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Apr 2020
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Quantification of RuBisCO Expression and Photosynthetic Oxygen Evolution in Cyanobacteria
蓝藻RuBisCO表达和光合氧释放的定量研究   

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Abstract

Phototrophic microorganisms are frequently engineered to regulate the expression and the activity of targeted enzymes of interest for specific biotechnological and agricultural applications. This protocol describes a method to evaluate the expression of RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) in the model cyanobacterium Synechococcus elongatus PCC 7942, at both the transcript and protein levels by quantitative PCR and Western blot, respectively. We further describe an experimental method to determine photosynthetic activity using an oxygen electrode that measures the rate of molecular oxygen production by cyanobacterial cultures. Our protocol can be utilized to assess the effects of RuBisCO engineering at the metabolic and physiological levels.

Keywords: RuBisCO (RuBisCO), Cyanobacteria (蓝藻细菌), Synechococcus elongatus (聚球藻), Gene expression (基因表达), Oxygen evolution (析氧)

Background

RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) is a central enzyme involved in fixation of atmospheric CO2 into biomass of its photoautotrophic producers (Nisbet et al., 2007; Tabita et al., 2008; Kacar et al., 2017; Erb and Zarzycki, 2018). Improving the efficiency of the RuBisCO carboxylation activity might increase the yield of produced biomass and drive cost-effective, ecologically friendly ways to biosynthesize diverse carbon-based compounds (Simkin et al., 2019; Kubis et al., 2019). Therefore, RuBisCO has become one of the most extensively studied and engineered enzymes (Bainbridge et al., 1995; Whitney et al., 2011). Different approaches have been utilized to manipulate the total activity of RuBisCO, such as improving its activation by activases (Bhat et al., 2017), incorporating beneficial mutations in the enzyme substrate-binding or active sites (Andersson, 2008), and upregulating RuBisCO expression (Parry et al., 2003; Carmo-Silva et al., 2015; Liang and Lindblad, 2017; Salesse-Smith et al., 2018; Durall et al., 2020; Garcia et al., 2021), as well genomically replacing the native RuBisCO with its ancient counterpart (Kedzior et al., 2021).


The effects of engineering strategies to increase the expression of RuBisCO are typically evaluated at both the transcript and protein levels. Here, we present a detailed protocol that builds and expands upon methods applied by others to analyze the expression of RuBisCO in cyanobacteria. The approach detailed here permits the assessment of the potential metabolic effect of altered enzyme expression by measuring the photosynthetic activity of engineered phototrophs. In particular, this protocol uses the model cyanobacterium Synechococcus elongatus PCC 7942 (hereafter S. elongatus) that is broadly employed in basic and applied research as it is naturally competent and its genome can easily be modified using standard engineering techniques (Atsumi et al., 2009; Taton et al., 2020). As an example of a modified strain that can be evaluated relative to wild-type S. elongatus (WT), we used a mutant strain “Syn02” that harbors the native rbc operon and a second copy inserted in the chromosome neutral site, as described and studied by our laboratory (Garcia et al., 2021).


Here, we have combined disparate pieces of operative methodological information in a single, readily available protocol. Furthermore, we adjusted methods reported elsewhere specifically to increase target yield and purity of isolated total RNA – required to generate cDNA templates used in qPCR analysis. Overall, our analysis of RuBisCO expression at the transcript level employs adapted protocols, including those recommended by different manufacturers (QIAGEN, Invitrogen, Applied Biosystems) to extract and purify total RNA from bacteria, reverse transcribe to cDNA, and analyze gene expression by qPCR. Specifically, we use reference gene secA for normalization of rbcL expression based on previous studies, showing its stable expression under diverse growth conditions in S. elongatus (Szekeres et al., 2014; Luo et al., 2019). To analyze expression at the protein level, total proteins were isolated from crude cell extracts under denaturing conditions, building on methods by Ivleva and Golden (2007) with modifications. The SDS-PAGE loading buffer containing 100 mM dithiothreitol and 2% (w/v) SDS is replaced with the hot Denaturing Lysis Buffer containing 1% (w/v) SDS without a reducing agent that would otherwise prohibit precise quantification of isolated proteins. The method for physical cell disruption by freeze-thaw cycles and glass beads is replaced with sonication. The procedure to perform Western blot is elaborated based on standard polyacrylamide gel electrophoresis and protein transfer protocols (Bio-Rad). Finally, directions for the use of a primary anti-RbcL antibody are adapted from Agrisera, and instructions on total protein stain and near-infrared secondary antibody-mediated immunodetection are provided by LI-COR Biosciences. Our protocol enables the isolation of ~5 mg/ml total protein. The procedure for oxygen evolution rate measurement follows manufacturer guidelines for the Oxygraph+ System (Hansatech Instruments) with some adjustments specific to cyanobacterial cultures based on previous methods (Liang and Lindblad, 2016; De Porcellinis et al., 2018). We additionally modified cyanobacteria preparation for the analysis (step E8 of this protocol) to decrease fluctuation during oxygen measurements and, thus, to improve reproducibility. Figure 1 illustrates all the major steps described in this protocol.



Figure 1. Schematic diagram of the experimental steps presented in this protocol

Materials and Reagents

  1. 96-well low-profile skirted PCR plates (Thermo Scientific, catalog number: AB0800W)

  2. Synechococcus elongatus PCC 7942 WT (wild-type strain with one RuBisCO operon copy in the native site)

  3. Synechococcus elongatus PCC 7942 Syn02 (engineered strain with two RuBisCO operon copies: one in the native site and the other in the neutral site; Garcia et al., 2021)

  4. RNeasy Protect Bacteria Mini Kit (QIAGEN, catalog number: 74524)

  5. Deoxyribonuclease I, amplification grade (Invitrogen, catalog number: 18068-015)

  6. SuperScriptTM IV First-Strand Synthesis System (Invitrogen, catalog number: 18091050)

  7. Q5® High-Fidelity DNA Polymerase (New England BioLabs, catalog number: M0491)

  8. PowerUpTM SYBRTM Green Master Mix (Applied Biosystems, catalog number: A25742)

  9. Agarose, LE, analytical grade (Promega, catalog number: V3125)

  10. SYBRTM Safe DNA Gel Stain (Invitrogen, catalog number: S33102)

  11. Bovine serum albumin (BSA) standards (Lee BioSolutions, catalog number: 100-10-0.25); concentrations [µg/ml]: 25, 125, 250, 500, 750, 1,000, 1,500, 2,000

  12. PierceTM BCA Protein Assay Kit (Thermo Scientific, catalog number: 23225)

  13. Tris (Thermo Fisher Scientific, catalog number: PRH5131)

  14. Ammonium persulfate, APS (Sigma-Aldrich, catalog number: A3678)

  15. TEMED (Bio-Rad, catalog number: 1610801)

  16. 30% acrylamide/bis solution (Bio-Rad, catalog number: 1610158)

  17. Precision Plus ProteinTM Dual Color Standards (Bio-Rad, catalog number: 1610374)

  18. RevertTM 700 Total Protein Stain (LI-COR Biosciences, catalog number: 926-11011)

  19. Rabbit anti-RbcL antibody (Agrisera, catalog number: AS03 037)

  20. Non-fat dry milk (LabScientific, catalog number: M0841)

  21. IRDye® 800CW goat anti-rabbit IgG secondary antibody (LI-COR Biosciences, catalog number: 926-32211)

  22. Potassium chloride (Sigma-Aldrich, catalog number: P9541)

  23. Sodium dithionite/sodium hydrosulfite (Sigma-Aldrich, catalog number: 157953)

  24. Methanol, anhydrous (Sigma-Aldrich, catalog number: 322415)

  25. Sodium chloride (Sigma-Aldrich, catalog number: S9888)

  26. Tween® 20 (Sigma-Aldrich, catalog number: P9416)

  27. Ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, catalog number: ED)

  28. Lysozyme from chicken egg white (Sigma-Aldrich, catalog number: L4919)

  29. Proteinase K (QIAGEN, catalog number: 19131)

  30. Glycine (Thermo Fisher Scientific, catalog number: PRH5073)

  31. Sodium dodecyl sulfate, SDS (Sigma-Aldrich, catalog number: L3771)

  32. Acetic acid (Sigma-Aldrich, catalog number: 695092)

  33. Intercept® (TBS) blocking buffer (LI-COR Biosciences, catalog number: 927-60001)

  34. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148)

  35. Ethanol, anhydrous (Sigma-Aldrich, catalog number: 443611)

  36. Bromophenol blue (Sigma-Aldrich, catalog number: B0126)

  37. Glycerol (Sigma-Aldrich, catalog number: G5516)

  38. Sodium nitrate, NaNO3 (Thermo Fisher Scientific, catalog number: BP360-500)

  39. Magnesium sulfate heptahydrate, MgSO4·7H2O (Sigma-Aldrich, catalog number: 230391)

  40. Calcium chloride dihydrate, CaCl2·2H2O (Sigma-Aldrich, catalog number: 223506)

  41. Potassium phosphate dibasic, K2HPO4 (Sigma-Aldrich, catalog number: P8281)

  42. Sodium carbonate, Na2CO3 (Supelco, catalog number: SX0395)

  43. Citric acid monohydrate, C6H8O7·H2O (J.T. Baker, catalog number: 0115-01)

  44. Ammonium iron(III) citrate, C6H11FeNO7 (Sigma-Aldrich, catalog number: F5879)

  45. Ethylenediaminetetraacetic acid disodium salt, Na2EDTA·2H2O (Alfa Aesar, catalog number: A15161)

  46. Boric acid, H3BO3 (J.T. Baker, catalog number: 0084-01)

  47. Manganese(II) chloride tetrahydrate, MnCl2·4H2O (J.T. Baker, catalog number: 2540-01)

  48. Zinc sulfate heptahydrate, ZnSO4·7H2O (Sigma-Aldrich, catalog number: Z0251)

  49. Sodium molybdate dihydrate, Na2MoO4·2H2O (J.T. Baker, catalog number: 3764-01)

  50. Copper(II) sulfate pentahydrate, CuSO4·5H2O (Sigma-Aldrich, catalog number: C8027)

  51. Cobalt(II) nitrate hexahydrate, Co(NO3)2·6H2O (Sigma-Aldrich, catalog number: 239267)

  52. BG-11 medium (see Recipes)

  53. Denaturing Lysis Buffer (see Recipes)

  54. Laemmli Sample Buffer (see Recipes)

  55. Polyacrylamide resolving gel (see Recipes)

  56. Polyacrylamide stacking gel (see Recipes)

  57. Sodium dithionite saturated solution (see Recipes)

  58. TAE Buffer (see Recipes)

  59. TBS Buffer (see Recipes)

  60. TBST Buffer (see Recipes)

  61. TE Buffer (see Recipes)

  62. TE Lysis Buffer (see Recipes)

  63. TGS Buffer (see Recipes)

  64. Transfer Buffer (see Recipes)

Equipment

  1. 250-ml glass flasks with sponge caps (Thermo Fisher Scientific)

  2. Controlled environment chamber equipped with light source (Percival, model: I36LLVLC8)

  3. UV-Vis Spectrophotometer (Agilent, model: Cary 60)

  4. Heraeus Multifuge Centrifuge (Thermo Scientific, model: X3 FR)

  5. Sorvall Legend Micro 17 Centrifuge (Thermo Scientific, catalog number: 75002431)

  6. NanoDrop 2000c Spectrophotometer (Thermo Scientific, catalog number: ND-2000C)

  7. Dry Bath Digital Heat Block (Benchmark, model: BSH1002)

  8. Digital Shaker (VWR, model: 5000)

  9. Analog Vortex Mixer (Fisher Scientific, model: 02215414)

  10. Thermal Cycler (Applied Biosystems, model: 2720)

  11. ABsolute qPCR plate seals (Thermo Scientific, catalog number: AB1170)

  12. Real-Time Thermal Cycler (Analytik Jena AG, model: qTOWER3 G)

  13. Microwave

  14. 250-ml beaker

  15. Sonicator (Qsonica, model: Q125)

  16. Nitrocellulose membrane (Thermo Scientific, catalog number: 88018)

  17. Mini-PROTEAN® Tetra Vertical Electrophoresis Cell system (Bio-Rad, catalog number: 1658004)

  18. Mini Trans-Blot Electrophoretic Transfer Cell system (Bio-Rad, catalog number: 1703930)

  19. Odyssey® Fc Imaging System (LI-COR Biosciences, model: 2800-03)

  20. Oxygraph+ System (Hansatech Instruments, catalog number: OXY1+)

Software

  1. qPCRsoft (Analytik Jena AG, version: 3.1)

  2. Image StudioTM (LI-COR Biosciences, version: 5.0)

  3. Quantity One® 1-D Analysis (Bio-Rad, version: 4.6.7)

  4. OxyTrace+ (Hansatech Instruments)

  5. Microsoft 365 Excel

Procedure

  1. Total RNA isolation and preparation for reverse transcription

    1. Prepare the cultures of two S. elongatus strains (PCC 7942 WT and Syn02) in 50 ml of BG-11 medium inside 250-ml glass flasks capped with sponges. Incubate in the controlled environment chamber (Percival) under the desired selected CO2 concentration at 30°C, slowly agitating under 80 µmol·m-2·s-1 illumination.

      Note: Culture time depends on the growth phase at which RuBisCO expression needs to be measured. Here, we collected cells at the early exponential phase.

    2. Once the cultures reach the desired OD750 value that indicates the anticipated growth phase, harvest the cells.

      Note: To normalize the cyanobacteria number and isolated RNA yield, we harvest 6 ml of cultures diluted to ~OD750 = 1.0.

    3. Centrifuge the collected cultures at 4,700 × g for 10 min.

      Note: Because the mRNA turnover is very rapid, we recommend pouring the cells directly over ice in a Falcon tube and centrifuging at 4°C. In certain cases, using a stop solution prior to harvesting the cells is also recommended (95% ethanol, 5% phenol – 1/5 of the total volume).

    4. Decant the supernatant and resuspend the pelleted bacteria in 2 ml of TE buffer.

    5. Extract RNA by using the RNeasy Protect Bacteria Mini Kit, following the RNAprotect Bacteria Reagent Handbook – Protocol 4: Enzymatic Lysis and Proteinase K Digestion of Bacteria, with several adjustments to ensure the high yield of isolated RNA, as specified below:

      1. Mix 4 ml of RNA Protect Bacteria Reagent and 2 ml of the cell suspension (prepared in Step A4) in a 15-ml conical tube.

      2. Centrifuge at 4,700 × g for 10 min at room temperature.

      3. Decant the supernatant and re-centrifuge the pellet at 4,700 × g for 2 min at room temperature to discard the residual supernatant with a pipette tip.

        Note: Pellets can be frozen at -80°C to proceed on another day if necessary.

      4. Add 200 µl of TE buffer containing 15 mg/ml lysozyme and 100 µg/ml proteinase K and resuspend the pellet by pipetting up and down.

        Note: Inclusion of lysozyme in TE buffer is sufficient to lyse cyanobacterial cells, but addition of proteinase K enables extraction of higher amounts of total RNA.

      5. Mix by vortexing for 10 s and incubate cells resuspended in TE Lysis Buffer for 10 to 20 min at room temperature while constantly rocking the suspension on a shaker.

        Notes:

        1. We extended the incubation time from the original RNeasy Protect Bacteria Mini Kit protocol to ensure that the rigid cyanobacterial cell walls are sufficiently disintegrated.

        2. After eluting the total RNA, measure its concentration using a NanoDrop and store RNA samples at -80°C or proceed immediately to DNase I treatment (next step). Alternatively, use a Qubit fluorometer (Invitrogen) instead of a NanoDrop to measure RNA concentration. Qubit is recommended for the precise quantification of nucleic acids, but it is not utilized in this protocol.

      6. Add 700 µl of RLT buffer (supplemented with 10 µl β-mercaptoethanol per 1 ml of RLT buffer) and vortex vigorously for a few seconds.

      7. Add 500 µl of 96% ethanol (molecular grade) and mix by pipetting up and down (do not centrifuge).

      8. Transfer 700 µl of the lysate to an RNeasy Mini Spin Column placed in a 2 ml collection tube and centrifuge for 15 s at 10,000 × g at room temperature.

      9. Discard the flow-through, add the remaining lysate, and centrifuge as in the previous step.

      10. Add 700 µl of buffer RW1, centrifuge at 10,000 × g for 15 s at room temperature to wash the membrane, and discard the flow-through.

      11. Add 500 µl of buffer RPE (prepared by adding 44 ml of 100% ethanol to 11 ml of the RPE concentrate from the kit), centrifuge at 10,000 × g for 15 s at room temperature to wash the membrane, and discard the flow-through.

      12. Add another 500 µl of buffer RPE and centrifuge at 10,000 × g for 2 min at room temperature.

      13. Place the RNeasy Mini spin column in a new 2 ml collection tube and centrifuge at full speed (17,000 × g) for 1 min at room temperature.

      14. Place the spin column in a new 1.5 ml collection tube, add 40 µl of RNase-free water directly to the center of the membrane, and centrifuge at 17,000 × g for 1 min at room temperature to elute the total RNA.

      15. Measure RNA concentration in the eluates using a NanoDrop.

      16. Store the RNA samples at -80°C or proceed immediately to DNase I treatment (next step).

    6. Place the RNA samples on ice and prepare the reactions containing amplification grade DNase I according to the instructions provided by the manufacturer, with a few substantial adjustments to increase the purity of isolated RNA:

      1. Use 4 U of DNase I (4 µl) instead of 1 U (1 µl).

        Note: The additional amount of DNase I should help to completely eliminate the residual gDNA.

      2. Increase the total reaction volume to 20 µl. After adding 1 µg of RNA and 2 µl of 10× DNase I Reaction Buffer, add RNase-free water if necessary.

        Note: Provide a sufficient volume of each RNA sample to be used in reverse transcription (in both the positive reaction and the negative control; Step B2).

      3. Extend the incubation time from the recommended 15 min to 60 min at room temperature.

        Note: This is another step (besides the increased amount of DNase I) to ensure the complete elimination of gDNA. We did not observe any substantial adverse effects from increased DNase I concentration and incubation time on the quality (stability) of RNA.

      4. Add 2 µl of 25 mM EDTA to each reaction to inactivate DNase I prior to incubation at 65°C for 10 min.

    7. Measure RNA concentration in the DNase I-treated samples using a NanoDrop or Qubit fluorometer (we also recommend running the RNA on a gel to ensure it is not degraded). Store the samples at -80°C or proceed immediately to reverse transcription.

      Note: We usually obtain a final concentration of 50-100 ng/ml.


  2. Reverse transcription and qPCR

    1. Use SuperScriptTM IV First-Strand Synthesis System to perform reverse transcription (RT) of RNA samples. Prepare reactions to anneal primers to template RNA according to the instructions provided in the kit. Per reaction, use:

      1 µl of 50 µM random hexamers

      1 µl of 10 mM dNTP mix

      1 µl of DEPC-treated water

      10 µl of RNA sample

    2. Prepare two identical reactions as described in Step B1 for each RNA sample. One reaction will be positive (reverse transcriptase will be added to obtain cDNA for qPCR analysis), and another reaction will be negative (reverse transcriptase will not be added; thus, only RNA should remain in the sample). Heat the reactions at 65°C for 5 min and then incubate on ice for at least 1 min.

    3. Prepare the reverse transcription (RT) master mix for all the RNA-primer samples obtained in Step B2. Per sample, use:

      4 µl of 5× SSIV Buffer

      1 µl of 100 mM DTT

      1 µl of RNase Inhibitor

    4. Aliquot the RT master mix into the tubes containing the RNA-primer samples prepared in Step B2; transfer 6 µl of the mix to each RNA-primer sample.

    5. Add 1 µl of SuperScript IV Reverse Transcriptase from the kit to each RT positive reaction prepared in Step B2 (total reaction volume will be 20 µl).

    6. Add 1 µl of DEPC-treated water to each negative control prepared in Step B2 (total reaction volume will be 20 µl).

    7. Incubate all the reactions sequentially:

      1. At room temperature for 10 min.

      2. At 50-55°C for 10 min.

      3. At 80°C for 10 min.

      4. Store the reactions at -80°C or proceed immediately to Step B8.

    8. Check the quality of the cDNA templates generated in Step B7 by PCR (Tables 1 and 2). Use the primers (Table 4) that will also be used in subsequent qPCR. They should enable the generation of the products of expected sizes in positive reactions. Assess the purity of cDNA templates. There should be no PCR products generated from negative controls. Otherwise, cDNA templates might be contaminated with gDNA.


      Table 1. Composition of PCR samples to check the quality of generated cDNA templates

      PCR component Volume per reaction [µl]
      5× Q5 Reaction Buffer 4
      10 mM dNTP mix 0.4
      10 µM forward primer 1
      10 µM reverse primer 1
      RT positive/negative reaction (obtained in Step B7) 1
      Q5 High-Fidelity DNA Polymerase 0.5
      DNase/RNase-free water 12.1

      Table 2. Parameters of PCR to check the quality of generated cDNA templates

      PCR step Temperature [°C] Time [min:s] Number of cycles
      1 98 00:30 1
      2 98 00:10 35× steps 2 to 4
      3 67 00:30
      4 72 00:30
      5 72 2:00 1
      6 12 hold 1

    9. Electrophorese the PCR products in a TAE + 2% (w/v) agarose gel. If the specific PCR products are generated only in RT positive reactions, and there are no products in negative controls, proceed to qPCR (Step B10).

    10. Prepare a separate qPCR master mix for each RT positive reaction that was obtained in Step B7 and quality-checked in Steps B8 and B9. The total volume of each master mix should be sufficient to test the expression of the desired number of genes for each individual qPCR sample in triplicate (Table 3).

      Note: We prepared two qPCR master mixes (for each WT and Syn02 strain), which were sufficient for 6 reactions as we analyzed the expression of two genes in three technical replicates: rbcL and secA; Table 4).


      Table 3. Composition of qPCR samples

      qPCR component Volume per reaction [µl]
      PowerUpTM SYBRTM Green Master Mix (2×) 5
      RT positive reaction (Step B7) 1
      Nuclease-free water 3

      Table 4. Primers used in qPCR. F, forward; R, reverse.


    11. Aliquot 9 µl of each qPCR master mix into an appropriate number of wells (depending on the number of reactions) in a 96-well plate.

    12. Add 1 µl of each 5 µM primer pair (primers listed in the table below) into triplicate qPCR samples in a 96-well plate.

    13. Prepare NTC (no template control) samples in a 96-well plate similarly to qPCR samples (steps B10-B12) but add nuclease-free water instead of RT positive reactions.

    14. Spin down the plate briefly and start qPCR (Table 5) using the real-time thermal cycler qTOWER3 G equipped with qPCRsoft software.


      Table 5. Parameters of qPCR

    15. qPCR step Temperature [°C] Time [min:s] Number of cycles
      1 50 2:00 1
      2 95 2:00 1
      3 95 0:15 40× steps 3 and 4
      4 60 1:00

    16. Export the qPCR results to an Excel spreadsheet (Table 6) and calculate the relative expression of rbcL (i.e., average fold change) normalized to secA using the double delta Ct method (Table 7).


      Table 6. Example data obtained by qPCR. Samples (derived from the WT and Syn02 strains) were prepared in three biological replicates. The cycle threshold values (Ct) were obtained for three technical replicates and then averaged (Avg. Ct).

      Sample Reference gene (secA) Gene of interest (rbcL)
      Ct1 Ct2 Ct3 Avg. Ct Ct1 Ct2 Ct3 Avg. Ct
      WT 1 26.24 26.79 26.50 26.51 26.50 26.23 26.23 26.32
      WT 2 27.14 26.55 26.73 26.81 26.16 26.18 26.51 26.28
      WT 3 26.90 26.86 27.01 26.92 26.10 26.71 26.15 26.32
      Syn02 1 23.13 23.14 23.11 23.13 18.97 18.74 18.81 18.84
      Syn02 2 23.19 23.26 23.10 23.18 18.92 18.90 19.00 18.94
      Syn02 3 23.33 23.23 23.36 23.31 19.02 18.88 19.04 18.98


      Table 7. Example calculation of the relative rbcL expression using the double delta Ct method. The relative expression of rbcL was normalized to secA (ΔCt = Avg. CtrbcL – Avg. CtsecA). An example to calculate ΔΔCt: ΔΔCtSyn02#1 = ΔCtSyn02#1 – Avg. ΔCtWT.

      Sample ΔCt Avg. ΔCtWT ΔΔCt 2-ΔΔCt rbcL expression
      (avg. 2-ΔΔCt)
      WT 1 -0.19 -0.44 0.25 0.84 1.01 ± 0.15
      WT 2 -0.52 -0.08 1.06
      WT 3 -0.60 -0.16 1.12
      Syn02 1 -4.29 -3.85 14.40 14.39 ± 0.42
      Syn02 2 -4.24 -3.80 13.97
      Syn02 3 -4.33 -3.89 14.80


  3. Total protein extraction under denaturing conditions

    1. Prepare the cultures of two S. elongatus strains (PCC 7942 WT and Syn02) in 50 ml of BG-11 medium inside of 250-ml glass flasks capped with sponges. Incubate in the controlled environment chamber (Percival) under the desired CO2 concentration at 30°C, agitation at 120 rpm, and illumination of 80 µmol·m-2·s-1.

      Note: Depends on the growth phase at which RuBisCO expression needs to be measured. Here, we collected cells at the early exponential phase.

      When the cultures reach the desired OD750 values, harvest the culture (normalization is important to ensure that similar amounts of cyanobacteria are processed in each sample to yield similar concentrations of extracted total protein; we used OD750 = ~1.0).

    2. Centrifuge the collected cultures at 4,700 × g for 10 min at room temperature.

    3. Decant the supernatant and re-centrifuge the pellet at 4,700 × g for 2 min at room temperature to discard the residual medium with a pipette tip.

    4. Preheat the Denaturing Lysis Buffer to 95°C in a heat block.

    5. Boil deionized water in a 250-ml beaker using a microwave.

    6. Add 500 µl of the hot Denaturing Lysis Buffer to the bacterial pellet. Immediately submerge the tube with the pellet in boiled deionized water and resuspend cyanobacterial cells by vigorously pipetting them up and down several times.

    7. Transfer the cell suspension to a new 1.5 ml tube and heat at 95°C for 10 min. Briefly vortex the tube midway through the incubation time.

    8. Sonicate the obtained lysate 3 × 10 s, with 10-s intervals, and at 40% amplitude (no incubation on ice is needed).

    9. Centrifuge the lysate at 17,000 × g for 10 min at room temperature and transfer the supernatant to a new 1.5 ml tube.

    10. Measure total protein concentration in the cleared lysate by using the PierceTM BCA Protein Assay Kit.

    11. Store the lysates at -80°C until proceeding to SDS-PAGE.


  4. Total protein separation by SDS-PAGE and immunodetection by Western blot

    1. Mix the prepared cyanobacterial cell lysates with the Laemmli Sample Buffer to obtain samples containing 5 µg of total protein.

      Note: Total protein amount to be used in SDS-PAGE depends on the expected RuBisCO signal intensity to be obtained by Western blot. We recommend preparing a calibration curve with RuBisCO signal intensity plotted as a function of total protein amount. This will allow estimation of the optimal range of total protein amount at which the signal intensity for RuBisCO correlates with the total protein level.

    2. Load the samples prepared in step 1 onto the 6% (v/v) polyacrylamide stacking gel assembled in the Mini-PROTEAN® Tetra Vertical Electrophoresis Cell system in TGS buffer.

    3. Run electrophoresis at 100-150 V until the samples migrate through the 12% (v/v) polyacrylamide resolving gel.

    4. Transfer total protein resolved in the gel onto a nitrocellulose membrane in Transfer Buffer using the Mini Trans-Blot Electrophoretic Transfer Cell system at 90 mA (constant) for 16 h at 4°C.

      Note: Alternatively, transfer at 400 mA (constant) for 1 h at 4°C. The advantage of a longer transfer is that it does not generate as much heat as a 1-h transfer, which might result in better-resolved bands.

    5. Optional: dry the membrane containing transferred proteins for approximately 20 min at room temperature.

      Note: This step may increase protein immobilization in the membrane, potentially resulting in clearer bands of target proteins during immunodetection (background signal may also be increased).

    6. Rinse the membrane briefly in deionized water.

    7. Pour 3-5 ml of RevertTM 700 Total Protein Stain over the membrane to cover it evenly and incubate for 5 min at room temperature with gentle agitation until clear bands of total protein stain become visible.

    8. Wash out the excess stain from the membrane with 3-5 ml of Wash Solution for 30 s at room temperature with gentle agitation.

    9. Rinse the washed membrane briefly in deionized water and acquire an image with the Odyssey® Fc Imaging System at 700 nm. The documented signal represents the total protein stain to normalize RuBisCO protein quantity.

      Note: To our knowledge, no particular cyanobacterial protein is used as a universal reference (loading control) in Western analyses. Therefore, the analysis of protein expression in cyanobacteria usually involves normalization to total protein stain.

    10. Rinse the membrane in deionized water.

    11. Block the membrane by incubation in 5% (w/v) non-fat milk in TBS for 1 h at room temperature with gentle agitation.

      Note: The incubation time can be shorted by 30 min if needed.

    12. Incubate the membrane in the antibody solution containing rabbit anti-RbcL antibody diluted 1:5,000 in TBST with 5% (w/v) non-fat milk and 0.03% (w/v) sodium azide, overnight at 4°C with gentle agitation.

      Notes: Alternatively, incubate the membrane in the antibody solution for 1 h at room temperature with gentle agitation. We do not recommend reusing the same antibody solution on another day (either after overnight or 1-h incubation).

    13. Discard the antibody solution and wash the membrane briefly in TBST, followed by 1 × 15 min and 3 × 5 min washes in TBST at room temperature with gentle agitation.

    14. Incubate the membrane in IRDye® 800CW goat anti-rabbit IgG secondary antibody diluted 1:20,000 in Intercept® (TBS) blocking buffer supplemented with 0.1% (v/v) Tween-20, for 1 h at room temperature with gentle agitation, protected from light.

    15. Discard the secondary antibody solution and wash the membrane briefly in TBST, followed by 1 × 15 min and 3 × 5 min washes in TBST at room temperature with gentle agitation.

    16. Rinse the membrane in deionized water and acquire images with the Odyssey® Fc Imaging System at 700 nm (to visualize protein mass standards) and 800 nm (to obtain the signal for RuBisCO large subunit, RbcL, with the expected band size of ~55 kDa).

      Note: An example of immunodetected RbcL is shown in Garcia et al., 2021.

    17. Perform the densitometric analysis of the obtained protein bands using appropriate software, such as Image Studio (LI-COR) or Quantity One (Bio-Rad). Normalize the RbcL signal to a corresponding total protein stain signal (acquired in Step D9). Compare the normalized RbcL signal intensity for each Syn02 strain replicate culture with the averaged normalized RbcL signal intensity across all WT replicate cultures. RbcL expression level in the Syn02 strain can be expressed WT expression level fold change.


  5. Determination of photosynthetic oxygen evolution rate

    1. Prepare the cultures of two S. elongatus strains (PCC 7942 WT and Syn02) in 50 ml of BG-11 medium inside of 250-ml glass flasks capped with sponges. Incubate in the controlled environment chamber (Percival) under desired CO2 concentration at 30°C, 120 × g (we used ambient air), and illumination of 80 µmol·m-2·s-1.

      Note: Depends on the growth phase at which RuBisCO expression needs to be measured. Here, we collected cells at the early exponential phase.

    2. Once the cultures reached an OD750 value indicating an anticipated growth phase, harvest the cells.

      Note: To normalize the number of cyanobacteria being analyzed in the reaction chamber of Oxygraph+ System, we harvest the same amount of bacterial culture as in Section A.

    3. Centrifuge the collected cultures at 5,000 × g for 10 min at room temperature.

    4. Decant the supernatant and resuspend cells in 1 ml of BG-11 medium.

    5. Meanwhile, assemble the Oxygraph+ System consisting of the Clark-type oxygen electrode mounted in the electrode disc, the oxygen electrode chamber, and the Oxygraph+ electrode control unit operated from the computer. Follow the steps according to the manual provided by the manufacturer (Oxygraph+ System Manual, version 2.0, by Hansatech Instruments Ltd, 2017).

      Note: The manual contains detailed pictures that may aid in the proper assembly of the instrument as well.

    6. Calibrate the Oxygraph+ System using the liquid phase calibration process. Follow the steps in the manual provided by the manufacturer (Liquid Phase Calibration Guide, version 2, by Hansatech Instruments Ltd, 2017).

    7. Measure the OD750 values of cell suspensions prepared in Step E4.

    8. Wrap the cell suspensions in aluminum foil to protect them from light and incubate with gentle agitation for 15 min at room temperature.

      Note: Shortening the 15-min incubation may result in higher background oxygen level upon starting the measurement using oxygraph. It may also introduce higher fluctuation of recorded oxygen, which, in turn, may lead to an increased deviation between replicates.

    9. Add the bacterial suspension into the reaction vessel of the oxygen electrode chamber.

      Note: Total volume of the bacterial suspension must be 0.2-2.5 ml. We used 1 ml.

    10. Insert the gas-tight plunger assembly that prevents the diffusion of atmospheric oxygen into the reaction vessel.

    11. Turn on stirring (set stirrer speed to 100 by using the OxyTrace+ software).

    12. Turn on the source of light to illuminate the reaction vessel during the measurement. The distance of the source of light from the reaction vessel must remain constant during the experiment – production of oxygen strictly depends on the amount of available light. Use high light intensity.

    13. Start recording oxygen evolution with OxyTrace+ for up to 10 min (the signal might not start to steadily increase until a few minutes have passed).

    14. Calculate oxygen evolution rate (expressed as nanomoles of O2 generated per ml of bacterial suspension) over three consecutive minutes while the level of produced oxygen grows steadily without interruption (Table 8).

    15. Measure the concentration of chlorophyll a in the bacterial suspension according to the protocol by Zavřel et al. (2015) .

      Note: We calculated the concentration of chlorophyll a using the equation: Chla [µg/ml] = 12.9447 (A665 – A720).

    16. Normalize oxygen evolution rates in cyanobacterial suspensions to their OD750 values measured in Step E7 and to chlorophyll a concentration measured in step 15 (Table 8).

      Note: Authors usually use one of the presented methods for normalization. The oxygen evolution rate normalized to OD750 is expressed as µM O2·min-1·OD750-1, and normalized to chlorophyll a – as nmol O2·h-1·µg chlorophyll a-1.


      Table 8. Example oxygen evolution rates measured with Oxygraph+ System and normalized to OD750 and chlorophyll a content


    17. Rinse the reaction vessel thoroughly with deionized water before starting the oxygen evolution rate measurement for another sample. Perform the analysis for each strain in three biological replicates.

Data analysis

The example results are presented as the mean and the sample standard deviation (SD) values of at least three independent technical and biological replicates unless indicated otherwise. The significance of the results is analyzed statistically with the two-tailed t-test performed by the Microsoft 365 Analysis ToolPak add-in. The unpaired sample t-test assuming equal variances is used to compare the values obtained for different cyanobacterial strains.

Recipes

  1. BG-11 medium (Rippka et al., 1979)

    17.6 mM NaNO3

    0.3 mM MgSO4·7H2O

    0.24 mM CaCl2·2H2O

    0.23 mM K2HPO4

    0.19 mM Na2CO3

    0.031 mM C6H8O7·H2O

    0.021 mM C6H11FeNO7

    0.0027 mM Na2EDTA·2H2O

    46 mM H3BO3

    9 mM MnCl2·4H2O

    0.77 mM ZnSO4·7H2O

    1.6 mM Na2MoO4·2H2O

    0.3 mM CuSO4·5H2O

    0.17 mM Co(NO3)2·6H2O

    Autoclave K2HPO4, Na2CO3, and C6H11FeNO7 separately from the other medium components to avoid the formation of precipitates. Combine after autoclaving and store the completed medium at 4°C for up to several months.

  2. Denaturing Lysis Buffer

    10 mM Tris, pH 8.0

    1 mM EDTA

    1% (w/v) SDS

  3. Laemmli Sample Buffer

    50 mM Tris, pH 6.8

    2% (w/v) SDS

    10% (v/v) glycerol

    5% (v/v) β-mercaptoethanol

    0.05% (w/v) bromophenol blue

    Prepare as a 4× concentrate and store refrigerated or at room temperature for up to several months.

  4. Polyacrylamide resolving gel

    12% (v/v) acrylamide/bis solution

    375 mM Tris, pH 8.8

    0.1% (w/v) APS

    0.1% (v/v) TEMED

  5. Polyacrylamide stacking gel

    6% (v/v) acrylamide/bis solution

    125 mM Tris, pH 6.8

    0.1% (w/v) APS

    0.1% (v/v) TEMED

  6. Sodium dithionite saturated solution

    Dissolve 18.2 g sodium dithionite in 100 ml deionized water.

  7. TAE Buffer

    40 mM Tris, pH 8.6

    20 mM acetic acid

    1 mM EDTA

  8. TBS Buffer

    50 mM Tris, pH 7.5

    150 mM NaCl

  9. TBST Buffer

    50 mM Tris, pH 7.5

    150 mM NaCl

    0.1% (v/v) Tween® 20

  10. TE Buffer

    10 mM Tris, pH 8.0

    1 mM EDTA

  11. TE Lysis Buffer

    30 mM Tris, pH 8.0

    1 mM EDTA

    15 mg/ml lysozyme

    10% (v/v) proteinase K

  12. TGS Buffer

    25 mM Tris, pH 8.3

    192 mM glycine

    0.1% (w/v) SDS

  13. Transfer Buffer

    25 mM Tris, pH 8.3

    192 mM glycine

    20% (v/v) methanol

Acknowledgments

We thank Amanda Garcia, Katie McGrath, and Arnaud Taton for feedback; and Susan Golden for providing the wild-type S. elongatus strain. This research was supported by the National Science Foundation (#1724090) and a NASA Early Career Faculty Award (#80NSSC19K1617).

Competing interests

The authors have no conflict of interest.

References

  1. Andersson, I. (2008). Catalysis and regulation in Rubisco. J Exp Bot 59(7): 1555-1568.
  2. Atsumi, S., Higashide, W. and Liao, J. C. (2009). Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat Biotechnol 27(12): 1177-1180.
  3. Bainbridge, G., Madgwick, P., Parmar, S., Mitchell, R., Paul, M., Pitts, J., Keys, A. J. and Parry, M. A. J. (1995). Engineering Rubisco to change its catalytic properties. J Exp Bot 46: 1269-1276.
  4. Bhat, J. Y., Thieulin-Pardo, G., Hartl, F. U. and Hayer-Hartl, M. (2017). Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair. Front Mol Biosci 4: 20.
  5. Carmo-Silva, E., Scales, J. C., Madgwick, P. J. and Parry, M. A. (2015). Optimizing Rubisco and its regulation for greater resource use efficiency. Plant Cell Environ 38(9): 1817-1832.
  6. De Porcellinis, A. J., Nørgaard, H., Brey, L. M. F., Erstad, S. M., Jones, P. R., Heazlewood, J. L. and Sakuragi, Y. (2018). Overexpression of bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase leads to enhanced photosynthesis and global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002. Metab Eng 47: 170-183.
  7. Durall, C., Lindberg, P., Yu, J. and Lindblad, P. (2020). Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803. Biotechnol Biofuels 13(1): 16.
  8. Erb, T. J. and Zarzycki, J. (2018). A short history of RubisCO: the rise and fall (?) of Nature's predominant CO2 fixing enzyme. Curr Opin Biotechnol 49: 100-107.
  9. Garcia, A. K., Kedzior, M., Taton, A., Li, M., Young, J. N. and Kaçar, B. (2021). System-level effects of CO2 and RuBisCO concentration on carbon isotope fractionation. bioRxiv 2021.04.20.440233.
  10. Ivleva, N. B. and Golden, S. S. (2007). Protein extraction, fractionation, and purification from cyanobacteria. Methods Mol Biol 362: 365-373.
  11. Kacar, B., Hanson-Smith, V., Adam, Z. R. and Boekelheide, N. (2017). Constraining the timing of the Great Oxidation Event within the Rubisco phylogenetic tree. Geobiology 15(5): 628-640.
  12. Kedzior, M., Garcia, A. K., Li, M., Taton, A., Adam, Z. R., Young, J. N. and Kaçar, B. (2021). Molecular foundations of Precambrian uniformitarianism. bioRxiv 2021.05.31.446354.
  13. Kubis, A. and Bar-Even, A. (2019). Synthetic biology approaches for improving photosynthesis. J Exp Bot 70(5): 1425-1433.
  14. Liang, F. and Lindblad, P. (2016). Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803. Metab Eng 38: 56-64.
  15. Liang, F. and Lindblad, P. (2017). Synechocystis PCC 6803 overexpressing RuBisCO grow faster with increased photosynthesis. Metab Eng Commun 4: 29-36.
  16. Luo, X., Li, J., Chang, T., He, H., Zhao, Y., Yang, X., Zhao, Y. and Xu, Y. (2019). Stable reference gene selection for RT-qPCR analysis in Synechococcus elongatus PCC 7942 under abiotic stresses. Biomed Res Int 2019: 7630601.
  17. Nisbet, E. G., Grassineau, N. V., Howe, C. J., Abell, P. I., Regelous, M. and Nisbet, R. E. R. (2007). The age of Rubisco: the evolution of oxygenic photosynthesis. Geobiology 5: 311-335.
  18. Parry, M. A. J., Andralojc, P. J., Mitchell, R. A. C., Madgwick, P. J. and Keys, A. J. (2003). Manipulation of Rubisco: the amount, activity, function and regulation. J Exp Bot 54(386): 1321-1333.
  19. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Microbiology 111(1): 1-61.
  20. Salesse-Smith, C. E., Sharwood, R. E., Busch, F. A., Kromdijk, J., Bardal, V. and Stern, D. B. (2018). Overexpression of Rubisco subunits with RAF1 increases Rubisco content in maize. Nat Plants 4(10): 802-810.
  21. Simkin, A. J., López-Calcagno, P. E. and Raines, C. A. (2019). Feeding the world: improving photosynthetic efficiency for sustainable crop production. J Exp Bot 70(4): 1119-1140.
  22. Szekeres, E., Sicora, C., Dragoş, N. and Drugă, B. (2014). Selection of proper reference genes for the cyanobacterium Synechococcus PCC 7002 using real-time quantitative PCR. FEMS Microbiol Lett 359(1): 102-109.
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  24. Taton, A., Erikson, C., Yang, Y., Rubin, B. E., Rifkin, S. A., Golden, J. W. and Golden, S. S. (2020). The circadian clock and darkness control natural competence in cyanobacteria. Nature Comm 11(1): 1688.
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简介

[摘要] 光养微生物经常被设计用于调节特定生物技术和农业应用中感兴趣的目标酶的表达和活性。该协议描述了一种评估 RuBisCO(核酮糖 1,5-二磷酸羧化酶/加氧酶)在模型蓝藻细长聚球藻PCC 7942 中的表达的方法,分别通过定量 PCR 和蛋白质印迹在转录物和蛋白质水平上进行。我们进一步描述了一种使用氧电极来确定光合活性的实验方法,该电极测量蓝藻培养物的分子氧产生率。我们的协议可用于评估 RuBisCO 工程在代谢和生理水平上的影响。


[背景] RuBisCO(核酮糖 1,5-二磷酸羧化酶/加氧酶)是一种中心酶,参与将大气 CO 2固定到其光合自养生产者的生物质中(Nisbet等人,2007 年;Tabita等人,2008 年;Kacar等人.,2017 年;Erb 和 Zarzycki,2018 年)。提高 RuBisCO 羧化活性的效率可能会增加生物质的产量,并推动具有成本效益、生态友好的方式来生物合成各种碳基化合物(Simkin等人,2019 年;Kubis等人,2019 年)。因此,RuBisCO 已成为研究最广泛和工程化的酶之一(Bainbridge等,1995;Whitney等,2011)。已使用不同的方法来操纵 RuBisCO 的总活性,例如通过激活酶提高其活化(Bhat等人,2017 年),在酶底物结合或活性位点中加入有益的突变(Andersson,2008 年),以及上调 RuBisCO表达(Parry等人,2003 年;Carmo-Silva等人,2015 年;Liang 和 Lindblad,2017 年;Salesse-Smith等人,2018 年;Durall等人,2020 年;Garcia等人,2021 年),以及在基因组上用其古老的对应物替换天然 RuBisCO(Kedzior等人,2021 年)。
通常在转录物和蛋白质水平上评估工程策略对增加 RuBisCO 表达的影响。在这里,我们提出了一个详细的协议,该协议建立和扩展了其他人应用的方法来分析 RuBisCO 在蓝藻中的表达。此处详述的方法允许通过测量工程光合生物的光合活性来评估改变的酶表达的潜在代谢影响。特别是,该协议使用模型蓝藻细长聚球藻PCC 7942(以下简称S. elongatus ),该模型广泛用于基础和应用研究,因为它具有天然能力,并且可以使用标准工程技术轻松修改其基因组(Atsumi等人, 2009 年;塔顿等人,2020 年)。作为修饰的菌株,可以相对于野生型进行评价的例子S.细长(WT)中,我们使用的是港口的天然突变株“Syn02” RBC操纵子和第二复制在染色体中性位点插入,如所描述的并由我们的实验室研究(Garcia等人,2021 年)。
在这里,我们将不同的操作方法信息组合在一个单一的、现成的协议中。此外,我们专门调整了其他地方报道的方法,以提高分离的总 RNA 的目标产量和纯度——这是生成 qPCR 分析中使用的 cDNA 模板所必需的。总体而言,我们在转录水平上对 RuBisCO 表达的分析采用了经过调整的方案,包括不同制造商(QIAGEN、Invitrogen、Applied Biosystems)推荐的方案,以从细菌中提取和纯化总 RNA,逆转录为 cDNA,并通过 qPCR 分析基因表达。具体来说,我们使用参考基因secA对基于先前研究的rbcL表达进行标准化,显示其在S. elongatus 的不同生长条件下稳定表达(Szekeres等人,2014 年;Luo等人,2019 年)。为了分析蛋白质水平的表达,在 Ivleva 和 Golden (2007) 的方法基础上进行了修改,在变性条件下从粗细胞提取物中分离了总蛋白质。含有 100 mM 二硫苏糖醇和 2% (w/v) SDS 的 SDS-PAGE 上样缓冲液被替换为含有 1% (w/v) SDS 的热变性裂解缓冲液,不含还原剂,否则会妨碍对分离的蛋白质进行精确定量。通过冻融循环和玻璃珠进行物理细胞破碎的方法被超声处理取代。基于标准聚丙烯酰胺凝胶电泳和蛋白质转移方案 (Bio-Rad) 详细阐述了执行蛋白质印迹的程序。最后,一抗 RbcL 抗体的使用说明改编自 Agrisera,总蛋白染色和近红外二抗介导的免疫检测说明由 LI-COR Biosciences 提供。我们的协议能够分离约 5 毫克/毫升的总蛋白质。析氧率测量程序遵循 Oxygraph+ 系统(Hansatech Instruments)的制造商指南,并根据以前的方法(Liang 和 Lindblad,2016 年;De Porcellinis等人,2018 年)对蓝藻培养进行了一些调整。我们还修改了用于分析的蓝藻准备工作(本协议的步骤 E8),以减少氧气测量过程中的波动,从而提高可重复性。图 1说明了本协议中描述的所有主要步骤。


图 1. 本协议中提出的实验步骤示意图

关键字:RuBisCO, 蓝藻细菌, 聚球藻, 基因表达, 析氧

 
材料和试剂
 
96孔低调裙边PCR板(Thermo Scientific,目录号:AB0800W)
细长聚球藻PCC 7942 WT(野生型菌株,在本机位点具有一个 RuBisCO 操纵子拷贝)
细长聚球藻PCC 7942 Syn02(具有两个 RuBisCO 操纵子拷贝的工程菌株:一个在天然位点,另一个在中性位点;Garcia等,2021)
RNeasy Protect Bacteria Mini Kit(QIAGEN,目录号:74524)
脱氧核糖核酸酶I,扩增级(Invitrogen,目录号:18068-015)
SuperScript TM IV 第一链合成系统(Invitrogen,目录号:18091050)
Q5 ®高保真DNA聚合酶(新英格兰生物实验室,目录号:M0491)
PowerUp TM SYBR TM Green Master Mix(Applied Biosystems,目录号:A25742)
琼脂糖,LE,分析级(Promega,目录号:V3125)
SYBR TM Safe DNA Gel Stain(Invitrogen,目录号:S33102)
牛血清白蛋白(BSA)标准品(Lee BioSolutions,目录号:100-10-0.25);浓度 [µg/ml]: 25, 125, 250, 500, 750, 1,000, 1,500, 2,000
Pierce TM BCA 蛋白质测定试剂盒(Thermo Scientific,目录号:23225)
Tris(Thermo Fisher Scientific,目录号:PRH5131)
过硫酸铵,APS(Sigma-Aldrich,目录号:A3678)
TEMED(Bio-Rad,目录号:1610801)
30%丙烯酰胺/双溶液(Bio-Rad,目录号:1610158)
Precision Plus Protein TM双色标准品(Bio-Rad,目录号:1610374)
Revert TM 700 Total Protein Stain(LI-COR Biosciences,目录号:926-11011)
兔抗 RbcL 抗体(Agrisera,目录号:AS03 037)
脱脂奶粉(LabScientific,目录号:M0841)
IRDye ® 800CW 山羊抗兔 IgG 二抗(LI-COR Biosciences,目录号:926-32211)
氯化钾(Sigma-Aldrich,目录号:P9541)
连二亚硫酸钠/连二亚硫酸钠(Sigma-Aldrich,目录号:157953)
无水甲醇(Sigma-Aldrich,目录号:322415)
氯化钠(Sigma-Aldrich,目录号:S9888)
Tween ® 20(Sigma-Aldrich,目录号:P9416)
乙二胺四乙酸,EDTA(Sigma-Aldrich,目录号:ED)
来自鸡蛋清的溶菌酶(Sigma-Aldrich,目录号:L4919)
蛋白酶 K(QIAGEN,目录号:19131)
甘氨酸(Thermo Fisher Scientific,目录号:PRH5073)
十二烷基硫酸钠,SDS(Sigma-Aldrich,目录号:L3771)
乙酸(Sigma-Aldrich,目录号:695092)
截距® (TBS)封闭缓冲液(LI-COR Biosciences公司,目录号:927-60001)
β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
无水乙醇(Sigma-Aldrich,目录号:443611)
溴酚蓝(Sigma-Aldrich,目录号:B0126)
甘油(Sigma-Aldrich,目录号:G5516)
硝酸钠,NaNO 3 (Thermo Fisher Scientific,目录号:BP360-500)
七水硫酸镁,MgSO 4 ·7H 2 O(Sigma-Aldrich,目录号:230391)
二水氯化钙,CaCl 2 ·2H 2 O(Sigma-Aldrich,目录号:223506)
磷酸二氢钾,K 2 HPO 4 (Sigma-Aldrich,目录号:P8281)
碳酸钠,Na 2 CO 3 (Supelco,目录号:SX0395)
柠檬酸一水合物,C 6 H 8 O 7 ·H 2 O(JT Baker,目录号:0115-01)
柠檬酸铵铁(III),C 6 H 11 FeNO 7 (Sigma-Aldrich,目录号:F5879)
乙二胺四乙酸二钠盐,Na 2 EDTA·2H 2 O(Alfa Aesar,目录号:A15161)
硼酸,H 3 BO 3 (JT Baker,目录号:0084-01)
四水氯化锰(II),MnCl 2 ·4H 2 O(JT Baker,目录号:2540-01)
七水硫酸锌,ZnSO 4 ·7H 2 O(Sigma-Aldrich,目录号:Z0251)
二水钼酸钠,Na 2 MoO 4 ·2H 2 O(JT Baker,目录号:3764-01)
五水硫酸铜(II),CuSO 4 ·5H 2 O(Sigma-Aldrich,目录号:C8027)
硝酸钴(II)六水合物,Co(NO 3 )2 ·6H 2 O(Sigma-Aldrich,目录号:239267)
BG-11 培养基(见配方)
变性裂解缓冲液(见配方)
Laemmli 样品缓冲液(见配方)
聚丙烯酰胺分离胶(见配方)
聚丙烯酰胺浓缩凝胶(见配方)
连二亚硫酸钠饱和溶液(见配方)
TAE 缓冲液(见配方)
TBS 缓冲液(见配方)
TBST 缓冲液(见配方)
TE 缓冲液(见配方)
TE 裂解缓冲液(见配方)
TGS 缓冲液(见配方)
转移缓冲液(见配方)
 
设备
 
250 毫升带海绵盖的玻璃烧瓶 (Thermo Fisher Scientific)
配备光源的受控环境室(Percival,型号:I36LLVLC8)
紫外-可见分光光度计(安捷伦,型号:Cary 60)
Heraeus Multifuge Centrifuge(Thermo Scientific,型号:X3 FR)
Sorvall Legend Micro 17 Centrifuge(Thermo Scientific,目录号:75002431)
NanoDrop 2000c 分光光度计(Thermo Scientific,目录号:ND-2000C)
干浴数字加热块(基准,型号:BSH1002)
数字摇床(VWR,型号:5000)
模拟涡流混合器(Fisher Scientific,型号:02215414)
热循环仪(Applied Biosystems,型号:2720)
绝对 qPCR 板密封件(Thermo Scientific,目录号:AB1170)
实时热循环仪(Analytik Jena AG,型号:qTOWER 3 G)
微波
250 毫升烧杯
声波器(Qsonica,型号:Q125)
硝酸纤维素膜(Thermo Scientific,目录号:88018)
Mini-PROTEAN ® Tetra 垂直电泳细胞系统(Bio-Rad,目录号:1658004)
Mini Trans-Blot电泳转移细胞系统(Bio-Rad,目录号:1703930)
Odyssey ® Fc 成像系统(LI-COR Biosciences,型号:2800-03)
Oxygraph+系统(Hansatech Instruments,目录号:OXY1+)
 
软件
 
qPCRsoft(Analytik Jena AG,版本:3.1)
Image Studio TM (LI-COR Biosciences, version: 5.0)
Quantity One ®一维分析(Bio-Rad,版本:4.6.7)
OxyTrace+ (Hansatech Instruments)
微软 365 Excel
 
程序
 
总 RNA 分离和逆转录制备
在用海绵盖住的 250 毫升玻璃烧瓶内,在 50 毫升 BG-11 培养基中制备两种S. elongatus菌株(PCC 7942 WT 和 Syn02)的培养物。在受控环境室 (Percival) 中于 30°C下在所需的选定 CO 2浓度下孵育,在 80 μmol·m -2 ·s -1光照下缓慢搅拌。
注意:培养时间取决于需要测量 RuBisCO 表达的生长阶段。在这里,我们收集了早期指数期的细胞。
一旦培养物达到指示预期生长期的所需 OD 750值,收获细胞。
注意:为了使蓝藻数量和分离的 RNA 产量正常化,我们收获了 6 ml 的培养物,稀释到 ~OD 750 = 1.0。
将收集的培养物以 4,700 × g离心10 分钟。
注意:由于 mRNA 转换非常快,我们建议将细胞直接倒在 Falcon 管中的冰上,并在 4°C 下离心。在某些情况下,还建议在收获细胞前使用终止液(95% 乙醇、5% 苯酚—— 总体积的 1/5)。
倒出上清液并将沉淀的细菌重新悬浮在 2 ml TE 缓冲液中。
使用 RNeasy Protect Bacteria Mini Kit 提取 RNA,遵循 RNAprotect Bacteria Reagent Handbook – Protocol 4: Enzymatic Lysis and Proteinase K Digestion of Bacteria,进行多项调整以确保分离 RNA 的高产量,如下所述:
将 4 ml RNA Protect Bacteria Reagent 和 2 ml 细胞悬液(在步骤 A4 中制备)混合在 15 ml 锥形管中。
在室温下以 4,700 × g离心10 分钟。
倒出上清液,并在室温下以4,700 x g重新离心沉淀2 分钟,用移液器吸头丢弃残留的上清液。
注意:如有必要,颗粒可以在 -80°C 下冷冻以在另一天进行。
加入 200 µl 含有 15 mg/ml 溶菌酶和 100 µg/ml 蛋白酶 K 的 TE 缓冲液,并通过上下吹打重悬沉淀。
注意:在 TE 缓冲液中加入溶菌酶足以裂解蓝藻细胞,但加入蛋白酶 K 可以提取更多的总 RNA。
涡旋混合 10 秒,然后在室温下孵育重悬在 TE 裂解缓冲液中的细胞 10 到 20 分钟,同时在振荡器上不断摇动悬浮液。
笔记:
我们延长了原始 RNeasy Protect Bacteria Mini Kit 方案的孵育时间,以确保刚性蓝藻细胞壁充分分解。
洗脱总 RNA 后,使用 NanoDrop 测量其浓度并将 RNA 样品储存在 -80°C 或立即进行 DNase I 处理(下一步)。或者,使用 Qubit 荧光计 (Invitrogen) 代替 NanoDrop 来测量 RNA 浓度。Qubit 被推荐用于核酸的精确定量,但在本协议中未使用。
加入 700 µl RLT 缓冲液(每 1 ml RLT 缓冲液补充 10 µl β-巯基乙醇)并剧烈涡旋几秒钟。
加入 500 µl 96% 乙醇(分子级),上下吹打混合(不要离心)。
将 700 µl 裂解液转移到 RNeasy Mini Spin Column 中,置于 2 ml 收集管中,并在室温下以 10,000 × g离心 15 秒。
弃去流出液,加入剩余的裂解液,并按照上一步进行离心。
加入 700 µl Buffer RW1,室温10,000 × g离心15 s 洗膜,弃去流出液。
加入 500 μl 缓冲液 RPE(通过将 44 ml 100% 乙醇加入试剂盒中的 11 ml RPE 浓缩液制备),室温下以10,000 × g离心15 秒以洗涤膜,并丢弃流过液.
再加入 500 µl 缓冲液 RPE 并在室温下以 10,000 × g离心2 分钟。
将 RNeasy Mini 离心柱置于新的 2 ml 收集管中,并在室温下全速 (17,000 × g )离心1 分钟。
将离心柱置于新的 1.5 ml 收集管中,将 40 µl RNase-free 水直接加入膜中心,室温下17,000 × g离心1 分钟以洗脱总 RNA。
使用 NanoDrop 测量洗脱液中的 RNA 浓度。
将 RNA 样品储存在 -80°C 或立即进行 DNase I 处理(下一步)。
将 RNA 样品放在冰上,根据制造商提供的说明准备含有扩增级 DNase I 的反应,并进行一些实质性调整以提高分离 RNA 的纯度:
使用 4 U 的 DNase I (4 µl) 而不是 1 U (1 µl)。
注意:额外的 DNase I 应该有助于完全消除残留的 gDNA。
将总反应体积增加到 20 µl。加入 1 µg RNA 和 2 µl 10×DNase I Reaction Buffer 后,必要时加入不含 RNase 的水。
注意:提供足够体积的每个 RNA 样本用于逆转录(在阳性反应和阴性对照中;步骤 B2)。
在室温下将孵育时间从推荐的 15 分钟延长至 60 分钟。
注意:这是确保完全消除 gDNA 的另一个步骤(除了增加 DNase I 的量)。我们没有观察到增加 DNase I 浓度和孵育时间对 RNA 质量(稳定性)的任何实质性不利影响。
在 65°C 孵育 10 分钟之前,向每个反应中加入 2 µl 25 mM EDTA 以灭活 DNase I。
使用 NanoDrop 或 Qubit 荧光计测量 DNase I 处理过的样品中的 RNA 浓度(我们还建议在凝胶上运行 RNA 以确保其不会降解)。将样品储存在 -80°C 或立即进行逆转录。
注意:我们通常会获得 50-100 ng/ml 的最终浓度。
 
逆转录和 qPCR
使用 SuperScript TM IV 第一链合成系统对 RNA 样品进行逆转录 (RT)。根据试剂盒中提供的说明,准备将引物退火到模板 RNA 的反应。每个反应,使用:
1 µl 50 µM 随机六聚体
1 µl 10 mM dNTP 混合物
1 µl DEPC 处理水
10 µl RNA 样本
为每个 RNA 样品准备两个相同的反应,如步骤 B1 中所述。一个反应为阳性(添加逆转录酶以获得用于 qPCR 分析的 cDNA),另一个反应为阴性(不添加逆转录酶;因此,样品中应仅保留 RNA)。将反应在 65°C 下加热 5 分钟,然后在冰上孵育至少 1 分钟。
为步骤 B2 中获得的所有 RNA 引物样品准备逆转录 (RT) 主混合物。每个样本,使用:
4 µl 5×SSIV 缓冲液
1 µl 100 mM DTT
1 µl RNase 抑制剂
将 RT 主混合物分装到含有步骤 B2 中制备的 RNA 引物样品的管中;将 6 µl 混合物转移到每个 RNA 引物样品中。
将 1 µl 试剂盒中的 SuperScript IV 逆转录酶添加到步骤 B2 中制备的每个 RT 阳性反应中(总反应体积为 20 µl)。
在步骤 B2 中制备的每个阴性对照中加入 1 µl DEPC 处理过的水(总反应体积为 20 µl)。
依次孵育所有反应:
在室温下放置 10 分钟。
在 50-55°C 下保持 10 分钟。
在 80°C 下保持 10 分钟。
将反应储存在 -80°C 或立即进行步骤 B8。
通过 PCR 检查步骤 B7 中生成的 cDNA 模板的质量(表 1和2 )。使用也将用于后续 qPCR的引物(表 4 )。它们应该能够在正反应中产生预期大小的产物。评估 cDNA 模板的纯度。阴性对照不应产生 PCR 产物。否则,cDNA 模板可能会被 gDNA 污染。
 
表 1. 检查生成的 cDNA 模板质量的 PCR 样品组成
 
表 2.检查生成的 cDNA 模板质量的 PCR 参数
 
在 TAE + 2% (w/v) 琼脂糖凝胶中对 PCR 产物进行电泳。如果特定 PCR 产物仅在 RT 阳性反应中产生,而阴性对照中没有产物,则进行 qPCR(步骤 B10)。
为在步骤 B7 中获得并在步骤 B8 和 B9 中进行质量检查的每个 RT 阳性反应准备单独的 qPCR 主混合物。每个主混合物的总体积应足以测试每个 qPCR 样本所需基因数量的表达,一式三份(表 3 )。
注意:我们准备了两个 qPCR 预混液(针对每个 WT 和 Syn02 菌株),足以进行 6 次反应,因为我们在三个技术重复中分析了两个基因的表达:rbcL 和 secA;表 4 )。
 


表 3. qPCR 样品的组成
 
表 4. qPCR 中使用的引物。F、前进;R,反转。
 
将 9 µl 的每个 qPCR 主混合物分装到 96 孔板中适当数量的孔中(取决于反应的数量)。
将 1 µl 每对 5 µM 引物(下表中列出的引物)添加到 96 孔板中的一式三份 qPCR 样品中。
在 96 孔板中制备 NTC(无模板控制)样品,类似于 qPCR 样品(步骤 B10-B12),但添加无核酸酶水而不是 RT 阳性反应。
使用配备 qPCRsoft 软件的实时热循环仪 qTOWER 3 G短暂旋转板并启动 qPCR(表 5 )。
 
表 5. qPCR 参数
 
将 qPCR 结果导出到 Excel 电子表格(表 6 ),并使用双增量 Ct 方法(表 7 )计算归一化为secA的rbcL (即平均倍数变化)的相对表达。


表 6. qPCR 获得的示例数据。样品(来自 WT 和 Syn02 菌株)以三个生物学重复制备。获得三个技术重复的循环阈值 (Ct),然后取平均值 (Avg. Ct)。
 
表 7.使用双 delta Ct 方法计算相对rbcL表达式的示例。rbcL的相对表达被标准化为secA (ΔCt = Avg. Ct rbcL – Avg. Ct secA )。计算 ΔΔCt 的示例:ΔΔCt Syn02#1 = ΔCt Syn02#1 – Avg. ΔCt WT 。
 
变性条件下的总蛋白提取
在用海绵盖住的 250 毫升玻璃烧瓶内,在 50 毫升 BG-11 培养基中制备两种S. elongatus菌株(PCC 7942 WT 和 Syn02)的培养物。在所需的 CO 2浓度下在 30°C、120 rpm 搅拌和 80 μmol·m -2 ·s -1光照下在受控环境室 (Percival) 中孵育。
注意:取决于需要测量 RuBisCO 表达的生长阶段。在这里,我们收集了早期指数期的细胞。
当培养物达到所需的 OD 750值时,收获培养物(标准化很重要,以确保在每个样品中处理相似数量的蓝藻以产生相似浓度的提取总蛋白;我们使用 OD 750 = ~1.0)。
在室温下以 4,700 × g离心收集的培养物10 分钟。
倒出上清液,并在室温下以4,700 x g重新离心沉淀2 分钟,用移液器吸头丢弃残留的培养基。
在加热块中将变性裂解缓冲液预热至 95°C。
使用微波炉在 250 毫升烧杯中煮沸去离子水。
向细菌沉淀中加入 500 µl 热变性裂解缓冲液。立即将带有沉淀物的管子浸入煮沸的去离子水中,并通过用力上下吹打几次来重新悬浮蓝藻细胞。
将细胞悬液转移到新的 1.5 ml 管中,并在 95°C 下加热 10 分钟。在孵化时间的中途短暂涡旋管子。
对获得的裂解物进行 3 × 10 s 超声处理,间隔为 10 s,振幅为 40%(无需在冰上孵育)。
在室温下以 17,000 × g离心裂解物10 分钟,并将上清液转移到新的 1.5 ml 管中。
使用 Pierce TM BCA 蛋白质检测试剂盒测量澄清裂解物中的总蛋白质浓度。
将裂解物储存在 -80°C,直到进行 SDS-PAGE。
 
SDS-PAGE 总蛋白分离和蛋白质印迹免疫检测
将制备的蓝藻细胞裂解物与 Laemmli 样品缓冲液混合,以获得含有 5 µg 总蛋白质的样品。
注意:SDS-PAGE 中使用的总蛋白量取决于通过蛋白质印迹获得的预期 RuBisCO 信号强度。我们建议用 RuBisCO 信号强度绘制校准曲线作为总蛋白质量的函数。这将允许估计总蛋白量的最佳范围,在该范围内 RuBisCO 的信号强度与总蛋白水平相关。
将步骤 1 中制备的样品上样到在 Mini-PROTEAN ® Tetra 垂直电泳槽系统中组装的 6% (v/v) 聚丙烯酰胺浓缩胶上,并置于 TGS 缓冲液中。
在 100-150 V 下运行电泳,直到样品迁移通过 12% (v/v) 聚丙烯酰胺分离胶。
使用 Mini Trans-Blot Electrophoretic Transfer Cell 系统以 90 mA(恒定)在 4°C 下将凝胶中溶解的总蛋白质转移到转移缓冲液中的硝酸纤维素膜上,持续 16 小时。
注意:或者,在 4°C 下以 400 mA(恒定)传输 1 小时。更长传输时间的优点是它不会产生与 1 小时传输一样多的热量,这可能会导致更好的分辨率条带。
可选:将含有转移蛋白的膜在室温下干燥约 20 分钟。
注意:此步骤可能会增加膜中的蛋白质固定,从而可能在免疫检测过程中产生更清晰的目标蛋白带(背景信号也可能会增加)。
用去离子水简单冲洗膜。
将 3-5 ml Revert TM 700 Total Protein Stain 倒在膜上,使其均匀覆盖,并在室温下轻轻搅拌孵育 5 分钟,直至可见清晰的总蛋白染色条带。
用 3-5 ml 洗涤液在室温下轻轻搅拌 30 秒,从膜上洗掉多余的污渍。
在去离子水中简单冲洗洗过的膜,并使用 Odyssey ® Fc 成像系统在 700 nm 处获取图像。记录的信号代表总蛋白质染色以标准化 RuBisCO 蛋白质数量。
注意:据我们所知,在西方分析中没有使用特定的蓝藻蛋白作为通用参考(加载控制)。因此,蓝藻中蛋白质表达的分析通常涉及对总蛋白质染色的归一化。
用去离子水冲洗膜。
通过在室温下在 5% (w/v) 脱脂牛奶中的 TBS 中孵育 1 小时并轻轻搅拌来封闭膜。
注意:如果需要,孵化时间可以缩短 30 分钟。 
在含有兔抗 RbcL 抗体的抗体溶液中孵育膜,该抗体溶液在 TBST 中以 1:5,000 稀释,含有 5% (w/v) 脱脂牛奶和 0.03% (w/v) 叠氮化钠,在 4°C 下轻轻搅拌过夜.
注意:或者,将膜在抗体溶液中在室温下轻轻搅拌孵育 1 小时。我们不建议在另一天(过夜或孵育 1 小时后)重复使用相同的抗体溶液。
丢弃抗体溶液并在 TBST 中短暂洗涤膜,然后在室温下在 TBST 中轻轻搅拌 1 × 15 分钟和 3 × 5 分钟洗涤。
将膜在 IRDye ® 800CW 山羊抗兔 IgG 二抗中孵育1 小时,该二抗在 Intercept ® (TBS) 封闭缓冲液中以1:20,000 稀释,并补充有 0.1% (v/v) Tween-20,在室温下轻轻搅拌,保护从光。
弃去二抗溶液并在 TBST 中短暂洗涤膜,然后在室温下轻轻搅拌在 TBST 中洗涤 1 × 15 分钟和 3 × 5 分钟。
在去离子水中冲洗膜并使用 Odyssey ® Fc 成像系统在 700 nm(以显示蛋白质质量标准)和 800 nm(以获取 RuBisCO 大亚基 RbcL 的信号,预期条带大小约为 55 kDa)下获取图像)。
注意:Garcia 等人,2021 年显示了免疫检测 RbcL 的示例。
使用适当的软件,如 Image Studio (LI-COR) 或 Quantity One (Bio-Rad),对获得的蛋白质条带进行光密度分析。将 RbcL 信号标准化为相应的总蛋白染色信号(在步骤 D9 中获得)。将每个 Syn02 菌株复制培养的归一化 RbcL 信号强度与所有 WT 复制培养的平均归一化 RbcL 信号强度进行比较。Syn02菌株中RbcL表达水平可以表达WT表达水平的倍数变化。
 
光合析氧速率的测定
在用海绵盖住的 250 毫升玻璃烧瓶内,在 50 毫升 BG-11 培养基中制备两种S. elongatus菌株(PCC 7942 WT 和 Syn02)的培养物。在受控环境室 (Percival) 中在 30°C、120 × g (我们使用环境空气)和 80 µmol·m -2 ·s -1 的照明下在所需的 CO 2浓度下孵育。
注意:取决于需要测量 RuBisCO 表达的生长阶段。在这里,我们收集了早期指数期的细胞。
一旦培养物达到指示预期生长期的 OD 750值,收获细胞。
注意:为了使 Oxygraph+ 系统反应室中被分析的蓝藻数量正常化,我们收获了与 A 部分相同数量的细菌培养物。
在室温下以 5,000 × g离心收集的培养物10 分钟。
倒出上清液并在 1 ml BG-11 培养基中重悬细胞。
同时,组装由安装在电极盘上的克拉克型氧电极、氧电极室和由计算机操作的 Oxygraph+ 电极控制单元组成的 Oxygraph+ 系统。按照制造商提供的手册(Oxygraph+ 系统手册,2.0 版,Hansatech Instruments Ltd,2017 年)执行步骤。
注意:手册包含详细的图片,也可能有助于正确组装仪器。
使用液相校准过程校准 Oxygraph+ 系统。按照制造商提供的手册中的步骤操作(Liquid Phase Calibration Guide,第 2 版,Hansatech Instruments Ltd,2017 年)。
测量步骤 E4 中制备的细胞悬浮液的 OD 750值。
用铝箔包裹细胞悬浮液以保护它们免受光照,并在室温下轻轻搅拌孵育 15 分钟。
注意:在开始使用 oxygraph 进行测量时,缩短 15 分钟的孵育时间可能会导致更高的背景氧气水平。它还可能会引入更高的记录氧波动,这反过来又可能导致重复之间的偏差增加。
将菌悬液加入氧电极室的反应容器中。
注意:细菌悬液的总体积必须为 0.2-2.5 毫升。我们用了 1 毫升。
插入防止大气氧气扩散到反应容器中的气密柱塞组件。
打开搅拌(使用 OxyTrace+ 软件将搅拌器速度设置为 100)。
在测量过程中打开光源照亮反应容器。在实验过程中,光源与反应容器的距离必须保持恒定——氧气的产生严格取决于可用光的数量。使用高光强度。
开始使用 OxyTrace+ 记录析氧长达 10 分钟(信号可能在几分钟后才开始稳定增加)。
在连续三分钟内计算析氧率(表示为每毫升细菌悬浮液产生的 O 2纳摩尔数),同时产生的氧气水平稳定增长而不会中断(表 8)。
根据 Zavřel等人的协议,测量细菌悬浮液中叶绿素a的浓度。(2015)。
注意:我们使用以下公式计算叶绿素 a 的浓度:Chl a [µg/ml] = 12.9447 (A 665 – A 720 )。
规格化氧释放速率在蓝藻混悬剂其OD 750值在步骤E7测定叶绿素一个在步骤15中测量的浓度(表8 )。
注意:作者通常使用所提出的方法之一进行规范化。以OD 750归一化的氧析出速率表示为μM O 2 ·min -1 ·OD 750 -1 ,归一化为叶绿素a – 表示为nmol O 2 ·h -1 ·μg 叶绿素a -1 。
 
表 8. 使用 Oxygraph+ 系统测量并标准化为 OD 750和叶绿素a含量的示例氧气析出率
 
在开始对另一个样品进行析氧速率测量之前,用去离子水彻底冲洗反应容器。在三个生物学重复中对每个菌株进行分析。
 
数据分析
 
除非另有说明,否则示例结果表示为至少三个独立的技术和生物学重复的平均值和样本标准偏差 (SD) 值。结果的显着性使用Microsoft 365 分析工具库加载项执行的双尾t检验进行统计分析。假设方差相等的未配对样本t检验用于比较不同蓝藻菌株获得的值。
 
食谱
 
BG-11 培养基(Rippka等,1979)
17.6 mM NaNO 3
0.3 mM MgSO 4 ·7H 2 O
0.24 mM CaCl 2 ·2H 2 O
0.23 mM K 2 HPO 4
0.19 mM Na 2 CO 3
0.031 mM C 6 H 8 O 7 ·H 2 O
0.021 mM C 6 H 11 FeNO 7
0.0027 mM Na 2 EDTA·2H 2 O
46 mM H 3 BO 3
9 mM MnCl 2 ·4H 2 O
0.77 mM ZnSO 4 ·7H 2 O
1.6 mM Na 2 MoO 4 ·2H 2 O
0.3 mM CuSO 4 ·5H 2 O
0.17 mM Co(NO 3 ) 2 ·6H 2 O
高压釜 K 2 HPO 4 、Na 2 CO 3和 C 6 H 11 FeNO 7与其他介质组分分开,以避免形成沉淀物。高压灭菌后合并,并将完成的培养基在 4°C 下储存长达数月。
变性裂解缓冲液
10 mM Tris,pH 8.0
1 mM EDTA
1% (w/v) SDS
Laemmli 样品缓冲液
50 mM Tris,pH 6.8
2% (w/v) SDS
10% (v/v) 甘油
5% (v/v) β-巯基乙醇
0.05% (w/v) 溴酚蓝
制备成 4 倍浓缩液,冷藏或在室温下储存长达数月。
聚丙烯酰胺分离胶
12% (v/v) 丙烯酰胺/双溶液
375 mM Tris,pH 8.8
0.1% (w/v) APS
0.1% (v/v) TEMED
聚丙烯酰胺浓缩凝胶
6% (v/v) 丙烯酰胺/双溶液
125 mM Tris,pH 6.8
0.1% (w/v) APS
0.1% (v/v) TEMED
连二亚硫酸钠饱和溶液
将 18.2 g 连二亚硫酸钠溶解在 100 ml 去离子水中。
TAE缓冲液
40 mM Tris,pH 8.6
20 mM 醋酸
1 mM EDTA
TBS 缓冲液
50 mM Tris,pH 7.5
150 毫米氯化钠
TBST 缓冲器
50 mM Tris,pH 7.5
150 毫米氯化钠
0.1% (v/v) 吐温® 20
TE缓冲液
10 mM Tris,pH 8.0
1 mM EDTA
TE 裂解缓冲液
30 mM Tris,pH 8.0
1 mM EDTA
15 毫克/毫升溶菌酶
10% (v/v) 蛋白酶 K
TGS缓冲液
25 mM Tris,pH 8.3
192 mM 甘氨酸
0.1% (w/v) SDS
传输缓冲区
25 mM Tris,pH 8.3
192 mM 甘氨酸
20% (v/v) 甲醇
 
致谢
 
我们感谢 Amanda Garcia、Katie McGrath 和 Arnaud Taton 的反馈;和 Susan Golden 提供野生型S. elongatus菌株。这项研究得到了美国国家科学基金会 (#1724090) 和 NASA 早期职业教师奖 (#80NSSC19K1617) 的支持。
 
利益争夺
 
作者没有利益冲突。
 
参考
 
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引用:Kędzior, M. and Kacar, B. (2021). Quantification of RuBisCO Expression and Photosynthetic Oxygen Evolution in Cyanobacteria. Bio-protocol 11(20): e4199. DOI: 10.21769/BioProtoc.4199.
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