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Feb 2020

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TetR Regulated in vivo Repression Technology to Identify Conditional Gene Silencing in Genetically Engineerable Bacteria Using Vibrio cholerae Murine Infections as Model System
以小鼠霍乱弧菌感染为模型系统的TetR调控体内抑制技术鉴定基因工程菌的条件基因沉默   

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Abstract

Investigation of bacterial gene regulation upon environmental changes is still a challenging task. For example, Vibrio cholerae, a pathogen of the human gastrointestinal tract, faces diverse transient conditions in different compartments upon oral ingestion. Genetic reporter systems have been demonstrated to be extremely powerful tools to unravel gene regulation events in complex conditions, but so far focused mainly on gene induction. Herein, we describe the TetR-controlled recombination-based in vivo expression technology TRIVET, which allows detection of gene silencing events. TRIVET resembles a modified variant of the in vivo expression technology (IVET) as well as recombination-based in vivo expression technology (RIVET), which were used to identify conditional gene induction in several bacteria during host colonization. Like its predecessors, TRIVET is a single cell based reporter system, which allows the analysis of bacterial gene repression in a spatiotemporal manner via phenotypical changes in the resistance profile. Briefly, a promoterless tetR (encoding the transcriptional repressor TetR) can be integrated randomly into the bacterial genome via transposon mutagenesis or site-specific downstream of a promoter of interest via homologous recombination. Reduction of transcriptional expression of TetR results in a de-repression of the TetR-controlled resolvase TnpR, which in turn leads to excision of an antibiotic resistance cassette (also known as res-cassette) and altered resistance profile observable via streaking on ampicillin and kanamycin plates. This alteration can then be quantified as the ratio between resistant and non-resistant isolates. Furthermore, the newly introduced second reporter gene, a promoterless phoA (encoding the alkaline phosphatase PhoA) offers an additional validation step of the results via an independent colorimetric assay to measure enzyme activity. The protocol presented herein also offers an approach to identify the gene locus in case of the random screen for gene repression as well as a quantification of the conditional repression of a gene of interest. Although the current protocol is established for gene repression during host colonization, it can likely be adapted to study gene silencing under various conditions faced by a bacterium.

Keywords: Gene expression (基因表达), Resolution (分辨率), Mouse (小鼠), Repression (抑制), Screen (筛选), Regulation (调节)

Background

Facultative bacterial pathogens constantly need to adapt to varying conditions during environmental passages and host colonization. Optimal survival fitness is assured by transient activation and repression of numerous genes. Unravelling these adaptation processes is key to understand bacterial physiology and identify potential targets for therapeutic intervention strategies. Diverse techniques have been established to study gene expression profiles in bacteria, including microarrays, RNA-Seq and qRT-PCR (Eisen and Brown, 1999; Bookout and Mangelsdorf, 2003; Wang et al., 2009). Such methods have to deal with the consequences of averaging heterogeneity in the bacterial population and only reflect transcription level at the time-point of harvest. Any unique patterns of gene expression related to specific regions in a subpopulation or transient regulation might be lost. Moreover, the requirement of relatively high quantity of RNA and purity restricts the above-mentioned technologies. In contrast, the TRIVET reporter system allows the detection of transient gene silencing events even in a subset of a complex, heterogenous bacterial population. TRIVET is a robust technique due to an irreversible change in the antibiotic resistance profile on a single cell level. On the contrary, TRIVET requires substantial, labor-intensive genetic engineering of the model organism and is therefore limited to bacteria with available genome sequence and tools for genetic modification.

Using the facultative human pathogen Vibrio cholerae as a representative example, we describe two potential applications of TRIVET: (i) a random approach to identify in vivo repressed genes during intestinal colonization in the murine model and (ii) a specific approach to study conditional transcriptional silencing of a gene of interest (Cakar et al., 2018; Zingl et al., 2020). The TRIVET system consists of three chromosomal elements, i.e., a tetR-phoA-cat (tpc) reporter cassette, a suicide vector system pTRIVET providing the TetR-controlled resolvase TnpR and the resolution (res)-cassette as target for TnpR.

The res-cassette of TRIVET is identical to the res-cassette of RIVET (Osorio et al., 2005) and is integrated in the lacZ-locus of the V. cholerae chromosome. The two selection markers [neo(KmR) and sacB (SucS)] are flanked by target sites for the TnpR resolvase also known as the res-sites.

The second part is the tpc-cassette. For the random approach, the tpc-cassette is subcloned between the IS10 sites of the mini Tn10-system using the pLOF vector delivery system (Herrero et al., 1990). Noteworthy, tetR and phoA encoding the TetR repressor and the alkaline phosphatase are promoterless, while the cat cassette was subcloned with its own constitutive promoter to allow selection of transposon mutants. Thus, transposon mutagenesis of res-cassette containing strains following chloramphenicol selection, generates random insertions of the tpc reporter cassette. A sub-population of these transposon mutants harbor transcriptional fusions of chromosomal genes to tetR and phoA and will express these genes upon activation of the respective promoter.

The third component of TRIVET is the pTRIVET suicide plasmid (ApR) containing an 800 bp intergenic region downstream of the lacZ locus and the tnpR (encoding the resolvase). Importantly, the promoter responsible for tnpR expression originates from the tetracycline-resistance gene tetA and is therefore tightly controlled by TetR. Mobilization of this pTRIVET into a res- and tpc-cassette containing V. cholerae strain results in homologous recombination downstream of lacZ in the genome. As the integration site is located downstream of the intrinsic transcriptional terminator of lacZ, undesired read-throughs of the RNA-Polymerase towards tnpR are prevented. Hence, the expression of tnpR solely relies on the TetR-controlled promoter. A comprehensive description of the system including a schematic overview of the genetic elements can be found in a recent publication by Cakar et al. (2019). In strains with tetR expression via the tpc-cassette, repression of tnpR will result in stable res-cassette containing strains, which can be isolated via their unique resistance profile (ApR, KmR and SucS). In contrast, no expression of tetR leads to induction of tnpR resulting in excision and irreversible loss of the res-cassette (ApR, KmS and SucR). This event is called resolution and results in a resolved strain. Thus, a relatively simple KmR-selection will identify strains with sufficient TetR expression with stabilized res-cassette. These strains can be used to infect infant mice. During in vivo colonization, silencing of the chromosomal promoter driving the expression of tetR may occur, which results in an induction of tnpR leading to excision and irreversible loss of the res-cassette. Hence, the loss of the res-cassette during in vivo colonization can be monitored by a phenotypic change to KmS/SucR, allowing the subsequent identification of these resolved strains similar to the original RIVET screens. Besides tetR, phoA acts as an independent transcriptional reporter and provides a refinement of the in vivo resolved strains via an alkaline phosphatase activity assay.

In case of gene specific approach, TRIVET uses the identical res-cassette and pTRIVET elements, while the tpc-cassette needs to be subcloned into the pCVD442 suicide vector flanked by up- and downstream regions of the desired integration site to ensure homologous recombination (Donnenberg and Kaper, 1991). Once the tpc-cassette has been fused to the chromosomal promotor of interest, the res-cassette and the pTRIVET can be subsequently mobilized and integrated into the genome. Such strains harboring a tpc-cassette downstream of a promotor of interest can be used to assay the repression of each fusion under specific conditions (e.g., in vitro and in vivo as described for the yrb-fusion strain). For example, the amount of resolution upon in vitro and in vivo cultivation can be determined by plating appropriate dilutions on LB-Kn (unresolved CFUs) and LB-Ap (total CFUs) plates. The resolved CFUs can be calculated by subtracting total unresolved CFUs from the total CFUs [(ApR CFU) minus (KmR CFU)]. The extent of resolution is given by the resolution frequency (% resolution), calculated as the amount of resolved CFUs divided by total CFUs multiplied by 100. If applicable, phoA acts as an independent transcriptional reporter and allows an additional assessment of the transcriptional activity of the tpc-fusion strain via an alkaline phosphatase assay, which will be described herein for the irgA-fusion strain under iron-replete and iron-deplete conditions.

Although originally designed to identify gene repression during intestinal colonization, TRIVET can be likely adapted to diverse cultivation conditions (Seper et al., 2014; Cakar et al., 2018; List et al., 2018). This technology can thus help to expand our knowledge of bacterial gene regulation, adaptation processes and the regulatory cascades involved.

Materials and Reagents

  1. Construction of the TRIVET System
    1. Bacterial strains and plasmids
      1. Escherichia coli DH5αλpir F endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169 hsdR17(rK mK+) λpirRK6 (54)
      2. E. coli XL-1 F′::Tn10 proA + B + lacq Δ(lacZ)M15/recA1 endA1 gyrA46 (Nalr) thi hsdR17 (rK mK+) supE44 relA1 lac (New England Biolabs)
      3. E. coli SM10λpirthi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu λpirRK6 (Miller and Mekalanos, 1988)
      4. V. cholerae WT spontaneous streptomycin resistant (SmR) mutant of E7946 (O1 El Tor Inaba), SmR (Miller et al., 1989)
      5. pCVD442 Suicide vector, OriR6K, sacB, ApR (Donnenberg and Kaper, 1991)
      6. pA C1000 CmR (Hava et al., 2003)
      7. pGOA1193 pIVET5n tnpR, oriR6K mobRP4 lacZ tnpR, ApR (Osorio et al., 2005)
      8. pGP704 oriR6K mobRP4, ApR (Miller and Mekalanos, 1988)
      9. pTrc99A-Km pBR322 origin, KmR (Amann et al., 1988)
      10. pRR51 (Reed and Grindley, 1981)
      11. pSL134 pPCR-Script::(res1-res1) (Osorio et al., 2005)
      12. pPCR-Script Amp SK(+) (Stratagene)
      13. pGAO2 [pPCR-Script::(res) (Osorio et al., 2005)]
      14. pGAO3 [pPCR-Script::(res1) (Osorio et al., 2005)]
      15. pGAO4 [pPCR-Script::(res-res) (Osorio et al., 2005)]
      16. pGAO5 [pPCR-Script::(res1-res1) (Osorio et al., 2005)]
      17. pGAO6 [pGOA4::neo-sacB (Osorio et al., 2005)]
      18. pGAO7 [pGOA5::neo-sacB (Osorio et al., 2005)]
      19. pRes pSL111 with res-neo-sacB-res cassette, KmR (Osorio et al., 2005)
      20. pRes1 pSL111 with res1-neo-sacB-res1 cassette KmR (Osorio et al., 2005)
      21. pLOFKm Tn10 based delivery plasmid, KmR, ApR (Herrero et al., 1990)
    2. Oligonucleotides
      1. tetR-5′-BamHI AATGGATCCTAGAGTGTCAACAAAAATTAGGAATTA
      2. tetR-3′-Kpnl TTAGGTACCATCACGGAAAAAGGTTATGCT
      3. phoA-5′-KpnI TTTGGTACCTTTTTAATGTATTTGTACATGGAGAA
      4. phoA-3′-XbaI AAATCTAGACATTAAGTCTGGTTGCTAACAGCA
      5. cat-5′-XbaI AAATCTAGATAAGCTTGATGAAAATTTGTTTGA
      6. cat-3′-BamHI TTTGGATCCTTCTTCAACTAACGGGGCA
      7. tetRphoAcat-5′-NotI TATGCGGCCGCCCTAGGTAATTAGGATCCTAGAGT
      8. tetRphoAcat-3′-NotI TTTGCGGCCGCCCTAGGTCTCATCCGCCAAAACAGCCAA
      9. lacZ-5′-SacI TTTGAGCTCTGATTTACCGCCGCTGCCAA
      10. lacZ-3′-NheI TTTGCTAGCTTATTGTGGGTGATGACGCTTT
      11. tnpR-5′-BgIII CGACCCGGGAGATCTCAATTGTTCGAATTTAGGATACATTTTTAT
      12. tnpR-3′-XbaI TTTTCTAGATTAAGTTGGGTAACGCCAGGGT
      13. PtetA-5′-3′ CTAGCCAGAGAGCCTTAAGGCTCTCTTTTTTCTAATTTTTGTTGACACC
        CTATCAGTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGA
      14. PtetA-3′-5′ GATCTCTATCACTGATAGGGAGTGGTAAAATAACTCTATCACTGATAGG
        GTGTCAACAAAAATTAGAAAAAAGAGAGCCTTAAGGCTCTCTG G
      15. Ptet1-5′-3′ CTAGCAGAGAGCCTTAAGGCTCTCTTTTTTCTAATTTTTGTCCCTATCA
        GTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCA
      16. Ptet1-3′-5′ GATCTGATGTGCTCAGTATCTCTATCACTGATAGGGATGTCAATCTCTAT
        CACTGATAGGGACAAAAATTAGAAAAAAGAGAGCCTTAAGG CTCTCTG
      17. 1RES-F TCTATTGAATTCCGTCCGAAATATTATAAATTATCGCAC
      18. 1RES-R TCTAATCTCGAGTGTATCCTAAATCAAATATCGGACAAG
      19. 1RES1-F TCTAATGAATTCCGTCCGAAATATTACAAATTATCGCAC
      20. 2RES-F TCTAATCTCGAGTCTAGACGTCCGAAATATTATAAATTATCGCAC
      21. 2RES1-F TCTAATCTCGAGTCTAGACGTCCGAAATATTACAAATTATCGCAC
      22. 2RES-R TCTAATGGTACCTGTATCCTAAATCAAATATCGGACAAG
      23. T3 AATTAACCCTCACTAAAGGG
      24. T7 AATACGACTCACTATAGGGC
    3. Materials
      1. Pipette tip (Greiner Bio-One, catalog numbers: 740290 , 739290 and 771291)
      2. PCR tubes (Thermo Fisher Scientific, catalog number: AB0266 )
      3. Double distilled H2O (ddH2O)
      4. dNTPs, 10 mM (Thermo Fisher Scientific, catalog number: R0192 )
      5. DNA Loading Dye (6x) (Thermo Fisher Scientific, catalog number: R0611 )
      6. Q5 High-Fidelity DNA Polymerase (NEB, catalog number: M0491S )
      7. Taq DNA polymerase (NEB, catalog number: M0273S )
      8. GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific, catalog number: SM1331 )
      9. QIAquickGel Extraction Kit (QIAGEN, catalog number: 28704 )
      10. QIAquickPCR Purification Kit (QIAGEN, catalog number: 28104 )
      11. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
      12. Cut smart buffer (NEB, catalog number: B7204S )
      13. Appropriate Restriction Enzymes (NEB)
      14. Antarctic phosphatase (NEB, catalog number: M0289S )
      15. T4 DNA ligase (NEB, catalog number: M0202S )
      16. T4 DNA ligase buffer (NEB, catalog number: B0202S )
      17. Glycerol (AppliChem, catalog number: A0970 ,1000)
      18. Yeast extract (Applichem, catalog number: A1552 )
      19. Bacto Tryptone (Thermo Fisher Scientific, catalog number: 211701 )
      20. NaCl (VWR, catalog number: 27810.364 )
      21. Agar (Sigma-Aldrich, catalog number: A5054 )
      22. Appropriate antibiotic for the plasmid vector (e.g., Ampicillin, Carl Roth, catalog number: K029.2 )
      23. LB agar plates containing the appropriate antibiotic for the plasmid vector (see Recipes)
      24. LB medium (see Recipes)

  2. Construction of the TRIVET library
    1. Bacterial strains and plasmids
      1. E. coli DH5αλpir F endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169 hsdR17(rK mK+) λpirRK6
      2. E. coli SM10λpir thi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu λpirRK6
      3. V. cholerae WT spontaneous streptomycin resistant (SmR) mutant of E7946 (O1 El Tor Inaba), SmR (Miller et al., 1989)
      4. pCVD442 Suicide vector, OriR6K, sacB, ApR (Donnenberg and Kaper, 1991)
      5. pRes pSL111 with res-neo-sacB-res cassette, KmR (Osorio et al., 2005)
      6. pRes1 pSL111 with res1-neo-sacB-res1 cassette, KmR (Osorio et al., 2005)
      7. pLOF::tpc (Cakar et al., 2018)
      8. pTRIVET (Cakar et al., 2018)
      9. pTRIVET1 (Cakar et al., 2018)
      10. pRES [pSL111::(res-neo-sacB-res) (Osorio et al., 2005)]
      11. pRES1 [pSL111::(res1-neo-sacB-res1) (Osorio et al., 2005)]
    2. Oligonucleotides
      1. tetRphoAcat-5′-SacI ATAGAGCTCAGAGTGTCAACAAAAATTAGGAATTAA
      2. tetRphoAcat-3′-SalI TTTGTCGACTTCTTCAACTAACGGGGCA
      3. irgA-SphI-1 AATGCATGCTCCGAGTAAACCGCAAACACTT
      4. irgA-SacI-2 AATGAGCTCGTATCCCGCCGCAGTGACCA
      5. irgA-SalI-3 AAAAGTCGACCGAACTATTCCATGTGT
      6. irgA-XbaI-4 AATTCTAGACGTGAGGTTTGGCGCTTA
      7. yrb_SacI_1 AAAGAGCTCGCGATATTGGCAATGTTTGAAC
      8. yrb_SphI_2 AAAGCATGCGATAAGGATAATTAATTGGAATC
      9. yrb_SalI_3 AAAGAATTCTCAGGATGTCGACCTAACAG
      10. yrb_XbaI_4 TTTTCTAGAGATTAAGGTTACGGATCAGTTC
      11. seqPrimer-cat GAATTGTCAGATAGGCCTAATG
    3. Materials
      1. PCR tubes (Thermo Fisher Scientific, catalog number: AB1771 )
      2. Needle 26G (B. Braun, catalog number: 4657683 )
      3. Gavage tubing (Thermo Scientific, catalog number: 10793527 )
      4. Gavage syringe 1 ml (Henry Schein, catalog number: 9003016 )
      5. 5-d-old CD-1 mice
      6. Double distilled H2O (ddH2O)
      7. dNTPs, 10 mM (Thermo Fisher Scientific, catalog number: R0192 )
      8. DNA Loading Dye (6x) (Thermo Fisher Scientific, catalog number: R0611 )
      9. Q5 High-Fidelity DNA Polymerase (NEB, catalog number: M0491S )
      10. Taq DNA polymerase (NEB, catalog number: M0273S )
      11. GeneRuler 1 kb Plus DNA Ladder (Thermo Fisher Scientific, catalog number: SM1331 )
      12. QIAquickGel Extraction Kit (QIAGEN, catalog number: 28704 )
      13. QIAquickPCR Purification Kit (QIAGEN, catalog number: 28104 )
      14. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
      15. QIAprepMidiprep Kit (QIAGEN, catalog number: 12143 )
      16. Cut smart buffer (NEB, catalog number: B7204S )
      17. Appropriate Restriction Enzymes (NEB)
      18. Antarctic phosphatase (NEB, catalog number: M0289S )
      19. T4 DNA ligase (NEB, catalog number: M0202S )
      20. T4 DNA ligasebuffer (NEB, catalog number: B0202S )
      21. Yeast extract (Applichem, catalog number: A1552 )
      22. NaCl (VWR, catalog number: 27810.364 )
      23. Bacto Tryptone (Thermo Fisher Scientific, catalog number: 211701 )
      24. Agar (Sigma-Aldrich, catalog number: A5054 )
      25. Appropriate antibiotic for the plasmid vector (e.g., Ampicillin, Carl Roth, catalog number: K029.2 )
      26. Tris (Sigma-Aldrich, catalog number: 10708976001 )
      27. MgSO4 (Sigma-Aldrich, catalog number: M7506 )
      28. ZnCl2 (Sigma-Aldrich, catalog number: 746355 )
      29. SDS (Sigma-Aldrich, catalog number: L3771 )
      30. Chloroform (Sigma-Aldrich, catalog number: 372978 )
      31. KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
      32. p-nitrophenyl phosphate (Sigma-Aldrich, catalog number: 4876 )
      33. EDTA (Sigma-Aldrich, catalog number: 0 3620 )
      34. Isoflurane IsoFlo (Zoetis, catalog number: 50019100 )
      35. Agarose peqGOLD (VWR, catalog number: 732-2789 )
      36. 50x TAE buffer (Thermo scientific, catalog number: 10399519 )
      37. 2,2'-bipyridyl (Sigma-Aldrich, catalog number: D216305 )
      38. FeSO4 (Sigma-Aldrich, catalog number: 450278 )
      39. X-Gal (5-Bromo-4-chloro-3-indolyl beta-D-galactopyranoside; Thermo scientific, catalog number: R0404 )
      40. DMSO (Roth, catalog number: 4720 )
      41. LB agar plates containing the appropriate antibiotic for the plasmid vector (see Recipes)
      42. LB medium (see Recipes)
      43. Sucrose agar plates (see Recipes)

Equipment

  1. Pipettes (Eppendorf)
  2. Combi-vet isoflurane administration instrument (Rothacher, catalog number: CV 30301 )
  3. Scissors (Thermo scientific, catalog number: 10694962 )
  4. Tweezers (Thermo scientific, catalog number: 15809651 )
  5. Tissue homogenizer (Biospec, catalog number: 985370 )
  6. Vortexer (Thermo Fisher Scientific)
  7. Incubator (Thermo Fisher Scientific, ThermoScientificTM, model: Heraeus B12 Function Line, catalog number: 50042307)
  8. Microcentrifuge (Eppendorf, model: CentrifugeMiniSpin®, catalog number: 5452000018 )
  9. Thermocycler (Bio-Rad C1000 )
  10. NanoDropTM ( ND 2000 )
  11. Photometer (Beckman DU730 )

Software

  1. blastN (blast.jcvi.org/cmr-blast/)
  2. Tm Calculator Software (e.g., NEB Tm Calculator)

Procedure

  1. General Protocols
    1. PCR to amplify fragments for plasmid construction
      For PCR reactions follow the individual manufacturer's protocols. Briefly, the temperature was calculated as follows: 2 °C for every hybridized AT-pair and 4 °C for every GC-pair were added to the melting temperature. The elongation time was calculated after the length of the amplified fragment and the cycle amount was 30. A standard recipe for a PCR reaction to amplify fragments for plasmid construction based on the agents used herein would be:
      Reagents
      Volume (µl)
      chromosomal DNA
      8
      dNTP’s
      5
      Primer I (1 µg/ml)
      5
      Primer II (1 µg/ml)
      5
      GC enhancer
      20
      Q5 Polymerase
      3
      Q5 Polymerase buffer
      25
      ddH2O
      32
      1. Perform PCR reaction.
      2. Add appropriate volumes of loading dye to the PCR reaction and separate bands by horizontal gel electrophoresis using a 0.8 to 1% agarose gel and apply approx. 10 V per cm gel.
      3. Excise band from the agarose gel and purify respective PCR fragments via the QIAGEN gel extraction kit following the manufacturer's instructions.
    2. Colony PCR
      For PCR reactions follow the individual manufacturer's protocols. The template for the colony PCR originates from a colony picked form an agar plate using a pipette tip. The selected colony is resuspended in 50 µl ddH2O and boiled at 100 °C for 10 min. A standard recipe for a PCR reaction to amplify fragments for plasmid construction based on the agents used herein would be:
      Reagents
      Volume (µl)
      Template (boiled colony resuspension)
      3
      dNTP’s
      0.5
      Primer I (1 µg/ml)
      0.5
      Primer II (1 µg/ml)
      0.5
      Thermo Polymerase Buffer
      2.5
      Taq Polymerase
      0.25
      ddH2O
      17.75
      1. Perform PCR reaction.
      2. Add appropriate volumes of loading dye to the PCR reaction and separate bands by horizontal gel electrophoresis using a 0.8 to 1% agarose gel and apply approx. 10 V per cm gel.
    3. Restriction digestion
      For restriction digests follow the individual manufacturer's protocols. Standard recipes for restriction digests based on the agents used herein would be:
      Reagents
      Volume (µl)
      Plasmid (isolated via a QIAGEN Plasmid Midi Kit; 100 ng/µl)
      5
      Restriction Enzyme I
      1.5
      Restriction Enzyme II
      1.5
      10x buffer (appropriate to Enzyme)
      10
      ddH2O
      81
      or
      Reagents
      Volume (µl)
      PCR Fragment (gel-purified via a QIAGEN gel extraction kit; 100 ng/µl)
      15
      Restriction Enzyme I
      1.5
      Restriction Enzyme II
      1.5
      10x buffer (appropriate to enzyme)
      10
      ddH2O
      71
      1. Perform overnight (ON) digestion using the appropriate restriction enzyme combinations and temperatures according to the manufacturer's instructions. In case of the PCR fragments appropriate restriction enzymes herein are indicated by the name of the oligonucleotide (see oligonucleotide lists above).
      2. In case of restriction digestion using plasmids, dephosphorylate 5′-ends by adding 1 µl antarctic phosphatase to the mix and incubate for 1 h at 37 °C.
      3. Add appropriate volumes of loading dye to the samples and separate the bands by horizontal gel electrophoresis using a 0.8 to 1% agarose gel and apply approx. 10 V per cm gel.
      4. Excise band from the agarose gel and purify the linearized, dephosphorylated plasmid from the gel via the QIAGEN gel extraction kit following the manufacturer's instructions.
      5. In case of restriction digestion using the PCR fragments, inactivate the enzymes at 65 °C for 10 min.
      6. Subsequently purify the digested PCR fragments using the QIAGEN PCR purification kit following the manufacturer's instructions.
    4. Ligation
      For ligations follow the individual manufacturer's protocols. Standard recipes for restriction digestion based on the agents used herein would be:
      Reagents
      Volume (µl)
      Plasmid (digested + dephos.; 30 ng/µl)
      5
      PCR fragment I (digested; 30 ng/µl)
      5.75
      PCR fragment II (digested; 30 ng/µl)
      5.75
      10x DNA ligase buffer
      1.5
      T4 DNA ligase
      1
      1. Perform ligation at room temperature for 1 h.
      2. Inactivate the ligase at 65 °C for 10 min.
    5. PhoA Assay
      1. Cultivate strains to late logarithmic growth phase under in vitro test conditions [e.g., herein LB at 37 °C and 180 rpm (supplemented with 2,2'-bipyridyl or FeSO4 in case of the irgA-fusion strains)].
      2. Pellet 1 ml of each culture using a microcentrifuge (5 min, 6,000 x g, RT).
      3. Wash cells once in P1 buffer (10 mM Tris-HCl, pH 8.0, 10 mM MgSO4) and resuspend the pellet in 1 ml P1 buffer.
      4. Dilute 0.1 ml of resuspended cells from Step A5c in 0.9 ml P1 buffer and measure OD600.
      5. Add 0.1 ml of resuspended cells from Step A5c into 0.9 ml P2 buffer (1 M Tris-HCl pH 8.0, 0.1 mM ZnCl2). In parallel, prepare a negative control without cells.
      6. To each sample add 50 µl of 0.1% SDS and 50 µl of chloroform, vortex and incubate at RT for 10 min to permeabilize cells.
      7. To start the enzymatic reaction, add 0.1 ml of 0.4% p-nitrophenyl phosphate (in 1 M Tris-HCl, pH 8.0) to each tube, vortex, and note time.
      8. Incubate each tube until a light-yellow color appears, then add 120 µl of 0.1 M EDTA, pH 8.0, 1 M KH2PO4 to stop the reaction and note time.
      9. Measure OD420 for each sample, using the negative control sample as a blank.
      10. Calculate the duration between start and stop of the reaction.
      11. Calculate PhoA activity using the formula stated in the chapter data analysis.

  2. Random approach to identify in vivo repressed (ivr) genes of V. cholerae
    1. Construction of the TRIVET components
      The TRIVET system requires construction of several elements partially based on components of the RIVET (Osorio et al., 2005).
      1. Construction of the tetR-phoA-cat (tpc) cassette for random integration in the bacterial chromosome via transposon mutagenesis.
        1. For optimal PCR results, calculate optimal Tm for all given oligonucleotides.
        2. PCR amplify the promoterless tetR-fragment using chromosomal DNA from E. coli XL-1 and oligonucleotide pairs tetR-5′-BamHI and tetR-3′-Kpnl (see Step A1 for details).
        3. PCR amplify the promoterless phoA-fragment using chromosomal DNA from SM10λpir oligonucleotide pairs phoA-5′-KpnI and phoA-3′-XbaI (see Step A1 for details).
        4. PCR amplify cat from pA C1000 . The oligonucleotide pairs cat-5′ to XbaI and cat-3′ to BamHI (see Step A1 for details).
        5. Gel-purify PCR fragments followed by restriction digestion and second purification (see Steps A1 and A3 for details).
        6. Digest (using BamHI), dephosphorylate and gel-purify pTrc99A-Km (see Step A3 for details)
        7. Ligate the digested tetR-, phoA- and cat-fragments into a BamHI-digested and dephosphorylated pTrc99A-Km to obtain the pTrc-tpc plasmid (see Step A4 for details).
        8. Transform into competent E. coli DH5αλpir cells and select by plating on LB agar supplemented with Kanamycin (Km) and Chloramphenicol (Cm).
        9. Incubate plates overnight at 37 °C.
        10. Confirm correct pTrc-tpc construct via colony PCR using the oligonucleotide pairs tetR-5′-BamHI and cat-3′-BamHI.
        11. Grow verified colonies with correct pTrc-tpc construct in LB-Km/Cm + 2% glucose (Glc), overnight at 37 °C and 180 rpm.
        12. Isolate pTrc-tpc using a QIAprep MidiprepKit.
        13. PCR amplify the entire tetR-phoA-cat (tpc)-cassette using oligonucleotide pairs tetRphoAcat-5′-NotI and tetRphoAcat-3′-NotI (see Step A1 for details).
        14. Gel-purify PCR fragments followed by restriction digestion with NotI and second purification (see Steps A1 and A3 for details).
        15. Ligate the purified, NotI-digested tpc-cassette into a NotI-digested, dephosphorylated pLOFKm to obtain pLOF::tpc (see Steps A1, A3 and A4 for details).
        16. Transform ligation products into competent E. coli DH5αλpir cells and select by plating on LB agar supplemented with ampicillin (Ap) and Cm.
        17. Incubate plates overnight at 37 °C.
        18. Confirm correct pLOF::tpc construct via colony PCR using the oligonucleotide pairs tetR-5′-BamHI and cat-3′-BamHI (see Step A2 for details).
        19. Grow verified colonies with correct pLOF::tpc construct in LB-Ap/Cm + 2%Glc, overnight at 37 °C and180 rpm.
        20. Isolate pLOF::tpc using the QIAprep Spin Miniprep Kit following the manufacturer's instructions.
        21. Transform pRES and pRES1 into competent SM10λpir cells and select by plating on LB agar supplemented with Ap.
        22. Incubate plates overnight at 37 °C.
      2. Construction of the pTRIVET and pTRIVET1 plasmids
        1. PCR amplify the lacZ-fragment from V. cholerae WT chromosomal DNA using oligonucleotide pairs lacZ-5′-SacI and lacZ-3′-NheI.
        2. PCR amplify tnpR-fragment from pGOA1193 using oligonucleotide pairs tnpR-5′-BgIII and tnpR-3′-XbaI.
        3. Gel-purify PCR fragments followed by restriction digestion and second purification (see Steps A1 and A3 for details).
        4. Mix oligonucleotide pairs PtetA-5′-3′ and PtetA-3′-5′ or Ptet1-5′-3′ and Ptet1-3′-5′, boil oligonucleotide mix for 5 min to ensure denaturation and cool at RT. This will allow hybridization of the oligonucleotide pairs with a compatible NheI- and BglII-site on each end. The generated dsDNA sequence comprises the original tetA promoter sequence ptet (in case of PtetA-5′-3′ and PtetA-3′-5′) or for a less stringent transcriptional regulation a slightly altered tetA promoter sequence ptet1 with mutated TetR-binding sites (in case of Ptet1-5′-3′ and Ptet1-3′-5′).
        5. Ligate the digested lacZ-fragment, the digested tnpR-fragment and one of the two tetA promoter sequence versions (ptet or ptet1, obtained via Step B1bviii) into a SacI/XbaI-digested, dephosphorylated pGP704 resulting in either pTRIVET containing the promoter ptet or pTRIVET1 containing the promoter ptet1, respectively (see Step A4 for details).
        6. Transform ligation products into competent DH5αλpir cells and select by plating on LB agar supplemented with Ap.
        7. Incubate plates overnight at 37 °C.
        8. Confirm correct pTRIVET or pTRIVET1 constructs via colony PCR using the oligonucleotide pairs lacZ-3′-SacI and tnpR-3′-XbaI as well as restriction analyses.
        9. Grow verified colonies with correct pTRIVET or PTRIVET1 in LB-Ap + 2% Glc overnight at 37 °C and 180 rpm.
        10. Isolate the pTRIVET and pTRIVET1 plasmids via the QIAprep Spin Miniprep Kit by following the manufacturer’s protocol.
        11. Transform pTRIVET and pTRIVET1 into competent SM10λpir cells and select by plating on LB agar supplemented with Ap.
        12. Incubate plates overnight at 37 °C.
      3. Construction of the pRes and pRes1 according to Osorio et al. (2005)
        1. PCR amplify the res sequence via PCR using pRR51 as a template and oligonucleotide pairs1RES-F and 1RES-R (see Step A1 for details). Alternatively, PCR amplify the res1 sequences via PCR using pSL134 and oligonucleotide pairs1RES1-F and 1RES1-R (see Step A1 for details).
        2. Gel-purify PCR fragments followed by restriction digestion (using EcoRI and XhoI) and second purification (see Steps A1 and A3 for details).
        3. Ligate the digested PCR fragments into an EcoRI/XhoI-digested, dephosphorylated pPCR-Script Amp SK(+) (Stratagene) to generate plasmids pGOA2 and pGOA3 (see Step A4 for details).
        4. Use a 2nd set of oligonucleotide pairs (2RESF and 2RESR or 2RES1F and 2RESR) to PCR amplify a second copy of res or res1. Subclone them immediately next to their counterparts in pGOA2 and pGOA3 to generate plasmids pGOA4 and pGOA5, which harbor two res or res1 sequences, respectively.
        5. Generate pGOA6 and pGOA7 via subcloning of the sacB-neo genes from pGOA1 into XbaI-digested pGOA4 and pGOA5.
        6. Amplify the res- and res1-cassettes via PCR from pGOA6 and pGOA7 using oligonucleotide pairs T3 and T7 and ligate them into KpnI-digested pSL111 treated with T4 DNA polymerase to generate pRES and pRES1.
        7. Transform ligation products into competent DH5αλpir cells and select by plating on LB agar supplemented with Ap.
        8. Incubate plates overnight at 37 °C.
        9. Confirm correct pRES and pRES1 constructs via restriction analyses and sequencing.
        10. Isolate the pRES and pRES1 plasmids via the QIAprep Spin Miniprep Kit by following the manufacturer’s protocol.
        11. Transform pRES and pRES1 into competent SM10λpir cells and select by plating on LB agar supplemented with Ap.
        12. Incubate plates overnight at 37 °C.
    2. Construction of the TRIVET library
      1. Insertion of the res-cassette
        1. Mobilize suicide plasmids pRES and pRES1 into V. cholerae WT by conjugation and allelic exchange to place each cassette into the lacZ-locus to obtain Vc_res and Vc_res1 as described below.
        2. Mobilize suicide plasmids pRES or pRES1 into V. cholerae by conjugation of E. coli SM10λpir pRES or pRES1 obtained by Step B1c with V. cholerae WT [(streptomycin-resistant (SmR)]. This was performed by cross-streaking sufficient cell material of both strains on the same LB agar plate and incubating plate for 6 h at 37 °C. Via homologous regions present on pRES or pRES1 an allelic exchange of the suicide plasmids and the lacZ-locus on the V. cholerae chromosome is possible at low frequency via homologous regions present on pRES or pRES1.
        3. V. cholerae with integrated pRES and pRES1 are then selected by streaking the single colonies form the conjugation mixtures on LB-Sm/Ap/Km agar plates.
        4. Incubate plates overnight at 37 °C.
        5. Streak-purify colonies on LB-Sm/Ap/Km agar plates at least twice.
        6. Grow purified colonies in LB-Sm/Km overnight at 37 °C, 180 rpm and plate appropriate dilutions on LB-Sm/Km plates supplemented with 5-Bromo-4-chloro-3-indolyl beta-D-galactopyranoside (40 µg/ml dissolved in DMSO, X-Gal).
        7. Incubate plates overnight at 37 °C.
        8. White colonies are potential candidates for double recombination events and allelic exchange of the lacZ-locus with the res- or res1-cassette.
        9. Confirm loss of suicide vector backbone by streaking on LB-Sm/Km (growth) and LB-Ap (no growth).
        10. Incubate plates overnight at 37 °C and proceed with SmR/KmR, but ApS colonies.
        11. Validate correct insertion of the res or res1-cassette in the lacZ-locus via colony PCR and/or sequencing resulting in strains Vc_res and Vc_res1.
      2. Transposon mutagenesis in Vc_res and Vc_res1.
        1. Transposon mutagenesis is achieved by mobilization of the pLOF::tpc transposon plasmid into V. cholerae via conjugation as described below. Along the incubation period, transfer of plasmid results in spontaneous transposition events in V. cholerae.
        2. Mobilize suicide vector pLOF::tpc into V. cholerae by conjugation of E. coli SM10λpir pLOF::tpc obtained from Step B1a with either Vc_res and Vc_res1 obtained from Step B2a via filter mating on LB agar plates. To do so, grow strains to an OD600 of approx. 1 and mix recipient and donor in a 2:1 ratio with a final volume of 2 ml.
        3. Pellet mixture by centrifugation (5,000 x g, 5 min, RT) and gently resuspend cells in 100 µl LB broth.
        4. Place bacterial suspension in the center of a LB plate and incubate plate for 1.5 h at 37 °C to allow conjugation.
        5. Pick up the bacteria of the plate using a pipet tip and resuspend them in 1 ml LB broth.
        6. Select for transposon mutants by plating appropriate dilutions of the resuspended conjugation mixture to reach approximately 50-200 colonies per LB-Sm/Km/Cm plate.
        7. Incubate plates overnight at 37 °C.
        8. Purify colonies by streaking on LB-Sm/Km/Cm agar plates and in parallel on LB-Ap agar plates. After overnight incubation at 37 °C, pursue only with LB-Sm/Km/Cm-resistant, but Ap-sensitive colonies representing bona fide transposon mutants (loss of pLOF backbone).
        9. Pool ~500 bona fide colonies to generate heterogeneity by adding 1-2 ml of LB on top of the plate and rinsing multiple times over the colonies.
        10. Repeat Steps B2bi to B2bvi about 20 times for Vc_res as well as Vc_res1 to obtain several independent transposon pools for Vc_res or Vc_res1, respectively.
      3. Integration of pTRIVET and pTRIVET1
        1. Mobilize suicide plasmids pTRIVET or pTRIVET1 into V. cholerae transposon pools obtained by Step B2b to insert the TRIVET or pTRIVET1 via homologous recombination downstream of the lacZ-locus as described below.
        2. To do so, cross-streak sufficient cell material of E. coli SM10λpir pTRIVET or E. coli SM10λpir pTRIVET1 obtained from Step B1b with V. cholerae transposon pools obtained by Step B2b (see Step B2aii for details).
        3. Resuspend conjugation mixture in 1 ml LB broth and select for insertion of the pTRIVET or pTRIVET by plating appropriate dilutions of the resuspended conjugation mixture on LB-Sm/Km/Ap plates.
        4. Incubate plates overnight at 37 °C.
        5. Pool ~2,000 colonies to maintain heterogeneity by adding 1-2 ml of LB on top of the plate and rinsing multiple times over the colonies. These pools represent the final TRIVET-pools.
    3. Identification of ivr genes
      1. Prescreen for elimination of fusion strains with high resolution frequency due to low TetR expression.
        1. Let each TRIVET-pool grow to late log phase in LB-Sm/Ap broth without Km as a selection marker for maintenance of the res or res1-cassette. This allows resolution of the fusion strains with insufficient TetR-expression to silence tnpR.
        2. Plate serial dilutions on LB-Sm/Km/Ap plates and incubate overnight at 37 °C to select for non-resolved strains in the TRIVET pool (res- or res1 cassette is stably maintained under in vitro conditions).
        3. Pool ~1,000-2,000 colonies to maintain heterogeneity by adding 1-2 ml of LB on top of the plate and rinsing multiple times over the colonies. (Optional: To confirm heterogeneity, detect restriction fragment length polymorphism of the tpc-fusion loci from 50 randomly picked colonies using Southern blot analysis.)
      2. Screening for ivr genes of V. cholerae using the infant mouse model (in vivo).
        1. Spread an aliquot of each pool of the library in triplicate on LB-Sm/Km/Ap plates and incubate overnight at 37 °C.
        2. Collect ~5,000 colonies from each plate and dilute in LB to a concentration of ~106 cfu/ml.
        3. Use 50 µl of the bacterial suspension to intragastrically inoculate 5-d-old CD-1 mice (anesthetized by isoflurane).
        4. Euthanize mice 22 h post infection by cervical dislocation, remove small intestine and homogenize in 1 ml LB media containing 20% Glycerol using a tissue homogenizer at medium power for 30 s while moving up and down.
        5. Plate serial dilutions of the homogenate on sucrose agar to select for loss of sacB (selection for in vivo resolved strains now lacking the res or res1 cassette).
        6. Incubate up to 48 h at RT.
        7. Confirm resolution (loss of res or res1 cassette by streaking in parallel on LB-Sm/Ap (growth) and LB-Km (no growth).
        8. Incubate plates overnight at 37 °C and proceed to identify the tpc-cassette insertion site according to Step B3d with Sm/Km-resistant, but Km-sensitive colonies.
      3. PhoA assays as refinement step of in vivo resolved strains
        1. Based on previous studies using the resolution technology, the in vivo screen has a substantial false positive rate of spontaneous resolution due to low in vitro expression levels of TetR in some strains of the TRIVET library. The implementation of phoA as a second reporter enables confirmation of substantial expression levels during in vitro growth for each of the in vivo resolved strains identified in Step B3b. We highly recommend this refinement step to eliminate false positives.
        2. To reduce the relatively high false positive rate of resolvase based screens of approximately 15% a spontaneous in vitro resolution frequency has to be defined as a cut-off. Reanalysis of the combined data acquired by previous resolvase-based screens (Osorio et al., 2005; Schild et al., 2007; Seper et al., 2014) reveal that strains with in vitro resolution frequencies below 30% have only a 5% chance to be false positive. As the tpc cassette remains stably integrated in the chromosome PhoA activity of in vivo resolved strains can be measured under in vitro conditions and correlate the obtained activity to a resolution frequency. According to the results obtained for the irgA-fusion test strain, an in vitro resolution frequency of 30% or lower correlates with PhoA activities of 10 Miller units or higher. Thus, we used this cut-off in the original study describing TRIVET (Cakar et al., 2018).
        3. Identify a promoter, which is differentially regulated under controllable in vitro cultivation conditions. Herein, we use the iron-regulated irgA promoter of the V. cholerae, which is highly expressed at low iron levels and repressed at high concentrations of iron. Differential iron availability in vitro can be mimicked in LB by adding varying amounts of the iron chelator 2,2'-bipyridyl or FeSO4.
        4. Construct a V. cholerae TRIVET reporter strain Vc_res_TRIVETirgA::tpc with a specific transcriptional fusion of the tpc-cassette to the iron-regulated irgA promoter (see Step C1 for details).
        5. Assess in parallel the resolution frequency and PhoA activity of the Vc_res_TRIVETirgA::tpc reporter after growth for 8 h in LB supplemented with varying amounts of iron chelators such as 2,2'-bipyridyl or FeSO4 (see Steps C2 and C3 for details). We recommend to test a range of 0 to 150 µM final concentrations in 50 µM increments for both agents.
        6. Correlate PhoA activity and resolution frequency of the Vc_res_TRIVETirgA::tpc strain grown in LB with different iron-availability. Choose the growth condition closest to the cut-off (less than 30% resolution frequency) and use the associated PhoA activity as refinement criterion (in our case 10 Miller Units).
        7. Cultivate in vivo resolved strains in LB for 8 h at 37 °C and 180 rpm.
        8. Perform PhoA assay as described above (see Step A5 for details).
        9. Proceed only with in vivo resolved strains showing PhoA Activities equal or higher than the refinement criterion (determined above).
      4. Identification of tpc fusion by subcloning and sequencing
        1. Isolate chromosomal DNA from overnight cultures of the in vivo resolved strains of interest.
        2. Digest chromosomal DNA with EcoRI and use the digested chromosomal DNA for ligation with an EcoRI-digested, dephosphorylated pBR322 plasmid (see Steps A3 and A4 for details).
        3. Transform ligation product into E. coli DH5αλpir and plate on LB-Cm plates to select for the cat-gene of the tpc-cassette including downstream chromosomal DNA of the tpc-fusion site.
        4. Isolate plasmids of CmR-colonies using a QIAprep Spin Miniprep Kit.
        5. Sequence with oligonucleotide seqPrimer-cat to obtain downstream sequence tpc-fusion site.
        6. BLAST sequence against the V. cholerae N16961 genome database using blastN to identify insertion site of the tpc-cassette.

  3. Resolution assays to measure gene silencing
    1. Construction of suicide plasmids for transcriptional fusions of the tpc cassette to specific promoters, exemplified by an irgA-tpc or yrb-tpc fusion.
      1. Construction of suicide plasmids.
        1. In case of the irgA-tpc fusion, PCR amplify the tpc-cassette using the oligonucleotide pair tetRphoAcat-5′-SacI and tetRphoAcat-3′-SalI as well as pTRc-tpc as template (see Step A1 for details).
        2. PCR amplify 800 bp fragments located upstream and downstream of the irgA-insertion site (irgA promoter) using the oligonucleotide pairs irgA-SphI-1 and irgA-SacI-2 as well as irgA-SalI-3 and irgA-XbaI-4 (see Step A1 for details).
        3. In case of the yrb-tpc fusion, PCR amplify the tpc-cassette using the oligonucleotide pair tetRphoAcat-5′-SacI and tetRphoAcat-3′-SalI as well as pTRc-tpc as template (see Step A1 for details).
        4. PCR amplify 800 bp fragments located upstream and downstream of the yrb-insertion site (yrb promoter) of the tpc-cassette using the oligonucleotide pairs, yrb_SacI_1 and yrb_SphI_2 as well as yrb_SalI_3 and yrb_XbaI_4.
        5. Gel-purify PCR fragments followed by restriction digestion and second purification (see Steps A1 and A3 for details).
        6. In case of the irgA-tpc fusion, ligate the digested tpc-cassette as well as the up- and downstream fragments (see Steps C1ai and ii) into an SphI/XbaI-digested, dephosphorylated pCVD442.
        7. In case of the yrb-tpc fusion, ligate the digested tpc-cassette as well as the up- and downstream fragments (see Steps C1aii and iv) into an SphI/XbaI-digested, dephosphorylated pCVD442.
        8. Transform ligation products into competent DH5αλpir cells and select by plating on LB agar supplemented with Ap.
        9. Incubate plates overnight at 37 °C
        10. Confirm correct pCVD442irgA::tpc or pCVD442yrb::tpc constructs via colony PCR using the oligonucleotide pairs irgA-SphI-1 and irgA-XbaI-4 or yrb_SphI_2 and yrb_XbaI_4 , respectively.
        11. Grow verified colonies with correct pCVD442irgA::tpc or pCVD442yrb::tpc in LB-Ap overnight at 37 °C and 180 rpm.
        12. Isolate the pCVD442irgA::tpc or pCVD442yrb::tpc plasmid via the QIAprep Spin Miniprep Kit by following the manufacturer’s protocol.
        13. Transform pCVD442irgA::tpc or pCVD442yrb::tpc into competent SM10λpir cells and select by plating on LB agar supplemented with Ap.
        14. Incubate plates overnight at 37 °C.
      2. Construction of TRIVET strains harboring a specific tpc-fusion to a gene of interest.
        1. To obtain Vc irgA::tpc and Vc yrb::tpc, mobilize suicide plasmids pCVD442irgA::tpc or pCVD442yrb::tpc into V. cholerae by conjugation and allelic exchange to place the tpc-cassette downstream of the respective promoter.
        2. Mobilize suicide plasmids pCVD442irgA::tpc or pCVD442yrb::tpc into V. cholerae by conjugation of E. coli SM10λpir pCVD442irgA::tpc or pCVD442yrb::tpc obtained by Step C1a with V. cholerae WT [(streptomycin-resistant (SmR)]. To do so, cross-streak sufficient cell material of both strains on the same LB agar plate and incubate plate for 6 h at 37 °C. Via homologous regions present on pCVD442irgA::tpc or pCVD442yrb::tpc an allelic exchange of the suicide plasmids and the respective locus on the V. cholerae chromosome at low frequency is possible.
        3. Streaking of single colonies form the conjugation mixtures on LB-Sm/Ap agar plates selects for V. cholerae with integrated pCVD442irgA::tpc or pCVD442yrb::tpc. 
        4. Incubate plates overnight at 37 °C.
        5. Streak-purify colonies on LB-Sm/Ap agar plates at least twice.
        6. Grow purified colonies in LB-Sm overnight at 37 °C, 180 rpm and plate appropriate dilutions on sucrose agar.
        7. Incubate plates for up to 48 h at RT.
        8. Confirm loss of suicide vector backbone by streaking on LB-Sm (growth) and LB-Ap (no growth)
        9. Incubate plates overnight at 37 °C, proceed with SmR, but ApS colonies.
        10. Validate correct insertion of the tpc-cassette via colony PCR and/or sequencing resulting in Vc irgA::tpc and Vc yrb::tpc.
        11. Insert res- or res1-cassette into Vc irgA::tpc or Vc yrb::tpc to obtain Vc_res irgA::tpc, Vc_res1 irgA::tpc, Vc_res yrb::tpc or Vc_res1 yrb::tpc as described above (see Step B2a for details).
        12. Integrate pTRIVET or pTRIVET1 into Vc_res irgA::tpc, Vc_res1 irgA::tpc, Vc_res yrb::tpc or Vc_res1 yrb::tpc to obtain Vc_res_TRIVET irgA::tpc, Vc_res1_TRIVET irgA::tpc, Vc_res_TRIVET yrb::tpc or Vc_res1_TRIVET yrb::tpc as well as Vc_res_TRIVET1 irgA::tpc, Vc_res1_TRIVET1 irgA::tpc, Vc_res_TRIVET1 yrb::tpc or Vc_res1_TRIVET1 yrb::tpc as described above (see Step B2c for details).
    2. Quantification of gene silencing
      1. In vivo and in vitro resolution assay using
        1. Grow TRIVET strain with a transcriptional fusion of the tpc-cassette to the promoter of interest [e.g., Vc_res1_TRIVET yrb::tpc as used in Cakar et al. (2018)] on LB- Sm/Km/Ap plates; overnight at 37 °C.
        2. Harvest bacterial cells from plate and resuspend them in LB-Sm/Km/Ap.
        3. Measure OD600 and adjust OD600 to 1 using appropriate dilutions to generate the inoculum.
        4. To assess the in vivo resolution, infect 5-d-old CD-1 mice (anesthetized by isoflurane) with 50 µl of the inoculum (∼106 cfu). At a post infection time point of interest [e.g., 6 and 22 h as used in Zingl et al. (2020)] euthanize mice by cervical dislocation, remove small intestine and homogenize in 1 ml LB media containing 20% Glycerol. Plate appropriate serial dilutions of the homogenate on LB-Sm/Km and LB-Sm/Ap plates and incubate overnight at 37 °C.
        5. To assess the in vitro resolution, inoculate 5 ml of LB-Sm/Ap with 50 µl of the inoculum and incubate parallel to the mouse infection at 37 °C and 180 rpm. Plate appropriate serial dilutions of the homogenate on LB-Sm/Km and LB-Sm/Ap plates and incubate overnight at 37 °C.
        6. Assess the amount of single colonies on plates and calculate the Sm/Km-resistant and Sm/Ap-resistant cfu in the original sample.
        7. Calculate resolution (%) for in vivo and in vitro formulas described in the chapter data analysis.
      2. In vitro PhoA Assays as alternative quantification of transcriptional activity.
        1. Grow the strains using cultivation conditions of interest.
        2. Perform PhoA assay as described above (see Step A5 for details).

Data analysis

Results of the resolution assay will be expressed as the percent resolution, which is calculated using the determined cfu of the Sm/Km-resistant and Sm/Ap-resistant population within a given culture sample. The Sm/Ap-resistant colonies reflect the entire population and the Sm/Km-resistant colonies reflects the unresolved population, Subtraction of the unresolved population from the entire population gives the resolved population (loss of res-/res1-cassette).
Percent resolution is calculated as:

    Resolution (%) = [cfu(Sm/Ap-resistant) – cfu(Sm/Km-resistant)]/cfu(Sm/Ap-resistant)

For calculation of alkaline phosphatase activity use the formula:

    Activity (Miller Units) = (1,000 x OD420)/[reaction time (min) x OD600]

Recipes

  1. LB broth/agar (g/L)
    BactoTM Tryptone 10 g
    Yeast extract 5 g
    NaCl 10 g
    Agar 15 g
  2. Sucrose agar (g/L)
    BactoTM Tryptone 10 g
    Yeast extract 5 g
    Agar 15 g
    Sucrose 100 g

If appropriate add antibiotics after autoclaving in the following concentrations: Antibiotics and other supplements were used in the following final concentrations: streptomycin (Sm, 100 µg/ml), ampicillin (Ap, 50 µg/ml in combination with other antibiotics, otherwise 100 µg/ml), kanamycin (Km, 50 µg/ml) and chloramphenicol (Cm, 2 µg/ml for V. cholerae; 10 µg/ml for E. coli).

Acknowledgments

We thank Andrew Camilli (Tufts University, Boston) for providing the original RIVET components and helpful discussions to establish the TRIVET system. This work was supported by Austrian Science Fund (FWF) Grants W901 (DK Molecular Enzymology) (to F.G.Z. and S.S.), 27654 and P25691 (to S.S.).

Competing interests

The authors declare no competing interests.

Ethics

Animals were used in all experiments in accordance with the rules of the ethics committee at the University of Graz and the corresponding animal protocol, which has been approved by Austrian Federal Ministry of Science and Research Ref. II/10b. Mice were housed with food and water ad libitum and monitored under the care of full-time staff.

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  15. Seper, A., Pressler, K., Kariisa, A., Haid, A. G., Roier, S., Leitner, D. R., Reidl, J., Tamayo, R. and Schild, S. (2014). Identification of genes induced in Vibrio cholerae in a dynamic biofilm system. Int J Med Microbiol 304(5-6): 749-763. 
  16. Wang, Z., Gerstein, M. and Snyder, M. (2009). RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10(1): 57-63. 
  17. Zingl, F. G., Kohl, P., Cakar, F., Leitner, D. R., Mitterer, F., Bonnington, K. E., Rechberger, G. N., Kuehn, M. J., Guan, Z., Reidl, J. and Schild, S. (2020). Outer membrane vesiculation facilitates surface exchange and in vivo adaptation of Vibrio cholerae. Cell Host Microbe 27(2): 225-237 e228.

简介

[摘要]研究细菌基因对环境变化的调控仍然是一项艰巨的任务。例如,人胃肠道的病原体霍乱弧菌在口服后会在不同的隔室中遇到各种短暂的状况。事实证明,遗传报告系统是揭示复杂条件下基因调控事件的极有力工具,但到目前为止,它主要集中在基因诱导上。在本文中,我们描述了基于TetR控制的重组的体内表达技术TRIVET,该技术可检测基因沉默事件。TRIVET类似于体内表达技术(IVET)以及基于重组的体内变异体 表达技术(RIVET),用于鉴定宿主定殖过程中几种细菌的条件基因诱导。像它的前辈一样,TRIVET是一个基于单细胞的报告系统,可以通过耐药谱的表型变化以时空方式分析细菌基因的阻遏。简而言之,无启动子的tetR (编码转录阻遏物TetR)可通过转座子诱变随机地整合到细菌基因组中,或通过同源重组在目标启动子的下游特异性整合到细菌基因组中。的TetR导致的去阻遏的转录表达的减少的TetR控制解离TNPR,这反过来又导致切除ö F A Ñ抗生素抗性盒(也称为RES-盒)和改变的电阻曲线可观察到的通过划线上氨苄青霉素和卡那霉素板。然后可以将这种改变量化为抗性和非抗性分离株之间的比例。此外,新引入的第二报道基因,promot erless phoA启动(编码碱性磷酸酶的PhoA)提供的结果经由独立比色法来测量酶活性的附加验证步骤。本文提出的方案还提供了一种方法,可在随机筛选基因进行基因筛选的情况下鉴定基因基因座,以及对感兴趣基因的条件抑制进行定量。尽管当前协议是为宿主定殖过程中的基因抑制建立的,但它很可能适用于研究细菌所面临的各种条件下的基因沉默。


[背景]兼性细菌病原体在环境传代和宿主定殖过程中不断需要适应各种条件。多个基因的瞬时激活和抑制可确保最佳的生存适应性。弄清这些适应过程是了解细菌生理学并确定治疗干预策略潜在目标的关键。已经建立了多种技术来研究细菌中的基因表达谱,包括微阵列,RNA-Seq和qRT-PCR (Eisen和Brown,1999;Bookout和Mangelsdorf,2003; Wang等,2009)。此类方法必须处理平均细菌种群中异质性的后果,并且仅反映收获时间点的转录水平。与亚群或瞬时调控中的特定区域有关的任何独特的基因表达模式都可能丢失。而且,相对大量的RNA和纯度的要求限制了上述技术。相比之下,TRIVET报告系统甚至可以检测复杂的异源细菌种群的子集中的瞬时基因沉默事件。由于在单个细胞水平上抗生素耐药性谱的不可逆变化,因此TRIVET是一种可靠的技术。相反,TRIVET需要对模型生物进行大量的劳动密集型基因工程,因此仅限于具有可用基因组序列和遗传修饰工具的细菌。

兼性人类病原体霍乱弧菌为代表,我们描述了TRIVET的两个潜在应用:(i)在鼠模型中肠道定植过程中鉴定体内抑制基因的随机方法,以及(ii)研究条件转录的特定方法使感兴趣的基因沉默(Cakar等人,2018; Zingl等人,2020)。TRIVET系统由三个染色体元件组成,即一个tetR - phoA - cat (tpc )报告基因盒,一个自杀载体系统pTRIVET,提供TetR控制的分辨酶TnpR和分辨率(res)盒式磁带作为TnpR的靶标。

三脚架的RES-盒是相同的铆钉的RES-盒(奥索里奥等人,2005)和集成在lacZ启动所述的-locus霍乱弧菌染色体。两个选择标记[ neo (Km R )和sacB (Suc S )]的两侧是Tnp R解酶的靶位点,也称为res位点。

第二部分是tpc -cassette。对于随机方法中,TPC r -盒是IS10站点使用pLOF载体递送系统的迷你Tn10转系统之间亚克隆(雷罗等人,1990) 。值得注意的是,编码TetR阻遏物和碱性磷酸酶的tetR和phoA是无启动子的,而猫盒则用其自身的组成性启动子亚克隆,以选择转座子突变体。因此,含有菌株RES-盒以下ch的转座子诱变升oramphenicol选择,生成的随机插入TPC报告基因盒。这些转座子突变体的亚群包含染色体基因与tetR和phoA的转录融合,并在各自启动子激活后表达这些基因。

TRIVET的第三个组成部分是pTRIVET自杀质粒(Ap R ),其中包含lacZ基因座下游的一个n 800 bp基因间区域和tnpR (编码分辨酶)。重要的是,负责tnpR表达的启动子起源于四环素抗性基因tetA ,因此受到TetR的严格控制。此pTRIVET的动员到一个水库-和TPC r -盒含有霍乱弧菌在下游同源重组菌株的结果的lacZ的基因组中。由于整合位点位于lacZ内在的转录终止子的下游,可防止RNA聚合酶朝向tnpR的不希望的读通。因此,tnpR的表达仅依赖于TetR控制的启动子。Cakar等人在最近的出版物中可以找到该系统的全面描述,其中包括遗传元件的示意图。(2019)。与菌株tetR的通过表达TPC r -盒,压制TNPR将导致稳定的含RES-盒的菌株,它可以通过其独特的电阻档(A隔离p - [R ,公里ř和的Suc小号)。相反,没有tetR的表达导致tnpR的诱导,导致切除卡带(Ap R ,Km S和Suc R )的切除和不可逆的损失。此事件称为分辨率,并导致应变消除。因此,相对简单的Km R选择将鉴定具有足够的TetR表达且具有稳定的res盒的菌株。这些菌株可用于感染婴儿小鼠。在体内定植过程中,可能会发生驱动tetR表达的染色体启动子沉默,从而导致tnpR的诱导,从而导致卡带的切除和不可逆丢失。因此,可以通过Km S / Suc R的表型变化来监测体内定居过程中res盒的丢失,从而可以随后鉴定出与原始RIVET筛选相似的已分离菌株。除tetR以外,phoA还充当独立的转录报告基因,并通过碱性磷酸酶活性测定法对体内分离的菌株进行完善。

在基因特异性方法的情况下,三脚架使用相同的res-盒和pTRIVET元素,而TPC r -盒需要被亚克隆到侧翼通过上调和所需的整合位点,以确保同源重组的下游区域的pCVD442自杀载体(Donnenberg和Kaper,1991年)。一旦TPC r -盒已经融合到感兴趣的染色体启动子中,RES-CASS ETTE和pTRIVET随后可以动员和整合到基因组中。这样小号列车窝藏TPC r -盒的感兴趣的启动子的下游可用于测定特定条件下每种融合的抑制(例如,在体外和体内作为用于描述YRB -融合菌株)。例如,T他在相当于分辨率的体外和体内培养可以通过普拉蒂确定纳克上LB-KN(适当稀释未解决的CFU )和LB-AP(总的CFU )的板。可以通过从总CFU中减去总未分解CFU来计算已分解CFU [[Ap R CFU)减去(Km R CFU)] 。分辨率的程度由分辨率频率(给定%的分辨率),计算公式为解决CFU的量除以由总的CFU乘以100 (如果适用),phoA启动作为独立的转录报告并允许的转录活性的另外的评估所述TPC通过碱性磷酸酶试验,这将在本文中描述为-融合菌株IRGA下铁充足和铁-贫化的条件-融合菌株。

尽管TRIVET最初旨在识别肠道菌落定居过程中的基因阻抑,但仍可能适用于多种培养条件(Seper等人,2014 ; Cakar等人,2018; List等人,2018)。因此,这项技术可以帮助扩展我们对细菌基因调控,适应过程和调控级联的知识。

关键字:基因表达, 分辨率, 小鼠, 抑制, 筛选, 调节

材料和试剂
TRIVET系统的构建
细菌菌株和质粒
一种。大肠杆菌DH5αλ PIR ˚F -恩达1 glnV 44 THI -1的recA 1 RELA 1的gyrA 96 deoR nupG Φ80d的lacZ ΔM15Δ(lacZYA - ARGF )U 169 HSDR 17(R ķ -米ķ + )λ PIR RK6 (54)       

b。大肠杆菌XL-1个F ':: Tn10转的ProA + B + LAC q Δ(lacZ启动)M15 / recA基因1恩达1的gyrA 46(NAL - [R )THI HSDR 17(R ķ -米ķ + )SUPE 44 RELA 1个lac启动(新英格兰生物实验室)      

C。大肠杆菌SM10λ pirthi -1 THR亮氨酸TONA拉齐SUPE的recA :: RP4-2锝::穆λ PIR RK6 (Miller和Mekalanos,1988)       

d。E7946(O1 El Tor Inaba),Sm R的霍乱弧菌WT自发链霉素抗性(Sm R )突变体(Miller等人,1989)      

e。pCVD442自杀载体,OriR6K,sacB,Ap R (Donnenberg和Kaper,1991)       

F。pAC1000 Cm R (Hava et al。,2003)        

G。pGOA1193 pIVET5n tnpR,oriR6K mobRP4 lacZ tnpR,Ap R (Osorio et al。,2005)      

H。pGP704 oriR6K mobRP4,Ap R (Miller and Mekalanos,1988)      

一世。pTrc99A-Km pBR322来源,Km R (Amann等,1988)        

j。pRR51 (Reed和Grindley,1981年)        

k。pSL134 pPCR-Script::( res1 - res1 )(Osorio et al。,2005)      

l。pPCR-Script Amp SK(+)(Stratagene)        

米 pGAO2 [pPCR-Script::( res )(Osorio et al。,2005)]    

。pGAO3 [pPCR-Script::( res1 )(Osorio et al。,2005)]      

o。pGAO4 [pPCR-Script::( res - res )(Osorio et al。,2005)]      

p。pGAO5 [pPCR-Script::( res1 - res1 )(Osorio et al。,2005)]      

q。pGAO6 [pGOA4 :: neo - sacB (Osorio et al。,2005)]      

河 pGAO7 [pGOA5 :: neo - sacB (Osorio et al。,2005)]       

s。带res-neo-sacB-res盒的pRes pSL111,Km R (Osorio et al。,2005)       

t。带res1-neo-sacB-res1盒Km R的pRes1 pSL111 (Osorio et al。,2005)       

你 基于pLOFKm Tn10的传递质粒Km R ,Ap R (Herrero et al。,1990)      

寡核苷酸
一种。tetR-5'-BamHI AATGGATCCTAGAGTGTCAACAAAAATTAGGAATTA                                                                  

b。tetR-3'- Kpnl TTAGGTACCATCACGGAAAAAGGTTATGCT                                                                  

C。phoA-5'-KpnI TTTGGTACCTTTTTAATGTATTTGTACATGGAGAA                                                                   

d。phoA-3′-XbaI AAATCTAGACATTAAGTCTGGTTGCTAACAGCA                                                                  

e。猫5'-XbaI AAATCTAGATAAGCTTGATGAAAATTTGTTTGA                                                                  

F。cat-3'-BamHI TTTGGATCCTTCTTCAACTAACGGGGCA                                                                   

G。tetRphoAcat-5'-NotI TATGCGGCCGCCCCCTAGGTAATTAGGATCCTAGAGT                                                                  

H。tetRphoAcat-3'-NotI TTTGCGGCCGCCCTAGGTCTCATCCGCCAAAACAGCCAA                                                                  

一世。lacZ-5'-SacI TTTGAGCTCTGATTTACCGCCGCTGCCAA                                                                    

j。lacZ-3′-NheI TTTGCTAGCTTATTGTGGGGGAGATGACGCTTT                                                                    

k。tnpR-5′- BgIII CGACCCGGGAGATCTCAATTGTTCGAATTTAGGATACATTTTTAT                                                                  

l。tnpR-3′-XbaI TTTTCTAGATTAAGTTGGGTAACGCCAGGGT                                                                    

米 PtetA-5′- 3′CTAGCCAGAGAGCCTTAAGGCTCTCTTTTTTCTAATTTTTGTTGACACC                                                                

                            CTATCAGTGATAGAGTTATTTTACCACTCCCTATCAGTGATAGA

。PtetA-3′- 5′GATCTCTATCACTGATAGGGAGTGGTAAAATAACTCTATCACTGATAGG                                                                  

                            GTGTCAACAAAAATTAGAAAAAAGAGAGCCTTAAGGCTCTCTG G             

o。Ptet1-5′- 3′CTAGCAGAGAGCCTTAAGGCTCTCTTTTTTCTAATTTTTGTCCCTATCA                                                                  

                            GTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACATCA

p。Ptet1-3′- 5′GATCTGATGTGCTCAGTATCTCTATCACTGATAGGGATGTCAATCTCTAT                                                                  

                            CACTGATAGGGACAAAAATTAGAAAAAAGAGAGCCTTAAGG CTCTCTG             

q。1RES-F TCTATTGAATTCCGTCCGAAATATTATAAATTATCGCAC                                                                  

河 1RES-R TCTAATCTCGAGTGTATCCTAAATCAAATATCGGACAAG                                                                   

s。1RES1-F TCTAATGAATTCCGTCCGAAATATTACAAATTATCGCAC                                                                   

t。2RES-F TCTAATCTCGAGTCTAGACGTCCGAAATATTATAAATTATCGCAC                                                                   

你 2RES1-F TCTAATCTCGAGTCTAGACGTCCGAAATATTACAAATTATCGCAC                                                                  

诉2RES-R TCTAATGGTACCTGTATCCTAAATCAAATATCGGACAAG                                                                  

w。T3 AATTAA CCCTCACTAAAGGG                                                                 

X。T7 AATACGACTCACTATAGGGC                                                                  

用料
一种。移液器吸头(Greiner Bio-One,目录号:740290、739290和771291)       

b。PCR管(Thermo Fisher Scientific,目录号:AB0266)      

C。双重蒸馏H 2 O(ddH 2 O)       

d。dNTPs,10 mM(Thermo Fisher Scientific,目录号:R0192)      

e。DNA上染染料(6x)(Thermo Fisher Scientific,目录号:R0611)       

F。Q5高保真DNA聚合酶(NEB,货号:M0491S)        

G。Taq DNA聚合酶(NEB,货号:M0273S)      

H。GeneRuler 1 kb Plus DNA梯子(Thermo Fisher Scientific,目录号:SM1331)      

一世。QIAquickGel提取试剂盒(QIAGEN,目录号:28704)        

j。QIAquickPCR纯化试剂盒(QIAGEN,目录号:28104)        

k。QIAprep Spin Miniprep试剂盒(QIAGEN,目录号:27104)      

l。剪切智能缓冲区(NEB,目录号:B7204S)        

米 适当的限制酶(NEB)    

。南极磷酸酶(NEB,目录号:M0289S)      

o。T4 DNA连接酶(NEB,目录号:M0202S)      

p。T4 DNA连接酶缓冲液(NEB,目录号:B0202S)      

q。甘油(AppliChem,目录号:A0970,1000)      

河 酵母提取物(Applichem ,目录号:A1552)       

s。Bacto Tryptone(Thermo Fisher Scientific,目录号:211701)       

t。NaCl(VWR,目录号:27810.364)       

你 琼脂(Sigma-Aldrich,目录号:A5054)      

v。用于质粒载体的适当抗生素(例如,氨苄青霉素,卡尔·罗斯,目录号:K029.2)      

w。LB琼脂板含有用于质粒载体的适当抗生素(请参见食谱)     

X。LB培养基(请参阅食谱)      



TRIVET库的构建
细菌菌株和质粒
一种。大肠杆菌DH5αλ PIR ˚F -恩达1 glnV 44 THI -1的recA 1 RELA 1的gyrA 96 deoR nupG Φ80d的lacZ ΔM15Δ(lacZYA - ARGF )U 169 HSDR 17(R ķ -米ķ + )λ PIR RK6       

b。大肠杆菌SM10λ PIR THI -1 THR列伊托纳拉齐苏佩的recA :: RP4-2锝::穆λ PIR RK6      

C。E7946(O1 El Tor Inaba),Sm R的霍乱弧菌WT自发链霉素抗性(Sm R )突变体(Miller等人,1989)       

d。pCVD442自杀载体OriR6K,sacB和Ap R (Donnenberg和Kaper,1991)      

e。带res - neo - sacB - res盒的pRes pSL111 ,Km R (Osorio et al。,2005)       

F。带res1 - neo - sacB - res1盒的pRes1 pSL111 ,Km R (Osorio et al。,2005)        

G。pLOF :: tpc (Cakar等人,2018)      

H。pTRIVET (Cakar等人,2018)      

一世。pTRIVET1 (Cakar等人,2018)        

j。pRES [pSL111 ::((res - neo - sacB - res )(Osorio et al。,2005)]        

k。pRES1 [pSL111::( res1 - neo - sacB - res1 )(Osorio et al。,2005)]      

寡核苷酸
一种。tetRphoAcat-5'-SacI ATAGAGCTCAGAGTGTCAACAAAAATTAGGAATTAA                                                                       

b。tetRphoAcat-3'-SalI TTTGTCGACTTCTTCAACTAACGGGGCA                                                                       

C。irgA-SphI-1 AATGCATGCTCCGAGTAAACCGCAAACACTT                                                                       

d。irgA-SacI-2 AATGAGCTCGTATCCCGCCGCAGTGACCA                                                                       

e。irgA-SalI-3 AAAAGTCGACCGAACTATTCCATGTGT                                                                       

F。irgA-XbaI-4 AATTCTAGACGTGAGGTTTGGCGCTTA                                                                        

G。yrb_SacI_1 AAAGAGCTCGCGATATTGGCAATGTTTGAAC                                                                       

H。yrb_SphI_2 AAAGCATGCGATAAGGATAATTAATTGGAATC                                                                       

一世。yrb_SalI_3 AAAGAATTCTCAGGATGTCGACCTAACAG                                                                         

j。yrb_XbaI_4 TTTTCTAGAGATTAAGGTTACGGATCAGTTC                                                                         

k。seqPrimer-cat GAATTGTCAGATAGGCCTAATG                                                                       

用料
一种。PCR管(Thermo Fisher Scientific,目录号:AB1771)       

b。针26G(B 。布劳恩,目录号:4657683)      

C。管管(Thermo Scientific,目录号:10793527)       

d。针管注射器1毫升(Henry Schein,目录号:9003016)      

e。5日龄CD-1小鼠       

F。双重蒸馏H 2 O(ddH 2 O)        

G。dNTPs,10 mM(Thermo Fisher Scientific,目录号:R0192)      

H。DNA上染染料(6x)(Thermo Fisher Scientific,目录号:R0611)      

一世。Q5高保真DNA聚合酶(NEB,货号:M0491S)        

j。Taq DNA聚合酶(NEB,货号:M0273S)        

k。GeneRuler 1 kb Plus DNA梯子(Thermo Fisher Scientific,目录号:SM1331)      

l。QIAquickGel提取试剂盒(QIAGEN,目录号:28704)        

米 QIAquickPCR纯化试剂盒(QIAGEN,目录号:28104)    

。QIAprep Spin Miniprep试剂盒(QIAGEN,目录号:27104)      

o。QIAprepMidiprep套件(QIAGEN,目录号:12143)      

p。剪切智能缓冲区(NEB,目录号:B7204S)      

q。适当的限制酶(NEB)      

河 南极磷酸酶(NEB,目录号:M0289S)       

s。T4 DNA连接酶(NEB,目录号:M0202S)       

t。T4 DNA连接酶缓冲液(NEB,目录号:B0202S )       

你 酵母提取物(Applichem,目录号:A1552)      

诉的NaCl(VWR,目录号:27810.364)      

w。Bacto Tryptone(Thermo Fisher Scientific,目录号:211701)     

X。琼脂(Sigma-Aldrich,目录号:A5054)      

y。质粒载体的适当抗生素(例如氨苄青霉素,卡尔·罗斯,目录号:K029.2)      

z。Tris(Sigma-Aldrich,目录号:10708976001)       

啊 MgSO 4 (Sigma-Aldrich,目录号:M7506)   

bb。ZnCl 2 (西格玛奥德里奇,目录号:746355)   

抄送 SDS(Sigma-Aldrich,目录号:L3771)    

dd。氯仿(Sigma-Aldrich,目录号:372978)   

ee。KH 2 PO 4 (Sigma-Aldrich,目录号:P0662)   

ff。磷酸对硝基苯酯(Sigma-Aldrich,目录号:4876)     

gg。EDTA (Sigma-Aldrich,目录号:03620)   

嗯 异氟烷IsoFlo(Zoetis,目录号:50019100)  

ii。琼脂糖peqGOLD(VWR,目录号:732-2789)      

j 50x TAE缓冲液(Thermo Scientific,目录号:10399519)      

kk。2,2'-联吡啶(Sigma-Aldrich,目录号:D216305)   

二。FeSO 4 (Sigma-Aldrich,目录号:450278)      

毫米 X-Gal(5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷; Thermo Scientific,目录号:R0404)

nn。DMSO(Roth,目录号:4720)  

喔 LB琼脂板含有用于质粒载体的适当抗生素(请参见食谱)   

第LB培养基(见配方)  

qq。蔗糖琼脂平板(请参见食谱)   

 

设备

 

移液器(Eppendorf)
组合兽医异氟烷给药仪(Rothacher,目录号:CV 30301)
剪刀(Thermo Scientific,目录号:10694962)
镊子(Thermo Scientific,目录号:15809651)
组织匀浆器(Biospec,目录号:985370)
Vortexer(Thermo Fisher Scientific)
孵化器(Thermo Fisher Scientific,ThermoScientific TM ,型号:Heraeus B12功能线,目录号:50042307)
微量仪(Eppendorf,型号:CentrifugeMiniSpin ®,目录号:5452000018)
热循环仪(Bio-Rad C1000)
NanoDrop TM (ND 2000)
光度计(贝克曼DU730)
 

软件

 

blastN(blast.jcvi.org/cmr-blast/)
T m计算器软件(例如NEB T m计算器)
 

程序

 

通用协议
PCR扩增片段以构建质粒
对于PCR反应,请遵循各个制造商的规程。简而言之,温度计算如下:将每个杂交AT对的温度为2°C,将每个GC对的温度为4°C。在扩增的片段的长度之后计算延伸时间,并且循环量为30。基于本文使用的试剂,用于PCR扩增用于质粒质粒构建的片段的PCR反应的标准配方为:

试剂量(微升)                           

染色体DNA 8                                                                                                                                                                       

dNTP的5                                                                     

底漆I(1 µg / ml)5                           

底漆II(1 µg / ml)5                           

GC增强剂20                           

Q5聚合酶3                           

Q5聚合酶缓冲液25                           

ddH 2 O 32             

一种。进行PCR反应。       

b。向PCR反应中添加适量的加载染料,并使用0.8至1%的琼脂糖凝胶通过水平凝胶电泳分离条带。10 V每厘米凝胶。      

C。从琼脂糖凝胶中提取条带,并按照制造商的说明通过QIAGEN凝胶提取试剂盒纯化相应的PCR片段。       

菌落PCR
对于PCR反应,请遵循各个制造商的规程。用于菌落PCR的模板源自使用移液器尖端从琼脂板中挑选的菌落。将选定的菌落重悬于50 µl ddH 2 O中,并在100°C下煮沸10分钟。基于本文使用的试剂,用于PCR扩增片段以构建质粒的PCR反应的标准配方为:

试剂量(微升)                           

模板(殖民地重煮)3             

dNTP的0.5                           

底漆I(1 µg / ml)0.5                           

底漆II(1 µg / ml)0.5                           

热聚合酶缓冲液2.5                           

Taq聚合酶0.25                           

ddH 2 O 17.75                                                       

一种。进行PCR反应。       

b。向PCR反应中添加适量的上染染料,并使用0.8至1%的琼脂糖凝胶通过水平凝胶电泳分离条带,并施加约10,000倍。10 V每厘米凝胶。      

限制性消化
对于限制性摘要,请遵循各个制造商的规程。基于本文使用的试剂进行限制性消化的标准配方为:

试剂量(微升)                                         

质粒(通过QIAGEN质粒Midi试剂盒分离; 100 ng / µl)5                           

限制酶I 1.5                           

限制酶II 1.5                           

10x缓冲液(适合酶)10                           

ddH 2 O 81                           

要么

试剂量(微升)                           

PCR片段(通过QIAGEN凝胶提取试剂盒进行凝胶纯化; 100 ng / µl)15                            

限制酶I 1.5                                                       

限制酶II 1.5                           

10x缓冲液(适合酶)10                           

ddH 2 O 71             

一种。使用APPR执行过夜(ON)消化opriate限制性内切酶组合根据S和温度到制造商的指示。在PCR片段的情况下,本文合适的限制酶由寡核苷酸的名称指示(参见上面的寡核苷酸列表)。       

b。在限制性消化的情况下,使用PLA小号米IDS,去磷酸化5 ' -ends通过加入1在37℃微升南极磷酸酶的混合物,并培育1个小时下进行。      

C。向样品中添加适量的上样染料,并使用0.8%至1%的琼脂糖凝胶通过水平凝胶电泳分离条带,并施加约5%的染料。10 V每厘米凝胶。       

d。从琼脂糖凝胶中精确条带化,并按照制造商的说明通过QIAGEN凝胶提取试剂盒从凝胶中纯化线性化,去磷酸化的质粒。      

e。如果使用PCR片段进行限制性酶切,请在65°C灭活10分钟。       

F。随后按照制造商的说明使用QIAGEN PCR纯化试剂盒纯化消化的PCR片段。        

结扎
对于结扎,请遵循各个制造商的规程。基于本文使用的试剂进行限制性酶切的标准配方为:

试剂量(微升)                                         

质粒(消化+去磷酸酶; 30 ng / µl)5             

PCR片段I(已消化; 30 ng / µl )5.75             

PCR片段II(已消化; 30 ng / µl )5.75             

10x DNA连接酶缓冲液1.5             

T4 DNA连接酶1             

一种。在室温下连接1小时。       

b。在65°C灭活连接酶10分钟。      

PhoA分析
在体外测试条件下将菌株培养至对数生长期后期(例如,此处LB在37°C和180 rpm下(在irgA融合菌株的情况下补充2,2'-联吡啶或FeSO 4 ))。
使用微量离心机(5分钟,6,000 xg ,室温)沉淀每种培养物1 ml 。
在P1缓冲液(10 mM Tris-HCl,pH 8.0,10 mM MgSO 4 )中洗涤细胞一次,然后将沉淀重悬于1 ml P1缓冲液中。
在0.9 ml P1缓冲液中稀释来自步骤A5c的0.1 ml重悬细胞,并测量OD 600 。
将来自步骤A5c的0.1 ml重悬细胞添加到0.9 ml P2缓冲液(1 M Tris-HCl pH 8.0,0.1 mM ZnCl 2 )中。同时,准备没有细胞的阴性对照。
向每个样品中加入50 µl的0.1%SDS和50 µl的氯仿,涡旋振荡并在室温下孵育10分钟以使细胞通透。
要开始酶促反应,向每个试管,涡旋和记录时间添加0.1 ml的0.4%对硝基苯基磷酸酯(在1 M Tris-HCl中,pH 8.0)。
孵育每个试管直至出现浅黄色,然后加入120 µl 0.1 M EDTA,pH 8.0、1 M KH 2 PO 4终止反应并记下时间。
使用阴性对照样品作为空白,测量每个样品的OD 420 。
计算反应开始和停止之间的持续时间。
使用数据分析一章中所述的公式计算PhoA活性。
 

      鉴定霍乱弧菌体内阻抑(ivr)基因的随机方法
TRIVET组件的构造
TRIVET系统需要部分基于RIVET的组件构造几个元素(Osorio等,2005)。

一种。通过转座子诱变构建用于随机整合到细菌染色体中的tetR-phoA-cat (tpc )盒。       

为了获得最佳的PCR结果,请计算所有给定寡核苷酸的最佳T m 。
PCR使用来自大肠杆菌XL-1的染色体DNA和寡核苷酸对tetR-5'-BamHI和tetR-3'-Kpnl扩增无启动子的tetR片段(有关详细信息,请参阅Ste p A1)。
PCR扩增的无启动子的phoA启动-Fragment使用染色体DNA从SM10λ PIR寡核苷酸对phoA启动-5'- KpnI和phoA启动-3'-的XbaI (见S TE p A1的详细信息)。
PCR从pAC1000扩增cat 。寡核苷酸将cat-5'与XbaI和cat-3'与BamHI配对(有关详细信息,请参阅Ste p A1)。
凝胶纯化PCR片段,然后进行限制性酶切和第二次纯化(有关详细信息,请参阅Ste ps A1和A3 )。
消化(使用BamHI),去磷酸化并凝胶纯化pTrc99A-Km(有关详细信息,请参阅Ste p A3)
将消化的tetR- ,phoA-和猫片段连接到BamHI消化并去磷酸化的pTrc99A-Km中,以获得pTrc-tpc质粒(有关详细信息,请参阅Ste p A4)。
转化到感受大肠杆菌DH5αλ PIR细胞并通过在LB琼脂电镀补充有卡那霉素(KM)和氯霉素(CM)中进行选择。
在37°C下孵育平板过夜。
使用寡核苷酸对tetR-5'-BamHI和cat-3'-BamHI通过菌落PCR确认正确的pTrc-tpc构建体。
用正确的pTrc-tpc构建体在LB-Km / Cm + 2%葡萄糖(Glc)中生长验证的菌落,在37°C和180 rpm下过夜。
使用QIAprep MidiprepKit隔离pTrc-tpc。
PCR扩增整个tetR的- phoA启动-猫(TPC使用寡核苷酸对tetRphoAcat -5'- NotI和tetRphoAcat -3'- NotI位)r -盒(见S TE p A1的详细信息)。
凝胶纯化PCR片段,然后用NotI进行限制性酶切消化,然后进行第二次纯化(有关详细信息,请参阅Ste ps A1和A3)。
结扎纯化,NotI位消化的TPC r -盒到NotI位消化,脱磷酸的pLOFKm获得pLOF :: TPC(见S TE PS A1,A3和A4的详细信息)。
变换连接产物到感受大肠杆菌DH5αλ PIR细胞和琼脂补充有氨苄青霉素(AP)与Cm通过电镀在LB选择。
在37°C下孵育平板过夜。
使用寡核苷酸对tetR-5'-BamHI和cat-3'-BamHI,通过菌落PCR确认正确的pLOF :: tpc构建体(有关详细信息,请参阅Ste p A2)。
在LB-Ap / Cm + 2%Glc中以正确的pLOF :: tpc构建物生长经过验证的菌落,在37°C和180 rpm下过夜。
按照制造商的说明,使用QIAprep Spin Miniprep Kit分离pLOF :: tpc 。
变换PRES和前S1到感受SM10λ PIR细胞,并通过电镀在LB琼脂中选择补充有鸭。
在37°C下孵育平板过夜。
b。pTRIVET和pTRIVET1质粒的构建      

使用寡核苷酸对lacZ-5'-SacI和lacZ-3'-NheI,PCR从霍乱弧菌WT染色体DNA扩增lacZ片段。
PCR使用寡核苷酸对tnpR-5'-BgIII和tnpR-3'-XbaI从pGOA1193扩增tnpR片段。
凝胶纯化的PCR片段,随后restric吨离子消化和第二纯化(见S TE PS A1和A3的详细信息)。
混合寡核苷酸对PtetA-5'-3'和PtetA-3'-5'或Ptet1-5'-3'和Ptet1-3'-5',煮沸寡核苷酸混合物5分钟以确保变性并在室温下冷却。这将允许寡核苷酸对在每个末端上具有相容的NheI和BglII位点杂交。生成的ds DNA序列包含原始的tetA启动子序列p tet (在PtetA-5'-3'和PtetA-3'-5'的情况下),或者对于不太严格的转录调控,稍微改变的tetA启动子序列p tet1具有突变TetR结合位点(在Ptet1-5'-3'和Ptet1-3'-5 '的情况下)。
将消化的lacZ片段,消化的tnpR片段和两个tetA启动子序列版本(p tet或p tet1 ,通过步骤B1bviii获得)之一连接到SacI / XbaI消化的,去磷酸化的pGP704中,从而得到包含启动子的pTRIVET p tet或pTRIVET1分别包含启动子p tet1 (有关详细信息,请参阅步骤A4)。
变换连接产物到感受DH5αλ PIR细胞,并通过电镀在LB琼脂中选择补充有鸭。
在37°C下孵育平板过夜。
确认正确pTRIVET或pTRIVET1 CON小号使用寡核苷酸对通过菌落PCR tructs lacZ启动-3'- SacI和TNPR -3'-的XbaI以及限制分析。
在37°C和180 rpm下,在LB-Ap + 2%Glc中用正确的pTRIVET或PTRIVET1培养经过验证的菌落过夜。
通过QIAprep Spin Miniprep试剂盒按照制造商的规程分离pTRIVET和pTRIVET1质粒。
变换pTRIVET和pTRIVET1到感受SM10λ PIR细胞,并通过电镀在LB琼脂中选择补充有鸭。
在37°C下孵育平板过夜。
C。根据Osorio等人的方法构建pRes和pRes1 。(2005年)       

PCR使用pRR51作为模板,通过寡核苷酸对1RES-F和1RES-R通过PCR扩增res序列(有关详细信息,请参见步骤A1)。或者,PCR使用pSL134和寡核苷酸对1RES1-F和1RES1-R通过PCR扩增res1序列(有关详细信息,请参见步骤A1)。
凝胶纯化PCR片段,然后进行限制性酶切(使用EcoRI和XhoI)和第二步纯化(有关详细信息,请参见步骤A1和A3)。
结扎消化的PCR片段引入到Ñ的EcoRI / XhoI位消化,脱磷酸的PPCR脚本放大器SK(+)(Stratagene)中以产生质粒pGOA2和pGOA3(详见步骤A4)。
使用第二组寡核苷酸对(2RESF和2RESR或2RES1F和2RESR)进行PCR扩增res或res1的第二个拷贝。将它们紧接着与它们在pGOA2和pGOA3中的对应物亚克隆以生成质粒pGOA4和pGOA5,它们分别具有两个res或res1序列。
通过将sacB-neo基因从pGOA1亚克隆到XbaI消化的pGOA4和pGOA5中,生成pGOA6和pGOA7。
使用寡核苷酸对T3和T7,通过PCR从pGOA6和pGOA7扩增res-和res1-cassettes,并将它们连接到经T4 DNA聚合酶处理的KpnI消化的pSL111中,以生成pRES和pRES1。
变换LIG通货膨胀产品到感受DH5αλ PIR细胞,并通过电镀在LB琼脂中选择补充有鸭。
在37°C下孵育平板过夜。
通过限制性酶切分析和测序确认正确的pRES和pRE S1构建体。
按照制造商的规程,通过QIAprep Spin Miniprep Kit分离pRES和pRES1质粒。
变换PRES和前S1到感受SM10λ PIR细胞,并通过电镀在LB琼脂中选择补充有鸭。
在37°C下孵育平板过夜。
TRIVET库的构建
一种。在插入资源-盒       

通过缀合和等位基因交换将抗性自杀质粒pRES和pRES1插入霍乱弧菌WT中,以将每个盒放入lacZ基因座中,从而获得Vc_res和Vc_res1,如下所述。
动员自杀质粒PRES或前S1到霍乱弧菌通过缀合大肠杆菌SM10λ PIR的pR ES或前S1与获得由步骤B1C霍乱弧菌WT [(链霉素抗性(SM - [R )。这是通过交叉划线足以进行两种菌株的细胞材料在同一LB琼脂平板上,于37°C孵育6小时。通过pRES或pRES1上存在的同源区域,自杀质粒和霍乱弧菌染色体上的lacZ基因座的等位基因交换是可能的通过存在于pRES或pRES1上的同源区域以低频率进行检测。
然后通过在LB- Sm / Ap / Km琼脂平板上划线形成结合混合物中的单个菌落,选择带有整合的pRES和pRES1的霍乱弧菌。
在37°C下孵育平板过夜。
在LB-Sm / Ap / Km琼脂平板上条纹纯化菌落至少两次。
生长在LB-SM /公里纯化的菌落在37℃下过夜,以180rpm和在LB-SM板适当稀释/公里PL茨补充有5-溴-4-氯-3-吲哚基的β-d吡喃半乳糖苷(40微克/ ml溶于DMSO,X-Gal)。
在37°C下孵育平板过夜。
白色菌落是双重组事件和lacZ基因座与res或res1盒的等位基因交换的潜在候选者。
通过在LB-Sm / Km(生长)和LB-Ap(无生长)上划线确认自杀载体主链的丢失。
将平板在37°C下孵育过夜,然后以Sm R / Km R进行培养,但出现Ap S菌落。
验证通过菌落PCR和/或测序确定res或res1-盒在lacZ基因座中的正确插入,产生菌株Vc_res和Vc_res1。
b。Vc_res和Vc_res1中的转座子诱变。      

转座诱变是通过将pLOF :: tpc转座子质粒动员到V中来实现的。如下所述通过缀合形成霍乱。在较长的潜伏期中,质粒的转移导致霍乱弧菌的自发转座事件。
动员自杀载体pLOF :: TPC入霍乱弧菌通过缀合大肠杆菌SM10λ PIR pLOF从步骤菌素B1a :: TPC得到任一Vc_res和Vc_res1从得到的步骤B 2a中通过过滤器的配合在LB琼脂平板上。为此,将菌株生长到OD 600约为。1,然后以2:1的比例混合受体和供体,最终体积为2 ml。
颗粒混合物通过离心(5 ,000 ×g下,5分钟,RT),并在100μl的LB肉汤轻轻重悬细胞。
将细菌悬液放置在LB板的中心,并在37°C下孵育1.5 h,以使其结合。
使用移液器吸头吸取板中的细菌,并将其重悬于1 ml LB肉汤中。
通过铺板适当稀释的重悬缀合混合物,以达到每个LB- Sm / Km / Cm平板约50-200个菌落,选择转座子突变体。
在37°C下孵育平板过夜。
通过在LB-Sm / Km / Cm琼脂平板上划线并平行在LB-Ap琼脂平板上纯化菌落。在37°C下孵育过夜后,仅对LB-Sm / Km / Cm耐药,但对Ap敏感的菌落代表真正的转座子突变体(pLOF主链丢失)。
通过在平板顶部添加1-2 ml LB,并在整个菌落上漂洗多次,合并约500个真正的菌落,以产生异质气源性。
重复步骤的B2Bi至B2bvi约20倍为Vc_res以及Vc_res1以获得用于多个独立的转座子池Vc_res分别或Vc_res1。
C。pTRIVET和pTRIVET1的集成       

动员自杀质粒pTRIVET或pTRIVET1入霍乱弧菌由步骤的B2B获得插入转座子池的三脚架或pTRIVET1经由下游同源重组的lacZ -locus如下所述。
为了这样做,的横条纹足够的细胞材料大肠杆菌SM10λ PIR pTRIVET或大肠杆菌SM10λ PIR从获得pTRIVET1步骤与B1b矮秆基因霍乱弧菌由步骤的B2B获得转座子池(见步骤B2aii细节)。
在1 ml LB肉汤中重悬缀合混合物,并通过将适当重悬的缀合混合物稀释液铺在LB- Sm / Km / Ap板上选择插入pTRIVET或pTRIVET 。
在37°C下孵育平板过夜。
通过在平板顶部添加1-2 ml LB并合并多个漂洗液,合并约2,000个克隆以维持异质性。这些池代表最终的TRIVET池。
鉴定IVR基因
一种。预筛选,以消除由于TetR表达低而导致的高分辨率频率的融合菌株。       

让每个TRIVET池在LB-Sm / Ap肉汤中生长到对数后期,而不用Km作为维持res或res1盒式磁带的选择标记。这允许以不足的TetR表达来沉默tnpR的融合菌株的解析。
在LB-Sm / Km / Ap平板上连续平板稀释,并在37°C下孵育过夜,以选择TRIVET池中未分离的菌株(res-或res1盒在体外条件下稳定保持)。
合并约1,000-2,000个菌落以通过在平板顶部添加1-2 ml LB并在菌落上漂洗多次来维持异质性。(可选:要确认异质性,请使用Southern印迹分析从50个随机选择的菌落中检测tpc- fusion基因座的限制性片段长度多态性。)
b。使用婴儿小鼠模型(体内)筛选霍乱弧菌的ivr基因。      

在LB-Sm / Km / Ap平板上一式三份地分散每个库的等分试样,并在37°C下孵育过夜。
从每个板中收集约5,000个菌落,并在LB中稀释至约10 6 cfu / ml的浓度。
使用50微升细菌悬液在胃内接种5 d大CD-1小鼠(由异氟烷麻醉)。
在感染后22 h通过颈椎脱位安乐死小鼠,去除小肠,并使用组织匀浆器在中等功率下上下移动,在含有20%G甘油的1 ml LB培养基中匀浆30 s。
在蔗糖琼脂上平板接种系列匀浆稀释液,以选择sacB的丢失(选择现在缺少res或res1盒的体内分离菌株)。
在室温下孵育48小时。
确认分辨率(通过在LB-SM /鸭(生长)的平行条纹水库或RES1盒的损失d LB-公里(无生长)。
在37°C下孵育平板过夜,然后根据S tep B 3d鉴定具有Sm / Km抗性但Km敏感菌落的tpc盒插入位点。
C。PhoA分析是体内分离菌株的完善步骤       

基于以前使用拆分技术的研究,由于某些TRIVET文库菌株中TetR的体外表达水平较低,因此体内筛选具有相当大的假阳性自发拆分率。实施phoA启动作为第二报告使得能够在相当大的表达水平的确认体外生长的每个的体内在步骤B3b上确定解决菌株。我们强烈建议您执行此优化步骤,以消除误报。
为了降低相对较高的基于分辨酶的筛选的假阳性率(约15%),必须将自发的体外分辨频率定义为临界值。重新分析以前基于分辨酶的筛选所获得的合并数据(Osorio等人,2005; Schild等人,2007; Seper等人,2014)发现,体外分离频率低于30%的菌株只有5%有机会是假阳性。由于tpc盒保持稳定整合在染色体中,可以在体外条件下测定体内分离株的PhoA活性,并将获得的活性与分离频率相关。根据针对irgA融合测试菌株获得的结果,体外解析频率为30%或更低与10 Miller单位或更高的PhoA活性相关。因此,我们在描述TRIVET的原始研究中使用了该临界值(Cakar等人,2018)。
鉴定在可控的体外培养条件下受到不同调控的启动子。在本文中,我们使用霍乱弧菌的铁调节的irgA启动子,该启动子在低铁水平下高表达,在高铁浓度下被抑制。通过添加不同量的铁螯合剂2,2'-联吡啶或FeSO 4可以在LB中模拟体外的差异铁可用性。
构建霍乱弧菌三脚架报告菌株Vc_res_多脚架IRGA :: TPC与的特定转录融合TPC r -盒的铁调节IRGA启动子(详见步骤C1)。
平行评估Vc_res_TRIVET irgA :: tpc报告基因在LB中生长8小时后的解析频率和PhoA活性,并补充不同量的铁螯合剂,例如2,2'-联吡啶或FeSO 4 (有关详细信息,请参见步骤C2和C3) )。对于两种试剂,我们建议以50 µM的增量测试0至150 µM的最终浓度范围。
所述Vc_res_的相关成分的PhoA活性和分辨率频率多脚架IRGA :: TPC在LB菌株生长的不同的铁的可用性。选择最接近临界值的生长条件(低于30%的分辨频率),并使用相关的PhoA活性作为精制标准(在我们的情况下为10 Miller单位)。
在LB中于37°C和180 rpm下培养体内分离的菌株8 h。
如上所述执行PhoA分析(有关详细信息,请参阅步骤A5)。
仅对显示PhoA活性等于或高于精制标准(如上确定)的体内分离菌株进行处理。
d。通过亚克隆和测序鉴定tpc融合蛋白      

从体内分离的目标菌株的过夜培养物中分离染色体DNA 。
用EcoRI消化染色体DNA,并使用消化的染色体DNA与EcoRI消化的去磷酸化pBR322质粒连接(有关详细信息,请参见步骤A3和A4)。
变换连接产物进大肠杆菌DH5αλ PIR和板在LB-CM平板以选择猫的TPC-盒包括下游染色体DNA的-基因TPC -融合位点。
使用QIAprep Spin Miniprep Kit分离Cm R-克隆的质粒。
用寡核苷酸seqPrimer-cat进行测序,以获得下游序列tpc-融合位点。
使用blastN识别霍乱弧菌N16961基因组数据库的BLAST序列,以鉴定tpc盒式磁带的插入位点。
 

      分辨率测定以测量基因沉默
自杀质粒的TPC盒到特异启动子的转录融合,通过举例说明的构建IRGA-TPC或YRB - TPC融合。
一种。自杀质粒的构建。       

在CAS E中的IRGA-TPC融合,PCR扩增TPC使用r -盒的寡核苷酸对tetRphoAcat -5'- SacI和tetRphoAcat -3'- SalI位以及子Ptrc-TPC作为模板(见步骤A1的详细信息)。
PCR使用寡核苷酸对irgA-SphI-1和irgA-SacI-2以及irgA-SalI-3和irgA-XbaI-4扩增位于irgA插入位点上游和下游(irgA启动子)的800 bp片段(请参见有关详细信息,请执行步骤A1。
在该情况下YRB-TPC融合,PCR扩增TPC r -盒用寡核苷酸对tetRphoAcat-5 ' -SacI和tetRphoAcat-3 ' -SalI以及子Ptrc-TPC作为模板(详见步骤A1)。
PCR扩增位于上游和下游的800bp的片段YRB -insertion位点(YRB所述的启动子)的TPC使用的寡核苷酸对,yrb_SacI_1和yrb_SphI_2 r -盒以及yrb_SalI_3和yrb_XbaI_4。
凝胶纯化PCR片段,然后进行限制性酶切和第二次纯化(有关详细信息,请参见步骤A1和A3)。
在的情况下,IRGA-TPC融合,结扎消化的TPC-盒以及所述上游和下游片段(参见步骤C1ai和ii)进入一个SphI位/ XbaI位消化,脱磷酸的pCVD442。
在该情况下YRB-TPC融合,结扎消化的TPC-盒以及所述上游和下游片段(参见步骤C1aii和iv)成一个SphI位/ XbaI位消化,脱磷酸的pCVD442。
变换连接产物到感受DH5αλ PIR细胞,并通过电镀在LB琼脂中选择补充有鸭。
在37°C下孵育平板过夜
确认正确pCVD442 IRGA :: TPC或pCVD442yrb :: TPC CON小号tructs通过使用寡核苷酸对菌落PCR IRGA-SphI位-1和IRGA-的XbaI-4或yrb_SphI_2和yrb_XbaI_4 ,分别。
在37°C和180 rpm下,在LB-Ap中用正确的pCVD442 irgA :: tpc或pCVD442yrb :: tpc生长经过验证的菌落过夜。
通过QIAprep Spin Miniprep试剂盒按照制造商的规程分离pCVD442 irgA :: tpc或pCVD442yrb :: tpc质粒。
变换pCVD442 IRGA :: TPC或pCVD442yrb :: TPC到感受SM10λ PIR细胞,并通过在LB琼脂电镀补充有鸭选择。
在37°C下孵育平板过夜。
b。三脚架携带的菌株具体的施工TPC -融合到感兴趣的基因。      

为了获得Vc的IRGA :: TPC和Vc YRB :: TPC ,发动自杀质粒pCVD442irgA :: TPC或pCVD442yrb :: TPC入霍乱弧菌通过接合和等位基因交换以将TPC r -盒的各启动子的下游。
动员自杀质粒pCVD442 IRGA :: TPC或pCVD442yrb :: TPC入霍乱弧菌通过缀合大肠杆菌SM10λ PIR pCVD442irgA :: TPC或pCVD442yrb :: TPC由S取得TE p庆大霉素C1a与霍乱弧菌WT [(链霉素抗性(Sm R )]。为此,在相同的LB琼脂平板上交叉划线两个菌株的足够细胞材料,并在37°C下孵育平板6 h。通过pCVD442irgA :: tpc或pCVD442yrb :: tpc上存在的同源区域自杀质粒与霍乱弧菌染色体上各个基因座的等位基因交换可能是低频的。
单个菌落的条纹形成在LB-Sm / Ap琼脂平板上的缀合混合物,选择带有整合的pCVD442 irgA :: tpc或pCVD442yrb :: tpc的霍乱弧菌。
在37°C下孵育平板过夜。
在LB-Sm / Ap琼脂平板上条纹纯化菌落至少两次。
将纯化的菌落在LB-Sm中于37°C,180 rpm培养过夜,然后将适当的稀释液铺在蔗糖琼脂上。
在室温下孵育板长达48小时。
通过在LB-Sm(生长)和LB-Ap(无生长)上划线确认自杀载体主链的丢失
将平板在37°C下孵育过夜,然后用Sm R克隆,但用Ap S克隆。
所述的验证正确的插入TPC r -盒通过菌落PCR和/或导致在VC测序IRGA :: TPC和Vc YRB :: TPC 。
将res-或res1-盒式磁带插入Vc irgA :: tpc或Vc yrb :: tpc中,以获得Vc_res irgA :: tpc ,Vc_res1 irgA :: tpc ,Vc_res yrb :: tpc或Vc_res1 yrb :: tpc如上所述(请参见步骤) B2a了解详情)。
整合pTRIVET或pTRIVET1成Vc_res IRGA :: TPC ,Vc_res1 IRGA :: TPC ,Vc_res YRB :: TPC或Vc_res1 YRB :: TPC以获得Vc_res_TRIVET IRGA :: TPC ,Vc_res1_TRIVET IRGA :: TPC ,Vc_res_TRIVET YRB :: TPC或Vc_res1_TRIVET YRB :: TPC以及Vc_res_TRIVET1 IRGA :: TPC ,Vc_res1_TRIVET1 IRGA :: TPC ,Vc_res_TRIVET1 YRB :: TPC或Vc_res1_TRIVET1 YRB :: TPC如上所述(详见步骤B2C)。
基因沉默的定量
一种。体内和体外分辨率测定       

用tpc盒与目标启动子的转录融合体生长TRIVET菌株[例如,在Cakar等人中使用的Vc_res1_TRIVET yrb :: tpc 。(2018)]在LB-Sm / Km / Ap板上; 在37°C下过夜。
从板中收获细菌细胞,并将其重悬于LB-Sm / Km / Ap中。
测量OD 600和OD调整600使用适当的稀释液,以产生接种物为1。
为了评估在体内的分辨率,感染5-d-旧CD-1小鼠(通过异氟烷麻醉)用50μl的接种物(的〜10 6 CFU)。在感兴趣的感染后时间点(例如Zingl等人所用的6和22 h)。(2020)通过使颈椎脱位使小鼠安乐死,去除小肠并在含有20%甘油的1 ml LB培养基中匀浆。在LB-Sm / Km和LB-Sm / Ap板上平板接种适当的系列匀浆稀释液,并在3 7°C下孵育过夜。
要评估体外分辨率,请在50 ml接种物中接种5 ml LB-Sm / Ap,并在37°C和180 rpm下与小鼠感染平行孵育。在LB-Sm / Km和LB-Sm / Ap平板上平板接种适当的系列匀浆稀释液,并在37°C下孵育过夜。
评估平板上单个菌落的数量,并计算原始样品中抗Sm / Km和抗Sm / Ap的cfu。
计算数据分析一章中描述的体内和体外公式的分辨率(%)。
b。体外的PhoA测定作为替代quantific一个转录活性的灰。      

使用感兴趣的培养条件培养菌株。
如上所述执行PhoA分析(有关详细信息,请参阅步骤A5)。
 

数据分析

 

分辨率测定的结果将表示为分辨率百分比,它是使用给定培养样品中Sm / Km抗性和Sm / Ap抗性群体的测定cfu计算得出的。抗Sm / Ap的菌落反映了整个种群,抗Sm / Km的菌落反映了未分离的种群。从整个种群中减去未分离的种群得到了分离的种群(res- / res1-cassette的损失)。

  分辨率的计算公式为:

 

分辨率(%)= [ cfu(耐Sm / Ap )– cfu(耐Sm / Km )] / cfu(耐Sm / Ap )

 

要计算碱性磷酸酶活性,请使用以下公式:

 

活性(米勒单位)=(1,000 x OD 420 )/ [反应时间(分钟)x OD 600 ]

 

菜谱

 

LB肉汤/琼脂(g / L)
Bacto TM胰蛋白10 10克                           

酵母提取物5克                           

氯化钠10克                           

琼脂15克                           

蔗糖琼脂(g / L)
Bacto TM胰蛋白10 10克             

酵母提取物5克             

琼脂15克             

蔗糖100克             

如果需要,在高压灭菌后添加以下浓度的抗生素:抗生素和其他补充剂的最终浓度如下:链霉素(Sm,100 µg / ml),氨苄青霉素(Ap,50 µg / ml与其他抗生素联合使用,否则100 µg / ml),卡那霉素(Km,50 µg / ml)和氯霉素(Cm,霍乱弧菌为2 µg / ml ;大肠杆菌为10 µg / ml )。

 

致谢

 

感谢波士顿大学塔夫茨大学的安德鲁·卡米利(Andrew Camilli)提供了原始的RIVET组件,并为建立TRIVET系统提供了有益的讨论。这项工作得到了奥地利科学基金(FWF)的拨款W901(DK分子酶学)(授予FGZ和SS),27654和P25691(授予SS)的支持。

 

利益争夺

 

作者宣称没有利益冲突。

 

伦理

 

根据格拉茨大学伦理委员会的规则以及相应的动物规程,所有实验均使用动物,该规程已获得奥地利联邦科学与研究部的批准。II / 10b。随意给小鼠饲养食物和水,并在专职人员的照顾下进行监测。

 

参考文献

 

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引用:Zingl, F. G., Mitterer, F., Thapa, H. B. and Schild, S. (2020). TetR Regulated in vivo Repression Technology to Identify Conditional Gene Silencing in Genetically Engineerable Bacteria Using Vibrio cholerae Murine Infections as Model System. Bio-protocol 10(19): e3774. DOI: 10.21769/BioProtoc.3774.
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