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Oct 2019
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Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale
食葡糖罗斯拜瑞氏菌和直肠真杆菌结合的优化方案   

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Abstract

Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic manipulation techniques. Conjugative transposons previously introduced into Roseburia species could not be easily modified, greatly limiting their applicability as genetic modification platforms. Modular plasmid shuttle vectors have previously been developed for Clostridium species, which share a taxonomic order with Roseburia and Eubacterium, raising the possibility that these vectors could be used in these organisms. Here, we describe an optimized conjugation protocol enabling the transfer of autonomously replicating plasmids from an E. coli donor strain into Roseburia inulinivorans and Eubacterium rectale. The modular nature of the plasmids and their ability to be maintained in the recipient bacterium by autonomous replication makes them ideal for investigating heterologous gene expression, and as a platform for other genetic tools including antisense RNA silencing or mobile group II interon gene disruption strategies.

Keywords: Conjugation (结合), Gut microbiota (肠道菌群), Lachnospiraceae (毛螺菌科), Gene transfer (基因转移), Shuttle vector (穿梭载体)

Background

Roseburia and Eubacterium species are among the most abundant bacteria in the human gut microbiota (Zhernakova et al., 2016), impacting human health by utilising dietary and host derived polysaccharides (Scott et al., 2006 and 2011; Cockburn et al., 2015; Sheridan et al., 2016) and producing the health promoting metabolite butyrate as a fermentation end product (Duncan et al., 2002 and 2006). Additionally, these species are capable of modulating host immunity via flagella (Neville et al., 2013). The lack of genetic modification techniques for these organisms has prevented a more complete understanding of the complex interactions between these bacteria and their human host.

Previously, conjugative transposons were successfully transferred into Roseburia inulinivorans from Eubacterium cellulosolvens and Clostridium cf. saccharolyticum (Scott et al., 2008). These large, novel mobile genetic elements could not be easily modified and thus were a suboptimal platform for detailed genetic modification. This work did however illustrate that conjugative mating was possible between Lachnospiraceae bacteria including Roseburia species. The development of easily modified conjugative plasmids for clostridial species (Purdy et al., 2002; Heap et al., 2009) raised the possibility that these techniques could be adapted for Roseburia and Eubacterium species.

The detailed protocol presented here is based on procedures established in Sheridan et al. (2019). In this work, the different conjugative plasmids developed for use in Clostridium species (Heap et al., 2009) were tested for transferability into the Roseburia and Eubacterium rectale species. Plasmid pMTL83151 was successfully transferred into two strains of E. rectale, while pMTL83151 and pMTL82151 were transferred into Roseburia inulinivorans A2-194. Transfer frequencies of 10-6-10-8 per potential recipient were obtained. These frequencies are similar to those observed when suicide vectors were introduced into other Gram-positive bacteria (Williams et al., 1990; Aquino de Muro and Priest, 2000). The ability to add exogenous DNA to bacterial species opens up opportunities for genetically manipulation, including knockout mutagenesis. Alternatively, these plasmids could be modified as expression vectors for mobile group II interon gene disruption strategies, as has been demonstrated in several clostridial species (Heap et al., 2007). Additionally, Plasmid pMTL83151 was shown to be a suitable vector for heterologous gene expression (Sheridan et al., 2019), producing an enzymatically active Streptococcus glycoside hydrolase in both species and thus proving the utility of this technique in studying researcher-selected functional gains in these important bacteria. The protocol below is a stepwise guide to introducing foreign DNA to these bacteria.

Materials and Reagents

  1. Pipette tips
  2. Cuvettes (Bio-Rad, catalog number: 1652083 )
  3. Petri dishes (Greiner Bio-One, catalog number: 633180 )
  4. Glass Pasteur pipettes (Fisher Scientific, catalog number: FB50261 )
  5. Nylon membranes (Roche, catalog number: 11417240001 )
  6. X-ray film (Fujifilm) (Fisher Scientific, catalog number: 12735325 )
  7. 50 ml conical centrifuge tubes (Corning, catalog number: 10038980 )
  8. Strains (Table 1)
  9. Plasmids (Table 1)
    Note: Modular plasmids can be obtained from CHAINbiotools (http://clostron.com/pMTL80000.php).
  10. Primers (Table 2)
  11. PBS tablets (Sigma, catalog number: P4417 )
  12. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 ) stock solution 10 µg/ml, stored at -20 °C
  13. HindIII restriction endonuclease (NEB, catalog number: R0104S )
  14. Wizard genomic DNA Purification kit (Promega, catalog number: A1120 )
  15. DIG High Prime DNA Labelling and Detection Starter Kit II (Roche, catalog number: 11585614910 )
  16. PCR reagents (Taq Polymerase kit Bioline, catalog number: BIO-21040 and dNTP’s Promega, catalog number: U1240 )
  17. Dipotassium phosphate, K2HPO4 (Fisher Scientific, catalog number: P/5240/53)
  18. Potassium dihydrogen phosphate, KH2PO4 (Fisher Scientific, catalog number: P/4800/60)
  19. Ammonium sulfate, (NH4)2SO4 (Fisher Scientific, catalog number: A/6480/53)
  20. Sodium chloride, NaCl (Fisher Scientific, catalog number: S/3160/53)
  21. Magnesium sulfate, MgSO4 (Sigma-Aldrich, catalog number: M7506 )
  22. Calcium chloride, CaCl2 (Sigma-Aldrich, catalog number: C1016 )
  23. Acetic acid (Fisher Scientific, catalog number: A/10400/PB17)
  24. Propionic acid (Sigma-Aldrich, catalog number: P1386 )
  25. n -Valeric acid (Sigma-Aldrich, catalog number: V9759 )
  26. Iso-Valeric acid (Sigma-Aldrich, catalog number: I7128)
  27. Iso-Butyric acid (Sigma-Aldrich, catalog number: I1754)
  28. Biotin (Sigma-Aldrich, catalog number: B4501 )
  29. Cobalamin (Sigma-Aldrich, catalog number: V2876 )
  30. p-Aminobenzoic acid (Sigma-Aldrich, catalog number: A9878 )
  31. Folic acid (Sigma-Aldrich, catalog number: F7876 )
  32. Pyridoxamine (Sigma-Aldrich, catalog number: P9755 )
  33. Potassium hydroxide, KOH (Sigma-Aldrich, catalog number: P5958 )
  34. Ethanol 95% (Fisher Scientific, catalog number: E/0650DF/17 )
  35. Haemin (Sigma-Aldrich, catalog number: H5533 )
  36. Bacto tryptone (BD Diagnostics Systems, catalog number: 211705 )
  37. Yeast Extract (BD Diagnostics Systems, catalog number: 212750 )
  38. Potassium chloride, KCl (Sigma-Aldrich, catalog number: P3911 )
  39. Magnesium chloride hexahydrate, MgCl2·6H2O (Sigma-Aldrich, catalog number: M9272 )
  40. Magnesium sulfate heptahydrate, MgSO4·7H2O (Sigma-Aldrich, catalog number: 230391 )
  41. Bacto casitone (BD Diagnostics Systems, catalog number: 225930 )
  42. Sodium bicarbonate, NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
  43. Glucose (Fisher Scientific, catalog number: G/0500/53)
  44. Soluble starch (Sigma-Aldrich, catalog number: S2004)
  45. Cellobiose (Sigma-Aldrich, catalog number: C7252 )
  46. Resazurine (Sigma-Aldrich, catalog number: R2127)
  47. L-cysteine (Sigma-Aldrich, catalog number: C1276 )
  48. Thiamin (Sigma-Aldrich, catalog number: T1270 )
  49. Riboflavin (Sigma-Aldrich, catalog number: R9504 )
  50. Gas mix 10% Carbon Dioxide, 10% Hydrogen balance Nitrogen (Anaerobic) Cylinder (BOC, catalog number: 290564-L)
  51. Agar (Oxoid, catalog number: LP0011 )
  52. Resazurin solution (see Recipes)
  53. Anaerobic phosphate buffered saline (PBS) (see Recipes)
  54. Mineral solution 1 (see Recipes)
  55. Mineral solution 2 (see Recipes)
  56. Short chain fatty acid solution (see Recipes)
  57. Vitamin solution 1 (see Recipes)
  58. Vitamin solution 2 (see Recipes)
  59. Haemin solution (see Recipes)
  60. SOC (see Recipes)
  61. LB (see Recipes)
  62. LA (see Recipes)
  63. YCFAGSC and AMM (see Recipes)

Equipment

  1. Pipettes Gilson P1000 (Gilson, catalog number: F123602 )
  2. Pipettes Gilson P200 (Gilson, catalog number: F123601 )
  3. Pipettes Gilson P20 (Gilson, catalog number: F123600 )
  4. Pipettes Gilson P2 (Gilson, catalog number: F144801 )
  5. Concept Plus Anaerobic Workstation, Ruskinn Technology
  6. Hungate tubes (Sciquip, catalog number: 2047-00125)
  7. Hungate lids butyl rubber septa (Sciquip, catalog numbers: 2047-11600 and 2047-16000)
  8. Wheaton bottles (Merck, catalog number: 33110-U )
  9. CO2 hooks (made in-house) and CO2 piped gas supply
  10. Shaking Incubator for E. coli growth (Sanyo Orbital incubator)
  11. Static Incubator for anaerobic bacteria (Sciquip incu-160S)
  12. Gene pulser (Bio-Rad, model: 1652078)
  13. Platform rocker (Stuart, model: STR6)
  14. UV-linker, Bio-Rad GS Gene linker UV chamber (UVP CL-1000 Ultraviolet Crosslinker)
  15. Hybridizer (UVP laboratory products HB 1000 hybridizer)
  16. Blot transfer pump (Hybaid Vacu-aid blot processing pump, DA7C.VAC/T)
  17. Centrifuge (Jouan MR1822)
  18. Transilluminator (UVtec BXT-20.L)

Procedure

  1. Electroporation of plasmids into E. coli CA434
    1. Pre-chill 1 mm electroporation cuvette in refrigerator overnight.
    2. Dilute 1 µl of plasmid solution (10 µg/ml) in 4 µl of dH2O (final concentration 2 µg/ml).
    3. Thaw 55 µl electrocompetent E. coli CA434 (prepared in house following standard protocols, https://www.protocols.io/view/Making-your-own-electrocompetent-cells-imsv6m) on ice and add the plasmid solution, mixing gently.
    4. Transfer the mixture to the cuvette and place in Gene Pulser.
    5. Electroporate at 1.8 kV, 200 Ohms, 25 µF.
    6. Transfer into 1 ml SOC medium, pre-warmed to 37 °C.
    7. Incubate for 1 h at 37 °C, 200 RPM.
    8. Dilute in sterile dH2O in ten-fold dilutions (101, 102 and 103).
    9. Spread 50 µl of these dilutions onto LA plates (see Recipes) supplemented with chloramphenicol 10 µg/ml (Cm10) and incubate at 37 °C for 24 h or until colonies appear.
    10. Pick individual colonies into LB Cm10 and incubate at 37 °C, 200 RPM for 24 h.

  2. Anaerobic culturing of recipient bacterium
    1. Prepare anaerobic media YCFAGSC and AMM in broth as 7.5 ml aliquots in Hungate tubes, sealed with butyl rubber septa and 2% agar solutions divided into 100 ml aliquots in Wheaton bottles, with all dispensing and inoculating carried out under CO2 using the Hungate technique as described by Bryant, 1972 (Figure 1).


      Figure 1. Culturing of strictly anaerobic bacteria using the Hungate methodology (growth in tubes) and anaerobic workstation (growth on Petri plates)

    2. Incubate anaerobic YCFAGSC liquid cultures at 37 °C, without agitation in Hungate tubes.
    3. Pour anaerobic agar media into Petri plate (20 ml per plate) within anaerobic workstation (gas mix CO2:N2:H2, ratio 7:11:2) approximately 24 h before use, enabling agar to adjust to anaerobic atmosphere.
    4. Incubate anaerobic agar cultures at 37 °C in an anaerobic workstation.

  3. Mating of donor E. coli CA434 and recipient bacterium
    1. Prepare 7.5 ml overnight culture of recipient. Donor, recipients and plasmids are described in Table 1.

      Table 1. Strains and plasmids

      *pBP1, pCB102, pCD6 and pIM13 (replicons of these plasmids). ColE1 (Gram-negative replicon), catP (chloramphenicol resistance gene), traJ (origin of transfer) and MCS (multiple cloning site).

    2. Inoculate donor E. coli CA434 in 40 ml of LB Cm10 in 50 ml conical centrifuge tubes and incubate at 37 °C, 200 RPM overnight.
    3. Centrifuge donor overnight culture at 1,200 × g for 10 min.
    4. Decant supernatant and resuspend pellet in 20 ml of anaerobic PBS.
    5. Centrifuge donor again at 1,200 × g for 10min.
    6. Decant supernatant in anaerobic cabinet.
    7. Transfer overnight culture of recipient bacterium (grown as described in Steps B1 and B2) to anaerobic cabinet.
    8. Resuspend donor pellet in 1 ml of recipient overnight culture in anaerobic cabinet.
    9. Spot 100 µl of this solution on the centre of AMM agar plate and incubate in the anaerobic cabinet at 37 °C for 48 h.
    10. Scrape mating mix off centre of AMM plate with sterile loop and suspend in 500 µl of anaerobic PBS.
    11. Spread 50 µl of this solution on to the YCFAGSC plates supplemented with either 5 or 7.5 µg/ml chloramphenicol and incubate anaerobically at 37 °C until colonies appear (usually 48 to 96 h).
    12. Restreak single colonies on fresh YCFAGSC plates supplemented with either 5 or 7.5 µg/ml chloramphenicol and incubate anaerobically at 37 °C until single colonies appear. This may take 3 days incubation.
    13. Diagrammatic representation of the optimised conjugation protocol shown in Figure 2.


      Figure 2. Detailed diagrammatic representation of the optimised conjugation protocol for Roseburia inulinivorans and Eubacterium rectale. Supplemented with 5 or 7.5 µg/ml chloramphenicol (Cm5 or Cm7.5).

  4. Verification of putative transconjugants
    It is good practice to perform various tests to confirm the validity of transconjugants. Some simple tests can readily eliminate bacteria that are not transconjugants, and can save time and expense.
    1. Incubate putative transconjugants aerobically on AMM agar at 37 °C. The recipient bacterium is incapable of aerobic growth while the donor E. coli bacterium will grow.
    2. Gram-stain using standard procedures. Coccus species are common contaminants in gut microbiology and are easily differentiated from the rod-like Roseburia and Eubacterium by Gram-staining.
    3. PCR amplify the 16S rRNA gene directly from colonies or liquid culture using the universal bacterial 16S rRNA gene primers FD1 and RP2 (Table 2) (annealing temp. 52 °C), generating an amplicon of 1,495 bp. PCR amplification conditions are provided in Table 3.

      Table 2. Primers

      Nucleotide code: Guanine (G), adenine (A), thymine (T), cytosine (C), adenine or thymine (W) and guanine or thymine (K)

      Table 3. PCR protocol


    4. Sanger sequence the resulting amplicon with the primers 519R and 926F (Table 2).
    5. BLASTn query each sequence against the NCBI 16S rRNA gene database to confirm identity.
    6. Confirm the presence of the plasmid in putative transconjugants by amplifying a nucleotide sequence common to all of the modular plasmids but absent in the recipient's chromosome. The primers PS#MTL-for and PS#MTL-rev (Table 2) (annealing temp 60 °C) amplify a 514 bp region incorporating sections of catP gene and ColE1 Gram-negative replicon.
    7. Expected results and comments are described in Table 4.

      Table 4. Verification procedures for putative transconjugants


  5. Verification of autonomous plasmid replication by Southern blotting
    1. Extract DNA from transconjugants using the Wizard Genomic DNA Purification kit, following the manufacturer's instructions.
    2. Digest 1 µg of extracted DNA with HindIII for 3 h at 37 °C to produce restriction fragments of various sizes.
      Note: HindIII only cuts the plasmid once, resulting in a single linear fragment, whereas restriction of the genomic DNA results in fragments of various sizes.
    3. Separate restriction fragments by size by gel electrophoresis (0.8% agarose, TBE).
    4. Photograph gel image with transilluminator to facilitate size inference in the final blot.
    5. Gently rock gel in depurination solution (0.25 M HCl) for 7 min and rinse in ddH2O (double distilled water).
    6. Gently rock the gel in denaturation solution (0.5 M NaOH, 1.5 M NaCl) for 30 min three times and rinse in ddH2O.
    7. Gently rock the gel in neutralisation solution (0.5 M Tris-HCl [pH 7.4], 3 M NaCl) for 30 min three times and rinse in ddH2O.
    8. Transfer DNA from gel to nylon membrane using the Hybaid blotter (1.5 h) and UV-crosslinked.
    9. Create probe by digesting pMTL83151 with HindIII and ApaLI. This produces two fragments (~3,000 bp and ~1,500 bp), the smaller of which is specific to a region common to all of the plasmids, but not present in the recipient chromosome. Gel purify the smaller fragment and use as probe template DNA.
    10. Perform Southern blotting with DIG High Prime DNA Labelling and Detection Starter Kit II, following the manufacturer's instructions.
    11. Visualise hybridizing bands by exposing membrane to x-ray film.
    This protocol enables the establishment and verification of autonomously replicating plasmids transferred into Roseburia inulinivorans and Eubacterium rectale and will provide an essential tool in investigating the biochemistry and physiology of these important organisms by genetic manipulation.

Recipes

  1. Resazurin solution
    Add 100 mg of powdered resazurin to 100 ml ddH2O
  2. Anaerobic phosphate buffered saline (PBS)
    1. Dissolve PBS tablets in 1 L of ddH2O and add 1 ml resazurin solution
    2. Place solution in boiling waterbath for 15 min
    3. Bubble solution with 100% CO2 until liquid turns from purple to clear
    4. Dispense in 100 ml aliquots in Wheaton bottles
  3. Mineral solution 1
    K2HPO4 3.0 g
    ddH2O to 1 L
    Store at 4 °C
  4. Mineral solution 2
    KH2PO4 3.0 g
    (NH4)2SO4 6.0 g
    NaCl 6.0 g
    MgSO4 0.6 g
    CaCl2 20.6 g
    ddH2O to 1 L
    Store at 4 °C
  5. Short chain fatty acid solution
    Acetic acid 17 ml
    Propionic acid 6 ml
    n -Valeric acid 1 ml
    Iso-Valeric acid 1 ml
    Iso-Butyric acid 1 ml
    Store at 4 °C
  6. Vitamin solution 1
    Biotin 1 mg
    Cobalamin 1 mg
    p-Aminobenzoic acid 3 mg
    Folic acid 5 mg
    Pyridoxamine 15 mg
    ddH2O to 100 ml
    Store at -20 °C
  7. Vitamin solution 2
    Thiamin 5.0 mg
    Riboflavin 5.0 mg
    ddH2O to 100 ml
  8. Haemin solution
    KOH 0.28 g
    Ethanol 95% 25 ml
    Haemin 100 mg
    ddH2O to 100 ml
    Store at 4 °C
  9. SOC
    Bacto tryptone 2 g
    Yeast Extract 0.5 g
    NaCl 200 μl of 5 M
    KCl 250 μl of 1 M
    ddH2O to 100 ml
    1. Stir to dissolve, autoclave, then cool to room temperature
    2. Add 1 ml of filter sterile 2 M Mg stock solution (1 M MgCl2·6H2O and 1 M MgSO4·7H2O) to give a final conc of 20 mM
    3. Before use add 20 µl of 1 M sterile glucose per 1 ml of SOC
  10. LB
    Bacto tryptone 1 g
    Yeast extract 0.5 g
    NaCl 1 g
    Deionized H2O to 100 ml
  11. LA
    Bacto tryptone 1 g
    Yeast extract 0.5 g
    NaCl 1 g
    Agar 1.5 g
    Deionized H2O to 100 ml
  12. YCFAGSC and AMM
    Bacto casitone 10.0 g
    Yeast extract 2.5 g
    NaHCO3 4.0g
    Glucose 2.0 g
    Soluble starch 2.0 g
    Cellobiose 2.0 g
    Mineral solution 1 150.0 ml
    Mineral solution 2 150.0 ml
    Haemin solution 10.0 ml
    Vitamin solution 1 (before autoclaving) 1.0 ml
    Vitamin solution 2 (after autoclaving) 1.0 ml
    Resazurine 1.0 ml
    L-cysteine 1.0 g
    Acetic acid (0.7 ml) to make AMM or short chain fatty acid solution (3.1 ml) to make YCFAGSC
    Add 20 g of agar to these recipes to make AMM or YCFAGSC agar
    Distilled water up to 1 L

Acknowledgments

The Rowett Institute (University of Aberdeen) receives financial support from the Scottish Government Rural and Environmental Sciences and Analytical Services (RESAS). The protocol is derived from work published in “Heterologous gene expression in the human gut bacteria Eubacterium rectale and Roseburia inulinivorans by means of conjugative plasmids” Anaerobe 59: 131-140 (2019) (Sheridan et al., 2019).

Competing interests

The authors state that there are no competing interests

References

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简介

[摘要 ] 人体肠道菌群中的玫瑰菌属和真细菌属在维持人类健康中起着重要作用,部分原因是产生丁酸盐,这是我们结肠上皮细胞的主要能源。但是,由于缺乏基因操作技术,我们对这些细菌的生物化学和生理学的认识受到限制。先前引入玫瑰花属物种的共轭转座子不容易被修饰,极大地限制了它们作为基因修饰平台的适用性。MOD ular质粒穿梭载体先前已经开发了用于梭菌物种,其与共享一个分类次序ř oseburia 和真杆菌,提高这些矢量可以在这些生物体中使用的可能性。在这里,我们描述了一种优化缀合协议使得能够自主复制的质粒的从转印大肠杆菌供体菌株为罗斯氏inulinivorans 和真杆菌rectale 。质粒的模块性质及其通过自主复制在受体细菌中得以维持的能力使其成为研究异源基因表达的理想之选,并成为其他遗传工具(包括反义RNA沉默或II 型移动子中断子基因破坏策略)的平台。

[背景 ] 玫瑰菌和真细菌属人类肠道菌群中含量最高的细菌(Zhernakova 等,2016),它们通过利用饮食和宿主衍生的多糖影响人类健康(Scott 等,2006和2011; Cockburn 等) 。,2015 ; 谢里登等人,2016 )并产生促进健康的代谢物丁酸作为发酵终产物(邓肯等人,2002和2006) 。另外,这些物种能够通过鞭毛调节宿主免疫(Neville 等,2013)。这些生物缺乏基因修饰技术,无法更全面地了解这些细菌与其人类宿主之间的复杂相互作用。

以前,结合转座子已成功地从纤维素解真杆菌和梭状芽胞杆菌cf. 转移到玫瑰蔷薇中。糖酵解糖(Scott 等,2008)。这些巨大的,新颖的移动遗传元件无法轻易地进行修饰,因此对于详细的遗传修饰而言,它是次佳的平台。然而,这项工作也说明了共轭匹配之间是可能的毛螺菌包括罗斯氏种。用于梭菌属物种的易于修饰的偶联质粒的开发(Purdy 等,2002; Heap 等,2009)增加了将这些技术应用于玫瑰菌和真细菌物种的可能性。

这里介绍的详细协议是基于Sheridan 等人建立的程序。(2019 )。在这项工作中,不同的接合质粒用于在使用中开发梭菌物种(堆等人,2009)对转印到被测试罗斯氏和真杆菌rectale 物种。质粒pMTL83151 被成功转移至的两个菌株E. rectale ,而pMTL83151和pMTL82151转移到罗斯氏 菊糖 A2-194。每个潜在接收者的转移频率为10 -6 -10 -8 。这些频率类似于将自杀载体引入其他革兰氏阳性细菌时观察到的频率(Williams 等,1990 ;Aquino d e Muro和Priest ,2000)。将外源DNA添加到细菌物种的能力为基因操作(包括敲除诱变)打开了机会。备选地,如在数个梭菌物种中已证明的那样,可以将这些质粒修饰为用于可移动的II 族interon 基因破坏策略的表达载体(Heap 等,2007)。此外,显示质粒pMTL83151是一种适合异源基因表达的载体(Sheridan 等,2019),在两种物种中均产生酶促活性链球菌糖苷水解酶,从而证明了该技术在研究人员选择的功能获得性方面的实用性这些重要的细菌。以下方案是将外源DNA引入这些细菌的逐步指南。

关键字:结合, 肠道菌群, 毛螺菌科, 基因转移, 穿梭载体

材料和试剂


 


P ipette提示
Cuvettes(Bio-Rad,目录号:1652083)
培养皿(Greinerbio -one,目录号633180)
玻璃巴斯德移液器(Fisher Scientific,目录号:FB50261)
尼龙膜(罗氏(Roche),目录号:11417240001)
X射线胶片(Fujifilm)(Fisher Scientific,目录号:12735325)
50 ml锥形离心管(Corning,目录号:10038980)
菌株(表1)
质粒(表1)
注意:模块化质粒可以从CHAINbiotools (http://clostron.com/pMTL80000.php)获得。


底漆(表2)
PBS片剂(Sigma,目录号:P4417)
氯苯丙泊酚(Sigma-Aldrich,目录号:C0378)储液10 µg / ml,储存在
-20°C
HindIII 限制性核酸内切酶(NEB,目录号:R0104S)
Wiza rd基因组DNA纯化试剂盒(Promega,目录号:A1120)
DIG High Prime DNA标记和检测入门工具包II(Roche ,目录号:11585614910)
PCR试剂(的Taq 聚合酶试剂盒生物在线,目录号:BIO-21040和的dNTP Promega公司,Ç atalog号:U1240)
磷酸氢二钾,K 2 HPO 4 (Fisher Scientific,目录号:P / 5240/53)
磷酸二氢钾,KH 2 PO 4 (Fisher Scientific,目录号:P / 4800/60)
硫酸铵(NH 4 )2 SO 4 (Fisher Scientific,目录号:A / 6480/53)
氯化钠,NaCl(Fisher Scientific ,目录号: S / 3160/53)
硫酸镁MgSO 4 (Sigma-Aldrich,目录号:M7506)
氯化钙C aCl 2 (Sigma-Aldrich,目录号:C1016)
醋酸(Fisher Scientific,目录号:A / 10400 / PB17)
丙酸(Sigma-Aldrich,目录号:P1386)
正戊酸(Sigma-Aldrich,目录号:V9759)             
异戊酸(Sigma-Aldrich,目录号:I7128)
异丁酸(Sigma-Aldrich,目录号:I1754)
生物素(Sigma-Aldrich,目录号:B4501)
钴胺素(Sigma-Aldrich,目录号:V2876)
对氨基苯甲酸(Sigma-Aldrich,目录号:A9878)
叶酸(西格玛奥德里奇,产品目录号:F7876)
吡rid胺(Sigma-Aldrich,目录号:P9755)
氢氧化钾,KOH(Sigma-Aldrich,目录号:P5958)
乙醇95%(Fisher Scientific ,目录号:E / 0650DF / 17)
Haemin (Sigma-Aldrich,目录号:H5533)
Bacto 胰蛋白((BD诊断系统,目录号:211705)
酵母提取物(BD诊断系统,目录号:212750)
氯化钾,KCl (Sigma-Aldrich,目录号:P3911)
六水合氯化镁,MgCl 2 ·6H 2 O(Sigma -Aldrich,目录号:M9272 )
七水硫酸镁,M gSO 4 ·7H 2 O(Sigma-Aldrich,目录号:230391)
Bacto casitone (BD Diag nostics Systems,目录号:225930)
碳酸氢钠NaHCO 3 (Sigma-Aldrich,目录号:S576 1 )
葡萄糖(Fisher Scientific ,目录号:G / 0500/53)
可溶性淀粉(Sigma-Aldrich,目录号:S2004)
纤维二糖(Sigma-Aldrich,目录号:C7252)
刃天青(Sigma-Aldrich,目录号:R2127)
L-半胱氨酸(Sigma-Aldrich,目录号:C1276)
硫胺(Sigma-Aldrich,目录号:T1270)
核黄素(Sigma-Aldrich,目录号:R9504)
混合气10%二氧化碳,10%氢气平衡氮气(厌氧)气瓶(BOC,货号:290564-L)
琼脂(Oxoid ,目录号LP0011)
刃天青溶液(参见配方)
厌氧磷酸盐缓冲盐水(PBS)(请参阅食谱)
矿物质溶液1(请参阅食谱)
矿物质溶液2(请参阅食谱)             
短链脂肪酸溶液(请参阅食谱)
维生素溶液1(请参阅食谱)
维生素溶液2 (请参阅食谱)
海敏解决方案(请参阅食谱)
SOC(请参阅食谱)
LB (请参阅食谱)
洛杉矶(请参阅食谱)
YCFAGSC和AMM (请参阅食谱)
 


设备


 


移液器Gilson P1000(Gilson,目录号:F123602)
移液器Gilson P200(Gilson,目录号:F123601)
移液器Gilson P20(Gilson,目录号:F123600)
移液器Gilson P2(Gilson,目录号:F144801)
Ruskin n Technology的Concept Plus厌氧工作站
的Hungate 管(Sciquip ,目录号:2047-00125)
饥饿的盖子丁基橡胶隔垫(Sciquip ,目录号s :2047-11600和2047-16000)
惠顿瓶(默克(Merck),货号:33110-U)
CO 2 钩(内部制备)和CO 2 p IPED气体供给
摇动大肠杆菌生长培养箱(三洋轨道培养箱)
厌氧菌静态培养箱(Sciquip incu-160S)
基因脉冲发生器(Bio - R ad,型号:1652078)
平台摇臂(Stuart,型号:STR6)
紫外线接头,Bio - R 和GS基因接头紫外线室(UVP CL-1000紫外线交联剂)
杂交仪(UVP实验室产品HB 1000杂交仪)
印迹转移泵(Hybaid Vacu 辅助印迹处理泵,DA7C.VAC / T)
离心机(Jouan MR1822)
透(UVtec BXT-20.L)
 


程序


 


将质粒电穿孔到大肠杆菌CA434中
将1毫米电穿孔试管在冰箱中预冷过夜。
在4 µl dH 2 O(终浓度2 µg / ml)中稀释1 µl质粒溶液(10 µg / ml)。
在冰上解冻55 µl具有电感受态的大肠杆菌CA434(按照标准协议在室内制备,https: //www.protocols.io/view/Making-your-own-electrocompetent-cells-imsv6m ),然后加入质粒溶液,轻轻混合。
将混合物转移到比色杯中,并置于Gene Pulser中。
在1.8 kV,200欧姆,25 µF时进行电穿孔。
转移至1 ml SOC介质中,预热至37°C。
在37°C,200 RPM下孵育1小时。
稀的无菌卫生署2 在10倍稀释ø (10 1 ,10 2 和10 3 )。
将这些稀释液的50 µl 分散在补充有10 µg / ml氯霉素(Cm10)的LA平板上(请参阅R ecipes),并在37°C下孵育24 h或直至出现菌落。
将单个菌落挑入LB Cm10中,并在37°C,200 RPM下孵育24 h 。
 


受体细菌的厌氧培养
将肉汤中的厌氧培养基YCFAGSC和AMM制成7.5毫升等分试样在Hungate 管中,用丁基橡胶隔垫和2%琼脂溶液密封,在Wheaton瓶中分成100毫升等分试样,所有分配和接种均采用Hungate 技术在CO 2 下进行于1972年由科比(Bryant)所描述(图1)。
 


D:\ Reformatting \ 2020-3-2 \ 1902849--1348 Karen Scott 810703 \ Figs jpg \ figure 1 .jpg


图1. 使用Hungate方法(管中生长)和厌氧工作站(在Petri板上生长)培养严格厌氧的细菌


 


厌氧孵育在37℃下YCFAGSC液体培养物,在不搅拌的Hungate 管中。
在使用前大约24小时,将厌氧琼脂培养基倒入厌氧工作站(混合气体CO 2 :N 2 :H 2 ,比率7:11:2)内的陪替氏培养皿中(每板20 ml),使琼脂适应厌氧气氛。
在厌氧工作站中于37°C孵育厌氧琼脂培养物。
 


马丁克供体的E. 大肠杆菌CA434和受体细菌
准备7.5毫升受体的过夜培养。表1描述了供体,受体和质粒。
 


 


 


表1.菌株和质粒


菌株/质粒


相对特征


来源/参考


菌株


 


 


真杆菌rectale A1-86


丁酸产生,严格的的厌氧菌毛螺法米尔ÿ 。


(Barcenilla et al。,2000)


真杆菌rectale T1-815


(Barcenilla et al。,2000)


玫瑰蔷薇A2-194


(Duncan et al。,2006)


真杆菌rectale EAM3


E. rectale A1-86 窝藏pMTL83151


(Sheridan et al。,2019)


真杆菌rectale ETM3


E. rectale T1-815 窝藏pMTL83151


(Sheridan et al。,2019)


真杆菌rectale ETBglu


E. rectale T1-815 窝藏pMTL3β- 谷氨酸


(Sheridan et al。,2019)


玫瑰蔷薇(Roseburia inulinivorans)RAM2


R. inulinivorans A2-194 窝藏pMTL82151


(Sheridan et al。,2019)


玫瑰蔷薇(Roseburia inulinivorans)RAM3


R. inulinivorans A2-194 窝藏pMTL83151


(Sheridan et al。,2019)


大肠杆菌CA434


共轭捐献者。基因型:HB101(THI-1 hsdS20(R- 乙,间- 乙)supE44 利甲ARA-14 leuB5proA2 lacY1的的galK rpsL20 (STR - [R )XYL-5 MTL-1 )携带R701(茶+,MOB +接合质粒)


(Williams 等,1990)


质粒*


 


 


pMTL82151


ColE1,cat P ,tra J ,MCS和pBP1


(Heap 等,2009)


pMTL83151


ColE1,cat P ,tra J ,MCS和pCB102


(Heap 等,2009)


* pBP1,pCB102,pCD6和pIM13(这些质粒的复制子)。ColE1(革兰氏阴性复制子),catP (氯霉素抗性基因),traJ (转移起点)和MCS(多个克隆位点)。


 


Inoculat Ë施主E. 大肠杆菌CA434在40毫升LB CM10的50 毫升锥形离心管中,并培育在37℃,200 RPM过夜。
离心供体以1200 × g 过夜培养10分钟。
倒出上清液,并将沉淀重悬于20 ml厌氧PBS中。
再次以1200 × g的速度离心供体10分钟。
在厌氧柜中倒出上清液。
将受体细菌的过夜培养物(按步骤B1和B 2 所述生长)转移到厌氧柜中。
将供体沉淀物在厌氧柜中的1 ml受体过夜培养物中重悬。
在AMM琼脂平板的中心点取100 µl该溶液,并在37°C的厌氧箱中孵育48 h。
              用无菌环将AMM板中心的混合混合物刮擦掉,并悬浮在500 µl厌氧PBS中。
              将50 µl此溶液铺在添加了5或7.5 µg / ml氯霉素的YCFAGSC平板上,并在37°C下厌氧孵育直至出现菌落(通常48至96 h)。
Restreak 唱在补充有5或7.5新鲜YCFAGSC板乐菌落微克/ ml氯霉素和孵化厌氧在37℃至单菌落出现。孵育可能需要3天。
所述的图解表示优化在图2中所示的缀合协议。
 


D:\ Reformatting \ 2020-3-2 \ 1902849--1348 Karen Scott 810703 \ Figs jpg \ figure 2 .jpg


图2 详细的的示意图优化用于缀合协议罗斯氏inulinivorans 和真杆菌rectale 。补充5或7.5 µg / ml氯霉素(Cm5或Cm7.5)。


 


验证推定的共轭物
优良作法是进行各种测试以确认转导结合剂的有效性。一些简单的测试可以轻松消除不是转导结合体的细菌,并且可以节省时间和费用。


在37°C的AMM琼脂上有氧孵育假定的转导结合剂。受体细菌不能有氧生长,而供体大肠杆菌将生长。
使用标准程序进行克染色。球菌是肠道微生物学中的常见污染物,通过革兰氏染色很容易将其与杆状Roseburia 和真细菌区分开来。
PCR扩增直接16S rRNA基因从使用通用细菌16S rRNA基因的引物和FD1 RP2(表2)(退火温度菌落或液体培养。52℃),产生1的扩增子,495 bp的。表3提供了P CR扩增条件。
 


表2.底漆


底漆


顺序(5'-3')


目标


参考


PS#MTL-用于


TATCTATGATACCGTGGTCAAC


pMTL80000系列质粒


(Sheridan et al。,2019)


PS#MTL-rev


CTGCTGAAGCCAGTTACC


pMTL80000系列质粒


(Sheridan et al。,2019)


FD1


AGAGTTTGATCCTGGCTCAG


完整的16S rRNA基因


(Wood 等,1998)


RP2


ACGGCTACCTTGTTACGACTT


完整的16S rRNA基因


(Wood 等,1998)


519R


GWATTACCGCGGCKGCTG


16S rRNA基因(通用)


(Turner 等,1999)


926F


ACTCAAAGGAATTGACGG


16S rRNA基因(通用)


(Muyzer et al。,1995)


核苷酸代码:鸟嘌呤(G),腺嘌呤(A),胸腺嘧啶(T),胞嘧啶(C),腺嘌呤或胸腺嘧啶(W)和鸟嘌呤或胸腺嘧啶(K)


 


表3. PCR方案


 


温度


时间


初始变性


94°摄氏度


5分钟


25个循环


95°摄氏度


30秒


取决于底漆


30秒


72°摄氏度


2分钟


最后扩展


72°摄氏度


8分钟


保持


4 °摄氏度


 


 


用引物519R和926F对所得扩增子进行Sanger测序(表2)。
BLASTn 针对NCBI 16S rRNA基因数据库查询每个序列,以确认身份。
通过扩增所有模块化质粒共有但在受体染色体中不存在的核苷酸序列,确认推定的转接合子中质粒的存在。PS#MTL-for和PS#MTL-rev引物(表2)(退火温度60°C)可扩增一个514 bp的区域,其中包含cat P 基因和ColE1革兰氏阴性复制子的片段。
预期结果和评论在表4中描述。
 


 


 


 


表4.假定的超偶联物的验证程序


 


确认书


预期结果


评论


革兰氏染色


没有污染


粉色或紫色杆菌


革兰氏染色很容易将常见的污染物(例如球菌)与玫瑰菌和真细菌区分开。


16S rRNA基因测序


没有污染


与受体DNA匹配


 


质粒特异性PCR


质粒的存在


514 bp扩增子


收件人染色体缺少DNA的扩增区域


无氧培养


捐助者缺席


无氧生长


收件人需氧菌不能有氧生长


南方印迹


自主复制


与线性质粒大小相同的单条带


染色体插入会改变探针杂交带的大小


 


通过Southern印迹验证自主质粒复制
按照制造商的说明,使用向导基因组DNA纯化试剂盒从转导结合物中提取DNA。
在37°C下用HindIII 消化1 µg提取的DNA 3小时,以产生各种大小的限制性片段。
注意:HindIII 仅将质粒切割一次,得到一个线性片段,而对基因组DNA的限制则产生了各种大小的片段。


通过凝胶电泳按大小分离限制性片段(0.8%琼脂糖,TBE)。
用透照器为凝胶图像拍照,以促进最终印迹中的大小推断。
在去纯化溶液(0.25 M HCl)中轻轻摇动凝胶7分钟,并在ddH 2 O(双蒸馏水)中冲洗。
在变性溶液(0.5 M NaOH,1.5 M NaCl)中轻轻摇动凝胶30分钟三遍,并在ddH 2 O中冲洗。
在中和溶液(0.5 M Tris-HCl [ pH 7.4 ] ,3 M NaCl)中轻轻摇动凝胶30分钟,三遍,并在ddH 2 O中冲洗。
使用Hybaid印迹仪(1.5 h)和UV交联将DNA从凝胶转移到尼龙膜上。
通过用HindIII 和ApaLI 消化pMTL83151来创建探针。这将产生两个片段(〜3 ,000 bp和〜1 ,500碱基对),其中较小的特定于区域共用于所有的质粒的,但非p 重新发送在受体染色体。凝胶纯化较小的片段,并用作探针模板DNA。
按照制造商的说明,使用DIG High Prime DNA标记和检测入门工具包II进行Southern印迹分析。
通过将膜暴露于X射线胶片来可视化杂交带。
 


该协议使得能够自主复制转移到质粒中的建立和验证罗斯氏inulinivorans 和真杆菌rectale ,将在通过遗传操作调查这些重要生物体的生物化学和生理学提供的必要工具。


 


菜谱


 


刃天青溶液
将100 mg刃天青粉加入100 ml ddH 2 O中


厌氧磷酸盐缓冲盐水(PBS)
将PBS片剂溶于1 L的ddH 2 O中,并加入1 ml刃天青溶液
将溶液放入沸水浴中15分钟
100%CO 2的气泡溶液,直到液体从紫色变为澄清
在Wheaton瓶中分装100毫升等分试样
矿物质溶液1
K 2 HPO 4 3.0克                                                                                                                                                                                                                                                           


ddH 2 O 至1 L                                                       


存放在4 °C


矿物质溶液2             
KH 2 PO 4 3.0克                                                                                                 


(NH 4 )2 SO 4 6.0克                           


氯化钠6.0克                                         


硫酸镁4 0.6克                           


氯化钙2 2 0.6 g                                         


ddH 2 O 至1 L                                                                                                                                           


储存在4 °C


短链脂肪酸溶液
醋酸17毫升                           


丙酸6毫升                                                       


正戊酸1毫升                           


异戊酸1毫升             


              异丁酸1毫升                           


储存在4 °C


维生素溶液1
生物素1毫克                                                       


钴胺素1毫克                                         


对氨基苯甲酸3 mg             


叶酸5毫克                                         


吡rid胺15毫克                           


ddH 2 O 至100 ml                                                       


储存在-20 °C


维生素溶液2
硫胺素5.0毫克


核黄素5.0毫克


ddH 2 O 至100 ml                                                       


海敏解决方案
KOH 0.28克                                         


乙醇95%25毫升                           


Haemin 100毫克


ddH 2 O 至100 ml             


储存在4 °C


SOC
Bacto 胰蛋白2 2克


酵母提取物0.5克


氯化钠200μ升的5M的


氯化钾250 μ 升1M的


ddH 2 O至100 ml


搅拌溶解,高压灭菌,然后冷却至室温
加入1 ml无菌过滤器2 M Mg储备溶液(1 M MgCl 2· 6H 2 O和1 M MgSO 4 ·7H 2 O )的最终浓度为20 mM
使用前,每1毫升SOC 添加20微升1 M无菌葡萄糖

Bacto 胰蛋白1 1克


酵母提取物0.5克


氯化钠1克


Deioni ž 编ħ 2 ö 至100毫升


洛杉矶
Bacto 胰蛋白1 1克


酵母提取物0.5克


氯化钠1克                           


琼脂1.5克


Deioni ž 编ħ 2 ö 吨ö百毫升


YCFAGSC和AMM
细菌培养用酪蛋白胨10.0克              


酵母提取物2.5克             


NaHCO 3 4.0 克              


葡萄糖2.0克             


可溶性淀粉2.0克             


纤维二糖2.0克             


矿物质溶液1 150.0毫升             


矿物质溶液2 150.0毫升


海敏溶液10.0毫升             


维生素溶液1(高压灭菌前)1.0毫升             


维生素溶液2(高压灭菌后)1.0毫升             


刃天青1.0毫升             


L-半胱氨酸1.0克             


用醋酸(0.7 ml)制成AMM或用短链脂肪酸溶液(3.1 ml)制成YCFAGSC             


在这些食谱中添加20克琼脂,制成AMM或YCFAGSC琼脂


蒸馏水至1 L             


 


致谢


 


该Rowett 研究所(阿伯丁大学)接收来自苏格兰政府农村和环境科学和分析服务(RESAS)的资金支持。该协议是由“异源基因表达在人肠道细菌发表在工作衍生真杆菌rectale 和罗斯氏inulinivorans 通过接合质粒的手段”厌氧菌59:131-140(2019) (谢里登等人,2019 )。


 


利益争夺


 


作者指出没有利益冲突


 


参考文献


 


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引用:Sheridan, P. O., Martin, J. C. and Scott, K. P. (2020). Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale. Bio-protocol 10(7): e3575. DOI: 10.21769/BioProtoc.3575.
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