177(14): 4121-4130.
  6. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166(4): 557-580.
  7. Jonas, K., Edwards, A. N., Simm, R., Romeo, T., Römling, U. and Melefors, O. (2008). The RNA binding protein CsrA controls cyclic di-GMP metabolism by directly regulating the expression of GGDEF proteins. Mol Microbiol 70(1): 236-257.
  8. Kellenberger, C. A., Chen, C., Whiteley, A. T., Portnoy, D. A. and Hammond, M. C. (2015). RNA-based fluorescent biosensors for live cell imaging of second messenger cyclic di-AMP. J Am Chem Soc 137(20): 6432-6435.
  9. Khan, S. R., Gaines, J., Roop, R. M., 2nd and Farrand, S. K. (2008). Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing. Appl Environ Microbiol 74(16): 5053-5062.
  10. McKenzie, G. J. and Craig, N. L. (2006). Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event. BMC Microbiol 6: 39.
  11. Römling, U., Galperin, M. Y. and Gomelsky, M. (2013). Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77(1): 1-52.
  12. Ryan, R. P., Fouhy, Y., Lucey, J. F., Crossman, L. C., Spiro, S., He, Y. W., Zhang, L. H., Heeb, S., Camara, M., Williams, P. and Dow, J. M. (2006). Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc Natl Acad Sci U S A 103(17): 6712-6717.
  13. Ryjenkov, D. A., Tarutina, M., Moskvin, O. V. and Gomelsky, M. (2005). Cyclic diguanylate is a ubiquitous signaling molecule in bacteria: insights into biochemistry of the GGDEF protein domain. J Bacteriol 187(5): 1792-1798.
  14. Schmidt, A. J., Ryjenkov, D. A. and Gomelsky, M. (2005). The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains. J Bacteriol 187(14): 4774-4781.
  15. Simm, R., Morr, M., Kader, A., Nimtz, M. and Römling, U. (2004). GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 53(4): 1123-1134.
  16. Sudarsan, N., Lee, E. R., Weinberg, Z., Moy, R. H., Kim, J. N., Link, K. H. and Breaker, R. R. (2008). Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science 321(5887): 411-413.
  17. Zhou, H., Zheng, C., Su, J., Chen, B., Fu, Y., Xie, Y., Tang, Q., Chou, S. H. and He, J. (2016). Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter. Sci Rep 6: 20871.

简介

环状二磷酸鸟苷(c-di-GMP)是一种无处不在的第二信使,它调节细菌生理学的不同方面。 它由diguanylate环化酶(DGC)合成并被磷酸二酯酶(PDE)水解。 迄今为止,通常通过表型分析,细胞内c-di-GMP浓度的质谱分析或基于核糖开关的荧光生物传感器来评估DGC和PDE的活性。 但是,其中一些方法需要尖端设备,而这些设备可能不适用于每个实验室。 在这里,我们报告了一个新的简单,方便和具有成本效益的系统,用于评估E中DGC和PDE的功能。大肠杆菌。 该系统利用核糖开关对c-di-GMP的高特异性及其响应于c-di-GMP浓度而调节下游β-半乳糖苷酶报道基因的表达的能力。 在该协议中,我们描述了该系统的构建及其用于评估DGC和PDE酶的活性。

【背景】Cyclic-di-GMP是细菌中重要且无处不在的第二信使,其调节各种过程,例如运动到衰退转变,生物膜形成,毒力和细胞周期进展(Römling等人, ,2013)。 GG(D / E)EF结构域具有二磷酸环化酶(DGC)活性,它负责从两个GTPs合成c-di-GMP,这是一个两步反应,以5'-pppGpG作为中间体和两个分子作为副产物的焦磷酸盐(Ryjenkov等人,2005)。具有EAL或HD-GYP结构域的磷酸二酯酶(PDE)将c-di-GMP水解为线性5'-pGpG(Schmidt等人,2005)和GMP(Ryan等人, ,2006)。

已经开发了几种工具来监测细胞内环状二核苷酸水平并鉴定涉及环二核苷酸信号传导的蛋白质,例如基于蛋白质的荧光共振能量转移(FRET)生物传感器(Christen等人 ,2010),基于核糖开关的荧光生物传感器(Kellenberger等人,2015)和基于核糖开关的双荧光记者(Zhou等人,2016)。然而,这些工具监测记者改变的荧光,并需要访问流式细胞仪或荧光显微镜。在这里,我们报告开发了另一种检测方法来监测细胞内c-di-GMP浓度,即通过监测琼脂生长细胞中β-半乳糖苷酶活性的变化。为此,Vc2核糖开关(Sudarsan等人,2008)被翻译融合到 lacZY 并整合到E染色体中。大肠杆菌菌株TOP10。 Vc2是来自霍乱弧菌的'off'核糖开关,因此当c-di-GMP结合时下调β-半乳糖苷酶的表达(图1)。稳定整合到E染色体中的Tn 7连接位点。大肠杆菌避免了拷贝数影响,并且不需要使用抗生素抗性标记。随后将c-di-GMP水平的变化转化为β-半乳糖苷酶表达的改变,这通过在含有5-溴-4-氯-3-吲哚基-β-半乳糖苷的琼脂平板上生长的菌落的颜色变化反映出来, D-吡喃半乳糖苷(X-gal)。例如,该测定法可用于揭示生理条件下蛋白质的功能并评估难以纯化和体外测试的蛋白质的酶活性。然而,此处描述的基于Vc2的测定是细胞内c-di-GMP浓度变化的定性评估。定量对于筛选测定来说不是关键的,但是在例如测量酶的活性中将是有利的。我们已经证明基于Vc2的测定可以用于验证体内DGC和PDE的活性(El Mouali等人,2017)。

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图1.基于核糖开关的筛选系统的原理 Vc2核糖开关位于β-半乳糖苷酶开放阅读框的上游以响应c-di-GMP浓度的变化来控制其表达。当c-di-GMP由于DGC的过度表达而以高丰度存在时,β-半乳糖苷酶的表达下调,导致含X-gal平板上的白色菌落。相反,当PDE过表达时,产生低细胞内c-di-GMP浓度,菌落呈蓝色。在 E。大肠杆菌TOP10野生型细胞中存在残余量的c-di-GMP,产生淡蓝色菌落(改编自El Mouali等人,2017)。

关键字:环鸟苷酸单磷酸酯(c-di-GMP), Diguanylate环化酶(DGC), 磷酸二酯酶(PDE), 核糖开关, β-半乳糖苷酶

材料和试剂

  1. PCR管
  2. 培养皿92 x 16毫米(SARSTEDT,目录号:82.1472.001)
  3. 注射器过滤器,0.2μm孔径(SARSTEDT,目录号:83.1826.001)
  4. 微量离心管1.5 ml(SARSTEDT,目录号:72.690.001)
  5. 移液器吸头(1,000μl:SARSTEDT,目录号:70.762.100; 1-200μl:Corning,目录号:4804; 0.1-10μl:Gilson,目录号:F171103)
  6. 分光光度计比色杯(SARSTEDT,目录号:67.742)
  7. 3部分一次性HSW SOFT-JECT注射器(5ml:Henke Sass Wolf,目录号:5050.X00V0; 10ml:Henke Sass Wolf,目录号:5100.X00V0)
  8. 铝箔
  9. 本研究中使用的质粒:

  10. 氯化钙感受态大肠杆菌TOP10细胞(Hanahan,1983)
  11. 本研究中使用的引物(从Sigma-Aldrich订购):

    a 限制性网站带下划线。
  12. GenElute质粒小量制备试剂盒(Sigma-Aldrich,目录号:PLN350)
  13. 无菌蒸馏去离子水(diH 2 O)
  14. Phusion高保真DNA聚合酶(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:F530S)
  15. dNTP混合物(各10mM)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:R0192)
  16. 琼脂糖(Sigma-Aldrich,目录号:A9539)
  17. 用于DNA的SmartLadder:200-10,000bp(Eurogentec,目录号:MW-1700-10)
  18. GelRed TM核酸凝胶染色剂(Biotium,目录号:41003)
  19. 不限制酶I(New England Biolabs,目录号:R0189S)
  20. PacI I限制酶(New England Biolabs,目录号:R0547S)
  21. PCR纯化试剂盒(QIAGEN,目录号:28106)
  22. 快速DNA连接酶试剂盒(Roche Diagnostics,目录号:10716359001)
  23. Taq DNA聚合酶与标准Taq缓冲液(New England Biolabs,目录号:M0273S)
  24. 氯化钠(Sigma-Aldrich,目录号:S7653)
  25. 胰蛋白胨(BD,Bacto TM TM,目录号:211705)
  26. 酵母提取物(BD,Bacto TM,目录号:212750)
  27. 琼脂(BD,Difco TM,产品目录号:281230)
  28. Tris碱(Sigma-Aldrich,目录号:93350)
  29. 冰醋酸(Sigma-Aldrich,目录号:ARK2183)
  30. EDTA(Sigma-Aldrich,目录号:E9884)
  31. 氨苄青霉素钠盐(Sigma-Aldrich,目录号:A9518)
  32. 硫酸庆大霉素盐(Sigma-Aldrich,目录号:G3632)
  33. L - (+) - 阿拉伯糖(Sigma-Aldrich,目录号:A3256)
  34. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Sigma-Aldrich,目录号:I6758)
  35. 二甲基亚砜(Sigma-Aldrich,目录号:D8418)
  36. 5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷(X-gal)(Roche Diagnostic,目录号:10703729001)
  37. LB(Luria-Bertani)培养基(见食谱)
  38. LB(Luria-Bertani)琼脂(见食谱)
  39. TAE缓冲液(见食谱)
  40. 100毫克/毫升氨苄青霉素储备(见食谱)
  41. 30毫克/毫升庆大霉素储备(见食谱)
  42. 100 mM IPTG库存(见食谱)
  43. 20毫克/毫升X-gal储备(见食谱)

设备

  1. 锥形瓶(50毫升/ 100毫升/ 250毫升)
  2. 例如PIPETMAN P20(Gilson,目录号:F123600),P200(Gilson,目录号:F123601)或P1000(Gilson,目录号:F123602) ]
  3. SureCycler 8800热循环仪(Agilent Technologies,型号:SureCycler 8800,目录号:G8800A)
  4. Heraeus TM Pico TM 17微量离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus TM Pico TM 17,目录号:75002410)
  5. Mini-Sub细胞GT水平电泳系统(Bio-Rad Laboratories,目录号:1704406)
  6. 15孔梳(Bio-Rad Laboratories,目录号:1704464)
  7. Sub-Cell GT UV-透明微型凝胶托盘(Bio-Rad Laboratories,目录号:1704436)
  8. 电泳电源
  9. Heraeus微生物培养箱(Thermo Fisherer Scientific,Thermo Scientific TM,型号:Heraeus微生物培养箱)。
  10. Multitron标准振动筛(INFORS HT)
  11. Gel Doc TM XR +凝胶文件系统(Bio-Rad Laboratories,目录号:1708195)
  12. 生物光度计(Eppendorf,型号:6131)
  13. NanoDrop TM 2000c分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000c,目录号:ND-2000C) >
  14. 紧凑型Thermomixer(Eppendorf,型号:5350)
  15. 数码相机

程序

实际上,工作流程可以分成两部分,如图2所示。第一部分是构建报告菌株,需要大约12-15天(协议的步骤1-3)。第二部分是通过报道菌株来评估DGC和PDE的活性(步骤4-5)。这将需要7-9天。


图2.工作流程概览。该程序分为两部分:1)记者菌株的构建和2)由记者评估c-di-GMP环化酶或磷酸二酯酶活性。构建报告菌株需要大约12-15天,并且需要7-9天来评估目标。

  1. 通过PCR扩增Vc2和 lacZY :
    1. 用于PCR的模板是Vc2-pRS414(由耶鲁大学RR Breaker教授接收),其中将含有核糖开关的片段及其天然启动子与框架内报道基因克隆在一起(
      。Sudarsan et。,2008)。
    2. 使用的引物是:
      Vc2 lacZY _F_ Pac I:GCG TTAATTAA ATAACGCCTATATTTGAAAGCTTG
      Vc2 lacZY _R_ 不是 I:GATCA GCGGCCGC TTAAGCGACTTCATTCACCTG
      限制性网站 Pac I和 Not I加下划线。
    3. 准备PCR反应混合物:


    4. 热循环条件:

    5. 通过使用1%TAE琼脂糖凝胶的凝胶电泳检查PCR产物。预计大小为4,733 bp。
  2. 克隆Vc2 lacZY 插入pGRG25载体:
    1. 在37℃下,用Pac< em-I和< em> I双重消化PCR产物Vc2 lacZY和质粒pGRG25过夜。为了减少星形活动,建议使用尽可能少的单位来达到消化。消化后,有必要使用琼脂糖凝胶电泳检查产物的大小。
      酶在65°C灭活20分钟
    2. 使用PCR纯化试剂盒去除限制酶。消化的Vc2 lacZY和pGRG25的浓度由NanoDrop测量。
    3. 按照制造商的说明书,使用T4 DNA连接酶在12°C过夜连接摩尔比为1:1的pGRG25和Vc2 lacZY 。为了验证载体是完全切割的,只需连接消化的载体。如果载体不能被两种限制酶有效切割,则会发生再循环,转化后会出现菌落。
    4. 将连接产物转化为化学感受态TOP10细胞,并在含有100μg/ ml氨苄青霉素的LB琼脂平板上选择重组质粒。 pGRG25载体对温度敏感,因此培养板应在32°C或更低的温度下培养。
    5. 通过菌落PCR验证回收的菌落。首先,按照制造商的说明书,用标准Taq缓冲液(来自New England Biolabs)为Taq DNA聚合酶制备主混合物。然后将25μl等分试样加入到PCR管中。使用无菌移液器吸头挑取单个菌落并与反应缓冲液混合。使用Taq DNA聚合酶进行菌落PCR时,建议在95°C预变性5 min。预期大小为518 bp。使用的引物是:
      pGRG25control_F:CACTTATCTGGTTGGTCGACACT
      Vc2 lacZY _R:CTGCAAGGCGATTAAGTTGG
      有关菌落PCR的细节可以在Azevedo等人的协议中找到(2017)。
    6. 使用相同的引物进行菌落PCR,通过DNA测序确认阳性克隆中核糖开关的序列完整性。
  3. 将Vc2 lacZY 集成到 E的Tn 7 附件网站中。大肠杆菌染色体(注1):
    1. 在补充有100μg/ ml氨苄青霉素的LB琼脂平板上条斑阳性菌落并在32℃孵育细胞。
    2. 在32℃,200rpm下将大约10个菌落接种到3ml不含氨苄青霉素的LB培养基中。加入0.1%L-阿拉伯糖(w / v)以促进Vc2 lacZY掺入大肠杆菌Tn 7 7附着位点。整合产生新的菌株E.coli TOP10 attTn7 :: Vc2 lacZY 。
    3. 将过夜培养物稀释10倍,并将50μl稀释的培养物平板接种于LB平板上以获得单菌落。在42°C孵育板以阻止温度敏感型pGRG25载体的复制。
    4. 在LB平板上条纹单个菌落并在42°C下重复孵育以确保质粒丢失。
    5. 使用Taq DNA聚合酶通过菌落PCR确认Vc2 lacZY 整合到染色体中。使用的引物是:
      pGRGcontrol_F:GATGCTGGTGGCGAAGCTGT
      pGRGcontrol_R:GATGACGGTTTGTCACATGGA

      。阳性克隆和对照的预期大小分别为6,328bp和678bp。
  4. 通过基于Vc2的筛选评估diguanylate环化酶的活性:以建立的DGC AdrA,YdeH和STM4551为例
    1. 准备氯化钙主管E。大肠杆菌 TOP10 attTn7 :: Vc2 lacZY 细胞。
    2. 通过在42处的热休克将含有DGC(AdrA,YdeH和STM4551)和催化失活突变体(STM4551E267A和YdeH-mut)的pBAD载体(注2)以及空载体转化到化学感受态细胞中°C持续40秒。将1ml不含抗生素的LB培养基加入微量离心管中,并在37℃,200rpm孵育1小时以使细胞恢复。
    3. 在含有100μg/ ml氨苄青霉素的LB琼脂平板上培养200μl细胞并在37℃培养平板过夜。
    4. 在含氨苄青霉素的LB琼脂平板上划线单菌落。
    5. 为每种菌株挑选一个菌落,并在37℃,200rpm过夜培养5 ml含100μg/ ml氨苄青霉素的LB培养基。
    6. 测量过夜培养物的OD 600,并将OD 600调至0.1。
    7. 在37℃,200转/分钟培养细胞。当OD 600达到0.6-1.0时,在加入100μg/ ml氨苄青霉素,80μg/ ml X-gal,0.1%(w / v)L-阿拉伯糖的LB平板上点3μl培养物(注3)。具有DGC,相应突变体和载体对照的细胞具有类似的OD 600是很重要的。
    8. 在28°C孵育板并监测集落颜色达72小时。 DGC活性将导致指示高c-di-GMP水平的白色菌落。使用数码相机拍摄照片。
  5. 研究PDE的活动:以已建立的PDE STM3611为例
    1. 细胞内c-di-GMP的浓度在TOP10细胞中低,因此Vc2 lacZY-harboring细胞具有β-半乳糖苷酶活性的基础水平,导致蓝色菌落,这使得不可能区分颜色由磷酸二酯酶进一步增强。然而,通过DGC的共表达可以可靠地研究PDE的活性。因此,将pWJB30(DGC AdrA克隆到pBAD30中)和pSTM3611(克隆到pSRKGm中的PDE STM3611)(注2和4)共转化到化学感受态TOP10 :: Vc2 lacZY细胞中。作为对照,空载体共转化或与DGC / PDE组合。表1列出了这些组合。

      表1.共转化到化学感受态TOP10 :: Vc2 lacZY细胞中的质粒组合


    2. 在含有氨苄青霉素(100μg/ ml)和庆大霉素(30μg/ ml)的LB琼脂平板上平板细胞。
      在37℃培养平板过夜
    3. 将含有抗生素的LB琼脂平板上的阳性菌落划线。
    4. 在含有氨苄青霉素和庆大霉素的5ml LB培养基中接种每菌株至少三个单独的菌落。
      在37°C,200转/分钟培养细胞过夜
    5. 测量过夜培养物的OD 600,并用含有抗生素的LB培养基将培养物稀释至0.1的OD 600。让细胞生长至指数期(OD 600〜0.6)。所有样品具有相似的OD <600>值是至关重要的。
    6. 在含有氨苄青霉素(100μg/ ml),庆大霉素(30μg/ ml),IPTG(0.75mM),L-阿拉伯糖(0.003%,w / v)和X-Gal(80μg/ ml)的LB琼脂平板上点出3μl培养物μg/ ml)(注3)。
    7. 在28°C孵育板并记录集落颜色达72小时。尽管单独的DGC表达导致白色菌落,但活性PDE的共表达会导致蓝色菌落。
    8. 使用数码相机拍摄照片。

数据分析

  1. 通过基于Vc2的测定来评估DGC的活性:在含有X-gal的平板上点样后,定期监测菌落的颜色。图3显示了在28℃下孵育24小时后的颜色。显然,与相应的突变体和载体对照相比,由于在高c-di-GMP水平下β-半乳糖苷酶表达的核糖开关抑制,表达DGC的细胞具有更白的颜色。


    图3.通过基于Vc2的测定检测已建立的diguanylate环化酶。分别将DGC AdrA,STM4551和无催化活性的STM4551E267A克隆到pBAD30中,分别产生pWJB30,p4551和p4551E267A。将另一个DGC YdeH及其突变体YdeH G206AG207A克隆到pBAD28中,产生pBAD28ydeH和pBAD28ydeH-mut。表达功能性DGC的细胞(AdrA / STM4551 / YdeH)是白色的,而具有载体对照或催化失活突变体(STM4551E267A / YdeH G206AG207A)的细胞是蓝色的。在补充有100μg/ ml氨苄青霉素,80μg/ ml X-gal和0.1%阿拉伯糖(w / v)的LB琼脂平板上28℃生长24小时。

  2. 通过基于Vc2的测定来评估PDE的酶活性:图4显示在28℃孵育48小时后菌落的颜色。 (第2行)或共表达AdrA和催化失活的PDE STM3611E136A(第5行)的细胞是白色的。然而,当野生型STM3611与AdrA(第4行)共表达时,细胞呈蓝色,说明STM3611的酶活性和细胞内c-di-GMP浓度对表达的降低。


    图4.通过基于Vc2的测定检测PDE STM3611的磷酸二酯酶活性将磷酸二酯酶STM3611和无催化活性的STM3611E136A克隆到pSRKGm中,产生pSTM3611和pSTM3611E136A ,分别。将Diguanylate环化酶AdrA克隆到pBAD30中,产生pWJB30。单独表达DGC AdrA的细胞具有白色。在用AdrA共表达PDE STM3611时,颜色变成蓝色,表明低c-di-GMP水平。然而,表达具有催化失活的PDE STM3611E136A的DGC AdrA的细胞是白色的。在补充有100μg/ ml氨苄青霉素,30μg/ ml庆大霉素,80μg/ ml X-gal,0.003%阿拉伯糖(wt / vol)和0.75mM IPTG(经改编)的LB琼脂平板上28℃生长48小时来自El Mouali et al。,2017)。

笔记

  1. 将目标基因整合进入 E的协议。大肠杆菌染色体Tn73附着位点由G.J.McKenzie和N.L.Craig(McKenzie和Craig,2006)描述。
  2. 我们将diguanylate环化酶克隆到pBAD28 / pBAD30载体中,并将磷酸二酯酶克隆到pSRKGm载体中。其他表达载体也可以使用,这取决于可用性。
  3. 不同的L - (+) - 阿拉伯糖和IPTG浓度需要进行测试,以获得diguanylate环化酶,磷酸二酯酶和对照之间最明显的颜色差异。
  4. 将diguanylate环化酶和磷酸二酯酶克隆到含有不同抗生素抗性标记和相容性复制起点的相容性载体中很重要。
  5. 基于Vc2的核糖开关对其他环状二核苷酸的特异性尚未在本文中进行测试。

食谱

  1. LB(Luria-Bertani)培养基400毫升
    4克NaCl
    4克胰蛋白胨
    2克酵母提取物
    将dH <2> O添加到400毫升

    在121°C高压灭菌15分钟
  2. LB(Luria-Bertani)琼脂400毫升
    4克NaCl
    4克胰蛋白胨
    2克酵母提取物
    6克琼脂
    将dH <2> O添加到400毫升

    在121°C高压灭菌15分钟 倒入培养皿(如果需要,加入抗生素,X-gal,L-阿拉伯糖或IPTG)
    让它完全冷却并在4°C储存。
  3. TAE缓冲液(50x储备液)
    将242克Tris碱溶于水中
    加入57.1毫升冰醋酸
    加100ml 0.5M EDTA(pH8.0)

    加水至1升

  4. 100毫克/毫升氨苄青霉素储备
    溶解5毫升去离子水中的500毫克氨苄青霉素钠盐
    用0.2μm无菌注射器过滤器过滤溶液 将1毫升等分试样溶液装入无菌离心管中。
    在-20°C储存
  5. 30毫克/毫升庆大霉素储备

    溶解150毫克硫酸庆大霉素盐溶于5毫升去离子水中
    用0.2μm无菌注射器过滤器过滤溶液 将1毫升等分试样溶液装入无菌离心管中。
    在-20°C储存
  6. 100 mM IPTG stock
    将1.191克IPTG溶于5毫升去离子水中

    用0.2μm无菌注射器过滤器过滤溶液 将1毫升等分试样溶液装入无菌离心管中。
    在-20°C储存

  7. 20毫克/毫升X-gal库存
    溶解5毫升DMSO中的100毫克X-gal
    用铝箔包裹管子避光 在-20°C储存

    该溶液在-20°C稳定6-12个月

致谢

作者想感谢Ronald R. Breaker博士教授共享Vc2-pRS414核糖开关结构。 UteRömling教授构思了这项研究。刘颖开发了该测定并评估了DGC活性。 Hyunhee Kim开发了测定来评估PDE活性。这项工作得到了瑞典自然科学和工程研究委员会(621-2013-4809)和卡罗林斯卡研究所的支持。此修改后的协议基于以前发表的作品(El Mouali等人,2017年)。作者声明不存在利益冲突或利益冲突。

参考

  1. Ahmad,I.,Lamprokostopoulou,A.,Le Guyon,S.,Streck,E.,Barthel,M.,Peters,V.,Hardt,W.D.和Römling,U。(2011)。 复杂的c-di-GMP信号网络介导沙门氏菌毒力特性与生物膜形成之间的转换enterica serovar typhimurium。 PLoS One 6(12):e28351。
  2. Azevedo,F.,Pereira,H.和Johansson,B。(2017)。 菌落PCR。 Methods Mol Biol 1620:129- 139。
  3. Christen,M.,Kulasekara,H. D.,Christen,B.,Kulasekara,B. R.,Hoffman,L. R. and Miller,S. I.(2010)。 细菌细胞分裂后第二信使c-di-GMP的不对称分布 <科学 328(5983):1295-1297。
  4. El Mouali,Y.,Kim,H.,Ahmad,I.,Brauner,A.,Liu,Y.,Skurnik,M.,Galperin,M.Y.和Römling,U。(2017)。 独立的EAL结构域蛋白形成参与调节细胞运动性和生物膜的EAL蛋白的不同亚类在细菌中形成。 J Bacteriol 199(18):pii:e0079-17。
  5. Guzman,L.M.,Belin,D.,Carson,M.J。和Beckwith,J。(1995)。 含有阿拉伯糖PBAD启动子的载体的严格调控,调节和高水平表达。 J Bacteriol 177(14):4121-4130。
  6. Hanahan,D。(1983)。 用质粒转化大肠杆菌的研究 < J Mol Biol 166(4):557-580。
  7. Jonas,K.,Edwards,A.N.,Simm,R.,Romeo,T.,Römling,U.和Melefors,O。(2008)。 RNA结合蛋白CsrA通过直接调节GGDEF蛋白的表达来控制环状二-GMP代谢。 / Mol> Microbiol 70(1):236-257。
  8. Kellenberger,C.A.,Chen,C.,Whiteley,A.T.,Portnoy,D.A。和Hammond,M.C。(2015)。 基于RNA的荧光生物传感器用于第二信使环状di-AMP的活细胞成像。 J Am Chem Soc 137(20):6432-6435。
  9. Khan,S.R.,Gaines,J.,Roop,R.M。,2nd和Farrand,S.K。(2008)。 具有严格调控启动子的广泛宿主范围表达载体及其用于检测TraR和TraM在Ti质粒群体感应中的表达 应用环境微生物 74(16):5053-5062。
  10. McKenzie,G.J。和Craig,N.L。(2006)。 快速,简单和高效:使用Tn 7 7将转基因插入到肠杆菌染色体中无需选择插入事件。 BMC Microbiol 6:39。
  11. Römling,U.,Galperin,M.Y.和Gomelsky,M.(2013)。 Cyclic di-GMP:通用细菌第二使者的第25年。 Microbiol Mol Biol Rev 77(1):1-52。
  12. Ryan,RP,Fouhy,Y.,Lucey,JF,Crossman,LC,Spiro,S.,He,YW,Zhang,LH,Heeb,S.,Camara,M.,Williams,P.and Dow,JM )。 野油菜黄单胞菌中的细胞信号传导涉及HD-GYP结构域蛋白它在循环的di-GMP营业额中起作用。美国国家科学院院刊103(17):6712-6717。
  13. Ryjenkov,D.A.,Tarutina,M.,Moskvin,O.V。和Gomelsky,M.(2005)。 环化diguanylate是细菌中无处不在的信号分子:洞察GGDEF蛋白结构域的生物化学。 a> Bacteriol 187(5):1792-1798。
  14. Schmidt,A.J。,Ryjenkov,D.A。和Gomelsky,M。(2005)。 普遍存在的蛋白质结构域EAL是环状二磷酸腺苷特异性磷酸二酯酶:具有酶活性和无活性的EAL结构域。 / a> J Bacteriol 187(14):4774-4781。
  15. Simm,R.,Morr,M.,Kader,A.,Nimtz,M。和Römling,U。(2004)。 GGDEF和EAL域反向调节循环di-GMP水平并从安定转变为运动。 Mol Microbiol 53(4):1123-1134。
  16. Sudarsan,N.,Lee,E.R.,Weinberg,Z.,Moy,R.H.,Kim,J.N。,Link,K.H。和Breaker,R.R。(2008)。 真细菌中的核糖开关意识到第二信使环状di-GMP。 科学 321(5887):411-413。
  17. Zhou,H.,Zheng,C.,Su,J.,Chen,B.,Fu,Y.,Xie,Y.,Tang,Q.,Chou,S.H.and He,J.(2016)。 表征天然三联串联c-di-GMP核糖开关和应用基于核糖开关的双重 - 荧光记者。 Sci Rep 6:20871.
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Liu, Y., Kim, H. and Römling, U. (2018). In vivo Analysis of Cyclic di-GMP Cyclase and Phosphodiesterase Activity in Escherichia coli Using a Vc2 Riboswitch-based Assay. Bio-protocol 8(5): e2753. DOI: 10.21769/BioProtoc.2753.
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