Chemicals
PFAS
| Compounds | Supplier | Associated Experiments |
| TFA (trifluoroacetic acid) | Sigma-Aldrich (Ref: 302031) | Substrate for the degradation of short-chain PFAS by the evolved dehalogenase
Toxicity tests on Pseudomonas putida |
| PFPrA (perfluoropropanoic acid) | Sigma-Aldrich (Ref: 245917) | Toxicity tests on Pseudomonas putida |
| PFOS (perfluorooctanesulfonic acid) | Carl ROTH (Ref: 20TA.1) | Degradation tests on Labrys portucalensis
Toxicity tests by Pseudomonas putida |
| PFOA (perfluorooctanoic acid) | Sigma-Aldrich (Ref: 171468) | Adsorbance tests of the material used to immobilize our bacteria on beads |
Other fluorinated chemicals
| Compounds | Supplier | Associated Experiments |
| NaF (sodium fluoride) | Sigma-Aldrich (Ref: 51504) | Toxicity tests on Pseudomonas putida
Functionality test of FluorMango |
| 2-fluoropropionic acid | Sigma-Aldrich (Ref: 694371) | Substrate to assay the degradation of a monofluorinated molecule by the dehalogenase. Functionality test of FluorMango. |
Biological Materials
Parent Plasmids
pCas: this plasmid was used as a part of the chromosomal insertion system of the replication operon in E. coli DH10B. It was introduced into E. coli prior to pTarget to activate the CRISPR and recombineering systems. It contains the Cas9 gene, the α, β, and γ recombination genes, all placed under the control of the arabinose-inducible araBAD promoter, and it includes a kanamycin resistance gene. This plasmid was kindly provided by Denis Jallet (Toulouse Biotechnology Institute, Toulouse, France).
pTarget: this plasmid was used as a part of the chromosomal insertion system of the replication operon in E. coli DH10B. It was introduced into E. coli after pCas to express the guide RNA targeting the chromosomal insertion site SS9. It contains the gRNA gene and a streptomycin resistance gene. This plasmid was kindly provided by Denis Jallet (Toulouse Biotechnology Institute, Toulouse, France).
pHD_SS9_eutC-mSCI: this plasmid was used as a vector DNA for chromosomal integration of the replication operon. It contains an ampicillin resistance gene. This plasmid was kindly provided by Denis Jallet (Toulouse Biotechnology Institute, Toulouse, France).
pUD1387: this one-copy plasmid, carrying a chloramphenicol resistance gene, was used as the backbone for the construction and expression of the replication operon. It was kindly provided by Laura Sierra Heras (Toulouse Biotechnology Institute, Toulouse, France).
pUC19: this plasmid is from Takara and was used as a backbone for the construct of the linear replicon. It contains an ampicillin resistance gene.
pSEVA438: this plasmid is from iGEM Toulouse team 2024 BioMoon. This medium copy number vector was used as a backbone for the overexpression of FluC. It contains a streptomycin resistance gene, a m-toluic acid inducible promoter Pm, and a multiple cloning site (MCS).
Constructed Plasmids
pUC19-Replicon: this plasmid was constructed by inserting the linear replicon sequence into the pUC19 backbone for amplification. The construct contains the ampicillin resistance gene from pUC19 and the kanamycin resistance gene from the linear replicon.
pUC19-pbbsg: this plasmid was constructed from pUC19-replicon by eliminating the dehalogenase gene from the linear replicon. It contains the ampicillin resistance gene from pUC19 and the kanamycin resistance gene from the linear replicon.
pUC19-sfGFP (also called pUC19-PJ23119_fluorriboswitch_sfGFP): this plasmid was constructed by inserting the fluor-sensitive riboswitch crcB prior to the sfGFP gene, into the pUC19 backbone. This design allows fluorescence-based detection by regulating sfGFP expression in response to fluoride.
pSEVA438-fluC: this plasmid was constructed by inserting fluC into the pSEVA438 backbone. The resulting construct allows overexpression of FluC under the control of the m-toluic acid-inducible promoter Pm. It contains a streptomycin resistance gene and a multiple cloning site (MCS) from the pSEVA438 backbone.
Strains
| Strain | Supplier | Associated Experiments |
| Escherichia coli DH10B | Kindly provided by Pascal Le Bourgeois from TBI | Chassis for orthogonal replication |
| Escherichia coli DH5α | Kindly provided by Laura Sierra from TBI | Plasmid amplification |
| Pseudomonas putida KT2440 | Kindly provided by Etienne Pujos from TBI | Chassis for overexpression of FluC |
| Labrys portucalensis F11 | From DSMZ (strain number DSM 17916) | Degradation of long-chain PFAS |
gBlocks
Replication Operon
gBlock A (see mutations N71D + Y127A in Design Page)
gBlock B
gBlock C
Linear Replicon
gBlock D
gBlock E
Primers
| Primer | Sequence (5' to 3') | Purpose |
| RepO-A FWD | TGGCGCAGTTGATATGTCAAACAGGT | Amplification of gBlock A |
| RepO-A REV | GGGGCATTAAAAACCCAATATCACGCG | |
| RepO-A' FWD | TGGAAAATCAGGAGAGCGTTTTCAATCCTACCTCTGGCGCAGTTG | |
| RepO-A' REV | GGCTTTCGGTTCCATATCCGGGGGCATTAAAAACC | |
| RepO-B FWD | TATTGGGTTTTTAATGCCCCCGGATATGG | Amplification of gBlock B |
| RepO-B REV | GCCTTGGCCTTGGCGGCGGG | |
| RepO-B' FWD | GCTAGTCTGGTTCCGCGTGATATTGGGTTTTTAATGC | |
| RepO-B' REV | GCCTTGGCCTTGGCGGCGGG | |
| RepO-C FWD | GCCCGCCGCCAAGGCCAA | Amplification of gBlock C |
| RepO-C' FWD | TGCCCCTGCCGCTGCCGGTGAAGCCGCTGCCGAAGCCAAGCCCGCCGCCAAGGCCAAGGC | |
| RepO-C REV | TAATCAACGCGATATAATAATTTTCGAAAAAACACCCTAACGGGTGTTT | Amplification of gBlock C without GFP |
| RepO-C' REV | CACATATATTAATTGCCGTTAAAACTAAAAACAGCATCAATAATCAACGCGATATAATAACGATTTTCGAAAAAACACCCTAACGG | |
| RepO-CGFP REV | CGTTAAAACTAAAAACAGCATCAATAATCAACGCGATATAATAA | Amplification of gBlock C with GFP |
| RepO-D FWD | ACTCATTAGGCACCCCAGGCG | Amplification of gBlock D |
| RepO-D REV | AATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAAC | |
| RepO-E FWD | GAGTTTTTCTAATCAGAATTGGTTAATTGGTTGCTCC | Amplification of gBlock E |
| RepO-E REV | GGGTGTCGGGGCTGGC | |
| RepO-rep FWD REV | GGGGATACGTGCCCCTCCC | Amplification of the linear replicon |
| RepO-SS9-L FWD | TTATTATATCGCGTTGATTATTGATGCTG | Amplification of the lower arm of pHD-SS9 |
| RepO-SS9-L REV | GCTACATCTTCCGTACTATGCTGTAGTCTCATGGTCGAGTTCTATTGCTGTTCGGCGGCAATAACCCGCCACAGTAGTTCC | |
| RepO-SS9-U FWD | TTAGGGAGCACATCCATGCCAATAGCTCGACAAGCGGCGAGAGCCTTGCACCTATGCTATCCGACGTCCATCCAGCCCAC | Amplification of the upper arm of pHD-SS9 |
| RepO-SS9-U REV | ACCTGTTTGACATATCAACTGCG | |
| FluR-FluC FWD | CCTAGGCCGCGGCCGCGCGAATTCATGATTGCACTCATCGCCGCG | Amplification of fluC by colony PCR on P. putida KT2440 |
| FluR-FluC REV | GAGGATCCCCGGGTACCGAGCTCTCAGAATCGGGTCAGGGACAGG | |
| FluR-pSEVA438 FWD | GAGCTCGGTACCCGGGGATC | pSEVA438 plasmid linearization |
| FluR-pSEVA438 REV | GAATTCGCGCGGCCGC | |
| RepO-pHD FWD | TTATTATATCGCGTTGATTATTGATGCTGTTTTTAGTTTTAACG | pHD SS9 plasmid linearization |
| RepO-pHD REV | ACCTGTTTGACATATCAACTGCGCCA | |
| RepO-pUC FWD | TTAAGCCAGCCCCGACACCC | pUC19 plasmid linearization |
| RepO-pUC REV | GCCTGGGGTGCCTAATGAGTGA | |
| RepO-OCP FWD | TTTTAGTTTTAACGGCAATTAATATATGTGGACAGCTTATCATCGAATTTCTGC | pUC1387 plasmid linearization |
| RepO-OCP REV | TAGGATTGAAAACGCTCTCCTGATTTTCCAGCAGGACACAGCAGCAATCC | |
| RepO-op FWD | CCGACGTCCATCCAGCCCAC | Replication operon extraction from pHD SS9 plasmid |
| RepO-op REV | ATAACCCGCCACAGTAGTTCCTTCTC | |
| Kit-pBBsg FWD 1 | GCTAGCGGATCCTTAATTAAGAGTTGGCTGCTGC | Plasmid linearization of biobrick, half one, without the DeHa gene |
| Kit-pBBsg REV 1 | CAAGACGATAGTTACCGGATAAGG | |
| Kit-pBBsg FWD 2 | CAACTCTTAATTAAGGATCCGCTAGCGGTTAATTCCTCC | Plasmid linearization of biobrick, half two, without the DeHa gene |
| Kit-pBBsg REV 2 | ATCCGGTAACTATCGTCTTGAGTCCAACC | |
| RepO-Mango1 | GGCCGGAATAATACGACTCACTATAGGGAGATGAGGCCCTCCCAAACTGCCAAGGAAGGATTGGTATGTGGTATATTGGATGATGGCCTCTACTG | Annealed to form a double-stranded DNA template for FluorMango aptamer synthesis by in vitro transcription |
| RepO-Mango2 | CAGTAGAGGCCATCATCCAATATACCACATACCAATCCTTCCTTGGCAGTTTGGGAGGGCCTCATCTCCCTATAGTGAGTCGTATTATTCCGGCC |