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
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
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
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 |