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Materials



Regolith

We used a Lunar South Pole (LSP-2) regolith simulant that was kindly provided by Space Resource Technologies (Exolith Lab®, Oviedo, Florida, USA).



Plasmids

pSEVA438: this medium copy number vector was used as a backbone for almost all our constructs. It contains a streptomycin/spectinomycin resistance gene, m-toluic acid inducible promoter Pm and a multiple cloning site (MCS). It was a kind gift from the Standard European Vector Architecture (SEVA) platform. See the plasmid map on Snapgene here.

pSEVA244: it contains a kanamycin/neomycin resistance gene, IPTG inducible promoter Ptrc and MCS. This is a medium copy number vector and was a kind gift from the Standard European Vector Architecture (SEVA) platform.See the plasmid map on Snapgene here.

pBx-Spas-sgRNA-Kan: this vector was used as a backbone for the insertion of guide RNA in CRISPRi experiments. It contains S. pasteurianus sgRNA without the 20-bp target (Empty control) from the anhydrotetracycline inducible Ptet promoter and a kanamycin/neomycin resistance gene.See the plasmid map on Snapgene here.

pUC18-mini-Tn7T-Plac-dCas9: this vector was used for the expression of S. pasteurianus deadCas9 in the CRISPRi experiments. It contains the IPTG inducible promoter Plac, gentamycin and ampicillin resistance genes. See the plasmid map on Snapgene here.

pHD_SS9_PLtetO1_TLt0_eutC-GFP: this plasmid was used for the amplification of the anhydrotetracycline inducible promoter Ptet for cloning into pSEVA438. It was kindly provided by our supervisor Denis Jallet.





Strains

Escherichia coli Stellar: HST08 strain [F-, endA1, supE44, thi-1, recA1, relA1, gyrA96, phoA, Φ80d lacZΔ M15, Δ(lacZYA-argF) U169, Δ(mrr-hsdRMS-mcrBC), ΔmcrA, λ-]. This strain was used in all transformations for In-Fusion® cloning (Stellar Competent cells from Takara, ref: 638952). See the strain here.

Pseudomonas fluorescens SBW25 strain: this strain was used as the BioMoon chassis strain. It was kindly provided by Patricia Bordes (Laboratoire de Microbiologie et Génétique Moléculaires, Toulouse, France).





gBlocks

The following genes were codon optimized for expression in Pseudomonas putida (the closest microorganism for our strain Pseudomonas fluorescens) using the Codon Optimization Tool from Integrated DNA Technologies (IDT) and gBlocks were obtained from the same company.

  • creA-crnA



  • hfq-rpoS





Primers

Insert amplification

List of primers used for gBlocks amplification:

DNA origins

DNA templates

Primers

Sequences (5’ to 3’)

gBlocks creA-crnA creA-crnA overhang backbone FWD ggccgcggccgcgcgaattccattgattacaaggaagtgtgtttat
gBlocks creA-crnA creA-crnA overhang Ptet REV tagagatactgagcacatcatgtgggtctaggtaggcgg
gBlocks hfq-rpoS hfq-rpoS FWD ggccgcggccgcgcgaattcttcatttttcacggatgaacccac
gBlocks hfq-rpoS hfq-rpos REV tagagatactgagcacatcaaaacacacattgattacaaggaagtg
Genomic P. fluorescens SBW25 genome katB FWD ggccgcggccgcgcgaattcaaaaaaaccagaggaaaccctgatg
Genomic P. fluorescens SBW25 genome katB REV cacgacgcggccgcaagctttcagtcagtcagtttttcagcca





Plasmid linearization

List of primers used for plasmid linearization:

Plasmids

Primers

Sequences (5’ to 3’)

pSEVA438 pSEVA backbone FWD aagcttgcggccgcgtcg
pSEVA438 pSEVA backbone REV gaattcgcgcggccgcg
pSEVA244 pSEVA backbone FWD aagcttgcggccgcgtcg
pSEVA244 pSEVA backbone REV gaattcgcgcggccgcg
pSEVA438-Ptet pSEVA438-Ptet backbone FWD tgatgtgctcagtatctctatcac
pSEVA438-Ptet pSEVA backbone REV gaattcgcgcggccgcg





Colony PCR and sequencing

List of primers used for colony PCR and sequencing:

Constructs

Primers

Sequences (5’ to 3’)

pSEVA438-Ptet
pSEVA438-Ptet-creA-crnA
pSEVA438-Ptet-hfq-rpoS
Control pSEVA438 FWD aggcctaccccttaggcttt
pSEVA244-katB Control pSEVA244 FWD tgcgccgacatcataacggt
pSEVA438-Ptet
pSEVA438-Ptet-creA-crnA
pSEVA438-Ptet-hfq-rpoS
pSEVA244-katB
Control pSEVA REV gccagggttttcccagtcac