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MATERIAL

materail

All materials used in the construction of Super BugBuster are indexed in this page.

Bacterial Strains

Strain Genotype Phenotype Reference/Origin
DH5a F– endA1 glnV44 thi-1 recA1 relA1 gyrA96 hsdR17(rK–mK+), Δ(lacZYA-argF)U169, deoR nupG purB20 φ80dlacZΔM15 λ– NalR NEB
DB3.1 F- gyrA462 endA1 ∆(sr1-recA) mcrB mrr hsdS20 glnV44 (=supE44) ara14 galK2 lacY1 proA2 rpsL20 xyl5 ∆leuB6 mtl1 Resistance to the CcdB toxin + SmR
TOP10/pOxa48 F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ- SmR AmpR Gived by C.Lesterlin
BL21/pLysE E. coli str. B F– ompT gal dcm lon hsdSB(rB–mB–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB+]K-12(λS) pLysS[T7p20 orip15A](CmR) CmR NEB
MFDpir MG1655 RP4-2-Tc::[ΔMu1::aac(3)IV-ΔaphA-Δnic35-ΔMu2::zeo] ΔdapA::(erm-pir) ΔrecA Auxotroph for DAP Contains genes encoding the RP4 conjugation machinery in its chromosome MAP collection Ferrières et al. (2010) https://doi.org/10.1128/jb.00621-10

Plasmid vectors

Name of plasmid Origin of replication Number of copies/cells Resistance Description Comment Provenance Séquences MAP
pEN-dcas9-CDA1-UGI pUC 100-500 KanR dCas9-CDA1-UGI expressed from the recA promoter, attR1 and attL4 recombination sites - Addgene
pdcas9 p15A 10-20 CmR dCas9 expressed from the anhydrotetracycline-inducible ptet promoter, TetR repressor - Addgene
pEN-sacB pUC 100-500 KanR Bacillus subtilis sacB gene encoding an enzyme that makes Gram-negative bacteria sensitive to sucrose, attL3 and attR2 recombination sites - Addgene
pEN-puro-R3-R4 pUC 100-500 KanR, PuroR AttR3 and attR4 recombination sites - Addgene
pMCF mScarlett pUC 100-500 KanR mScarlet gene encoding a red fluorescent protein expressed from the glpT promoter - Sobetzko P.
pT1 pUC 100-500 KanR ccdB, vioB, ptet promoter, gRNA backbone Replication in DB 3.1 strain Sobetzko P.
pH2-Cm ori2, pUC 1, 100-500 CmR ccdB, oriT RP4, repE for replication from ori2 of plasmid F, sopA, sopB, sopC for partition plasmid F Replication in DB 3.1 strain Sobetzko P.
pINS-Rif pUC 100-500 KanR, RifR rifR expressed from the synthetic promoter ptrc - Collection laboratory MAP
pENTR4-dual pUC 100-500 KanR, CmR ccdB, attL1 and attL2 recombination sites Replication in DB 3.1 strain Thermofisher
pUCIDTAmp-Fragment_1_ccdB_brick_H2 pUC 100-500 AmpR ccdB, attR4 recombination site - IDT
P206_MCF_EL_XE pUC 100-500 SmR Plasmid hosting the SmR gene, used to change the KanR gene into a SmR one in pV2-mScarlet and pV4-mScarlet to build pEDIT3a-oxa48-SmR - MAP collection
pENTR4-∆Bbs pUC 100-500 KanR, CmR ccdB, attL1 and attL2 recombination sites Replication in DB 3.1 strain This work
pEDIT1 ori2, pUC 1, 100-500 KanR, CmR oriT RP4, repE for replication from ori2 of plasmid F, attR3 and attR4 recombination sites - This work
pEDIT1-ccdB ori2, pUC 1, 100-500 KanR, CmR oriT RP4, repE for replication from ori2 of plasmid F, attR3 and attR4 recombination sites, ccdB - This work
pEDIT2-Cm pUC 100-500 KanR, CmR dCas9-CDA1-UGI expressed from ptet promoter, TetR repressor, attR1 and attL4 recombination sites - This work
pEDIT2 pUC 100-500 KanR dCas9-CDA1-UGI expressed from ptet promoter, TetR repressor, attR1 and attL4 recombination sites - This work
pHost-spacer1 pUC 100-500 KanR ccdB, ptet promoter, gRNA backbone, two BsaI sites to accommodate spacer 1 Replication in DB 3.1 strain This work
pHost-spacer2 pUC 100-500 KanR ccdB, ptet promoter, gRNA backbone, two BsaI sites to host spacer 2 Replication in DB 3.1 strain This work
pHost-spacer3 pUC 100-500 KanR ccdB, ptet promoter, gRNA backbone, two BsaI sites to accommodate spacer 3 Replication in DB 3.1 strain This work
pHost-spacer4 pUC 100-500 KanR ccdB, ptet promoter, gRNA backbone, two BsaI sites to accommodate spacer 4 Replication in DB 3.1 strain This work
pet28-Oxa pUC 100-500 plasmid used to overproduce oxa protein - This work
pEx-gRNA1-oxa48 pUC 100-500 KanR gRNA1-oxa48 expressed from the ptet promoter - This work
pEx-gRNA2-oxa48 pUC 100-500 KanR gRNA2-oxa48 expressed from the ptet promoter - This work
pEx-gRNA3-oxa48 pUC 100-500 KanR gRNA3-oxa48 expressed from the ptet promoter - This work
pEx-gRNA4-oxa48 pUC 100-500 KanR gRNA3-oxa48 expressed from the ptet promoter - This work
pV2-mScarlet pUC 100-500 KanR Plasmid hosting 2 tandem gRNAs, mScarlet flanked by two distinct BbsI sites, attL1 and attL2 recombination sites - This work
pV2-mScarlet pUC 100-500 Sm 4-gRNA host plasmid, tandem mScarlet flanked by two distinct BbsI sites, attL1 and attL2 recombination sites - This work
pV4-mScarlet pUC 100-500 KanR 4-gRNA host plasmid, tandem mScarlet flanked by two distinct BbsI sites, attL1 and attL2 recombination sites - This work
pV4-SmR-mScarlet pUC 100-500 SmR Plasmid hosting 2 tandem gRNAs, mScarlet flanked by two distinct BbsI sites, attL1 and attL2 recombination sites - This work
pEDIT3a-oxa48 pUC 100-500 KanR Plasmid expression of gRNA1-oxa48 and gRNA2-oxa48 in tandem from ptet promoters, attL1 and attL2 recombination sites - This work
pEDIT3a-oxa48 pUC 100-500 SmR Plasmid expression of gRNA1-oxa48 and gRNA2-oxa48 in tandem from ptet promoters, attL1 and attL2 recombination sites - This work

Primers

Primer number Primer name Primer sequence Matrix Usage
Pi1 ccdB_bbsI_fw_gRNA1 GCAGAAGACCATCCCTATCAG pT1 Construction pHost-spacer1
Pi2 ccdB_bbsI_rev_gRNA1 GCAGAAGACCAGGACCTGAAAAAAAACCCCGCCCT pT1 Construction pHost-spacer1
Pi3 ccdB_bbsI_fw_gRNA2 GCAGAAGACCAGTCCTCCCTATCAGTGATAGAGATTG pT1 Construction pHost-spacer2
Pi4 ccdB_bbsI_rev_gRNA2 GCAGAAGACCAAGAACTGAAAAAAAACCCCGCCCT pT1 Construction pHost-spacer2
Pi5 ccdB_bbsI_fw_gRNA3 GCAGAAGACCATTCTTCCCTATCAGTGATAGAGATTG pT1 Construction pHost-spacer3
Pi6 ccdB_bbsI_rev_gRNA3 GCAGAAGACCAACGGCTGAAAAAAAACCCCGCCCT pT1 Construction pHost-spacer3
Pi7 ccdB_bbsI_fw_gRNA4 GCAGAAGACCACCGTTCCCTATCAGTGATAGAGATTG pT1 Construction pHost-spacer4
Pi8 ccdB_bbsI_rev_gRNA4 GCAGAAGACCACTCCCTGAAAAAAAACCCCGCCCT pT1 Construction pHost-spacer4
Pi9 pINS_bbsI_fw_gRNA1 CGTCCCTGTCTTCCAGGACGAAAG pINS-Rif Construction pHost-spacer1
Pi10 pINS_bbsI_rev_gRNA1 CGGGATTGTCTTCGTCCAGACCAAG pINS-Rif Construction pHost-spacer1
Pi11 pINS_bbsI_fw_gRNA2 CTTCTCTGTCTTCCAGGACGAAAG pINS-Rif Construction pHost-spacer2
Pi12 pINS_bbsI_rev_gRNA2 CGGACTTGTCTTCGTCCAGACCAAG pINS-Rif Construction pHost-spacer2
Pi13 pINS_bbsI_fw_gRNA3 CAGAATTGTCTTCGTCCAGACCAAG pINS-Rif Construction pHost-spacer3
Pi14 pINS_bbsI_rev_gRNA3 CCCGTCTGTCTTCCAGGACGAAAG pINS-Rif Construction pHost-spacer3
Pi15 pINS_bbsI_rev_gRNA4 CACGGTTGTCTTCGTCCAGACCAAG pINS-Rif Construction pHost-spacer4
Pi16 pINS_bbsI_fw_gRNA4 CGGAGCTGTCTTCCAGGACGAAAG pINS-Rif Construction pHost-spacer4
Pi17 mScarlett2_bbsI_A1 GAGGTACCTCCCGAGTCTTCTATAAACGCAGAAAGGCCCACC pMCF mScarlett Construction pV2-mScarlet et pV4-mScarlet
Pi18 mScarlett2_bbsI_A2 TACTCGAGAGAATTGTCTTCGAAAGTGAAACGTGATTTCATG pMCF mScarlett Construction pV2-mScarlet
Pi19 mScarlett4_bbsI_A4 TACTCGAGCTCCTTGTCTTCGAAAGTGAAACGTGATTTCATG pMCF mScarlett Construction pV4-mScarlet
Pi20 S1_Oxa_fw GCACAATGGCAAGAAAACAAAAGT - Construction spacer S1-oxa48
Pi21 S1_Oxa_rev AAACACTTTTGTTTTCTTGCCATT - Construction spacer S1-oxa48
Pi22 S2_Oxa_fw GCACTGGTATTCGAATTTCGGCCA - Construction spacer S2-oxa48
Pi23 S2_Oxa_rev AAACTGGCCGAAATTCGAATACCA - Construction spacer S2-oxa48
Pi24 H2_CmR_OriT_fw GAGGTCTCAGGATGATAACAGGGTAATTCACGCCCCGCCCTGCCACTC pH2-Cm Construction pEDIT1
Pi25 H2_CmR_OriT_rev GAGGTCTCATCTCATCATCCTTAGCGAAAGCTTGCCTG pH2-Cm Construction pEDIT1
Pi26 H2_Ori2_RepE_fw GAGGTCTCAGAGAATAAATGCCTTGGCCTTTATATGG pH2-Cm Construction pEDIT1
Pi27 H2_Ori2_RepE_rev GAGGTCTCAGGAGATAAGAACACCACATAC pH2-Cm Construction pEDIT1
Pi28 H2_R3_R4_fw GAGGTCTCACTCCGTGGGCTTGTACTCGGTCAT pENTR puroR4-R3 Construction pEDIT1
Pi29 H2_R3_R4_rev GAGGTCTCAATCCCCCGCAAGCCCGGTGCCTGAAC pENTR puroR4-R3 Construction pEDIT1
Pi32 Oxa_pet28_BamH1_fw GAGGATCCaaggaatggcaagaaaacaaaagttg pOXA48 Oxa Overproduction
Pi33 Oxa_pet28_xhoI_rev GACTCGAGctagggaataattttttcctgtttgagc pOXA48 Oxa Overproduction
Pi36 attL3-speI-sspDI ACTAGTGAGGCGCCctagaatcgcgtatgcgagtg pEN-sacB To build the empty pEDIT4 plasmid
Pi37 attR2-SpeI caggtactagttactacctag pEN-sacB To build the empty pEDIT4 plasmid
pi62 attl_1-mscarlet-attL2-for gaggtctcagacacgggccagagctgcagc pV2-mScarlet Construction of new pV2-mScarlet and pV4-mScarlet with SmR
pi63 attl_1-mscarlet-attL2-rev gaggtctcaaccgctagcatggatctcgg pV2-mScarlet Construction of new pV2-mScarlet and pV4-mScarlet with SmR
pi64 p206-Sm-for gaggtctcatgtcagacgtcaggtggcacttttcg p206 Construction of new pV2-mScarlet and pV4-mScarlet with SmR
pi65 p206-Sm-rev gaggtctctcggtaacatgtgagcaaaaggccagc p206 Construction of new pV2-mScarlet and pV4-mScarlet with SmR
pi66 attR3-for GAGGTCTCACAGGAAACAGCTATGACCATG pENTR puroR4-R3 pENTR puroR4-R3
pi67 RifR-rev GAGGTCTCACCTGTCGTCCTGGAAGACAGGCGTG pINS_Rifr Construction of new pEDIT1
pi68 pUC-ori-for GAGGTCTCAAGGCCGCGTTGCTGGCGTT pINS_Rifr Construction of new pEDIT1
pi69 ccdB-rev GAGGTCTCAGCCTGGGGAAGACTGCCAGTCACG pUCIDTAmp-ccd-H2 Construction of new pEDIT1

Promoters

Promoter name Characteristic 5’ to 3’ sequence Length (bp)
PLtetO promoter Modified phage lambda PL promoter with tet operator sites tccctatcagtgatagagattgacatccctatcagtgatagagatactga 50
EM7 promoter Synthetic bacterial promoter gtcgtattatactatgccgatatactatgccgatgattaattgtcaac 48
tetR/tetA promoters overlapping promoters for bacterial tetR and tetA ttctctatcactgatagggagtggtaaaataactctatcaacgatagagtgtcaac 56

Bacterial culture media used:

  • LB (Lysogeny Broth medium) : (Casein peptides and peptones, Vitamins (including vitamin B), Trace elements (e.g. nitrogen, sulfur, magnesium), Minerals)

  • SOC (Super optimal medium with catabolic repressor medium) : (2% Tryptone, 0,5 % d'extrait de levure, 10 mM NaCl, 2,5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose)

  • SOB (Super Optimal Broth medium) : (1,5 % d'extrait de levure, 1% de Bacto-Tryptone, 10mM NaCl, 2mM KCl, 10 mM MgCl, 10 mM MgSO)

Chemicals used

  • For Oxa purification:
    • Tris
    • HCl (Hydrochloric acid)
    • NaCl (Sodium chloride)
    • MgCl2 (Magnesium chloride)
    • Glycine
    • SDS (Sodium dodecyl sulfate)
    • EDTA (Éthylènediaminetétraacétique)
    • Imidazole (ImH)
    • IPTG (Isopropyl ß-D-1-thiogalactopyranoside)
    • CB4X
    • Acrylamide
    • Temed (tetramethylethylenediamine)
    • APS (Ammonium persulfate)
    • Cracking Buffer
    • Ni-NTA resin
    • Imidazole
    • Coomassie blue dye
    • Technical ethanol
    • Acetic acid
  • For electrophoresis gels:
    • BET (ethidium bromide)
    • TAE
    • Agarose
  • Others:
    • DMF (Diméthylformamide)
    • NaOH (Sodium hydroxide)
    • Potassium acetate
    • Ethanol
  • Biomolecular KIT:
    • Plasmide extraction
      • Macherey-Nagel: Nucleospin plasmid
      • Promega: PureYield Plasmid
    • PCR clean-up
      • Macherey-Nagel: Nucleospin Gel and PCR clean-up
    • pGEM-T easy vector (Promega)
    • LR-Clonase KIT (Thermofisher)
    • Quick Blunting Kit (E1201)(NEB)

Machinery and observation devices:

  • Biomolecular KIT:
    • Plasmide extraction
      • Macherey-Nagel: Nucleospin plasmid
      • Promega: PureYield Plasmid
    • PCR clean-up
      • Macherey-Nagel: Nucleospin Gel and PCR clean-up
    • pGEM-T easy vector (Promega)
    • LR-Clonase KIT (Thermofisher)
    • Quick Blunting Kit (E1201)(NEB)

Software

Name Use Author/Society Version Date of Release Paper Link
Alpha Fold 2 Modeling protein interaction (OXA48 and nanobody CMY-2 specific) Milot Mirdita, Konstantin Schütze, Yoshitaka Moriwaki, Lim Heo, Sergey Ovchinnikov & Martin Steinegger/ Deep Mind ColabFold v1.5.2, google Colaboratory, Open source 2022 Jumper, J et al. Highly accurate protein structure prediction with AlphaFold. Nature (2021). Lien
RF Diffusion Creation of a new protein sequence (the nanobody OXA48 specific) from an other one, BacPROTACs Joseph L. Watson, David Juergens, Nathaniel R. Bennett and all/ Baker Lab First version, google Colaborator, Open source 31 March 2023 Joseph L. Watson, David Juergens, Nathaniel R. Bennett et al. Broadly applicable and accurate protein design by integrating structure prediction networks and diffusion generative models. arXiv preprint, doi: https://doi.org/10.1101/2022.12.09.519842. Lien
ChimeraX Visualization of the couple OXA48-nanobody, BacPROTACs Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. v 1.6.1, free of charge for academic, government, nonprofit, and personal use 09 May 2023 UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, Ferrin TE. Protein Sci. 2021 Jan;30(1):70-82. Lien
Jalview Sequence comparison, in order to select the best candidate for the nanobody, BacPROTACs Jim Procter, Mungo Carstairs, Ben Soares, Kira Mourao and all./ University of Dundee v 2.11.2.7 30 June 2023 Waterhouse, A.M., Procter, J.B., Martin, D.M.A, Clamp, M. and Barton, G. J. (2009) Jalview Version 2 - a multiple sequence alignment editor and analysis workbench Bioinformatics doi: 10.1093/bioinformatics/btp033 Lien
IMP, the Integrative Modeling Platform Linker’s modelisation, BacPROTACs D. Russel, K. Lasker, B. Webb, D. Schneidman, J. Velázquez-Muriel, A. Sali/ NCDIR v 2.19.0, Open source 19 June 2023 D. Russel, K. Lasker, B. Webb, D. Schneidman, J. Velázquez-Muriel, A. Sali, "Putting the pieces together: integrative structure determination of macromolecular assemblies", PLoS Biology, 2012. Lien
SnapGene® software (from Dotmatics; available at snapgene.com) Plasmids conception and visualization, sequence verification, manipulation simulation, CRISPR BacPROTACs Benjamin Glick/ Dotmatics v 7.0.1, need for a license June 2023 Dao, V. L., Chan, S., Zhang, J., Ngo, R. K. J., & Poh, C. L. (2022). Single 3′-exonuclease-based multifragment DNA assembly method (SENAX). Scientific Reports, 12(1), 4004. https://doi.org/10.1038/s41598-022-07878-x Lien
Image Lab Software (RRID:SCR_014210) Imaging software used to acquire and analyze images from specific Bio-Rad imaging systems. Used for analysis of electrophoresis gels, CRISPR Bio-Rad Laboratory v 6.1.0 build 7, standard edition 2020 Lien
GENEius Natural genes often contain bad motifs, like hairpins, that restrict the usability of the gene. Removal of bad motifs helps you to avoid ugly problems like poor PCR performance. The optimisation software GENEius improves the properties of your genes to enhance expression and usability. Furthermore, flaws are removed and good motifs like new restriction sites are introduced in the gene. GENEius software is designed and developed for Eurofins Genomics by BioLink Informationstechnologie GmbH. / Eurofins Genomics GENEius light — Lien

References

1. J. Du, J. Yuan, S. Yang, "Microbial Synthesis of Medium-Chain Chemicals from Renewable Resources," Biotechnology for Biofuels, vol. 9, no. 1, p. 105, 2016. [En ligne]. Disponible : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4857001/.

2. "SOB - OpenWetWare," [En ligne]. Disponible : https://openwetware.org/wiki/SOB.

3. "S.O.C Medium (SOB), InVitrogen," Clinisciences, [En ligne]. Disponible : https://www.clinisciences.com/autres-produits-186/s-o-c-medium-681000716.html.

4. "Milieu LB - Wikipédia," [En ligne]. Disponible : https://fr.wikipedia.org/wiki/Milieu_LB.

5. "New England Biolabs | NEB," [En ligne]. Disponible : https://www.neb-online.fr/.

6. "Thermo Fisher Scientific - FR," [En ligne]. Disponible : https://www.thermofisher.com/fr/fr/home.html.

7. "E. coli Genotypes - OpenWetWare," [En ligne]. Disponible : https://openwetware.org/wiki/E._coli_genotypes.

8. "Addgene | A Better Way to Share Science," [En ligne]. Disponible : https://www.addgene.org/.

9. "Eurofins France - Votre partenaire en biologie et en environnement - Eurofins France," [En ligne]. Disponible : https://www.eurofins.fr/.

10. "Promega Corporation," [En ligne]. Disponible : https://france.promega.com/.


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