Materials
Strains
| Strain | Use |
|---|---|
| Escherichia coli BL21 (DE3) | For protein expression |
| Escherichia coli DH5α | For the cloning of our plasmid of interest |
| Escherichia coli TOP10 | To increase the copy number of the plasmid |
| S. cerevisiae | Plasmid for MEV pathway |
| Chaetomium thermophilum var. thermophilum 1495 | Fungal assay in the presence of different Linalool conc. |
| Vibrio fischeri | Quorum sensing system |
| Pseudomonas sp. | AHL for Lux system induction |
Plasmids
| Plasmid | Speciality | Why Used? |
|---|---|---|
| pUC19 | Ori derived from pMB1, ampicillin, lacZa promoter | Cloning and propagation of the DH5alpha protein producing genes |
| C11 | Chloramphenicol selective marker, lux system can be expressed | |
| M20 | Chloramphenicol resistant pSB1C3SA, IPTG inducible pTac promoter | Inserted variable lengths of CCA (Antisense) sequence |
| pBbA5c-MevT(CO)-T1-MBIS(CO, ispA) | Chloramphenicol | Insertion of genes GPPS and LIS for linalool production |
Primers
For the PCM Circuit:
| Primers | Sequence | Used For |
|---|---|---|
| PL-RP | AGGGATGTCAATCTCTATCACTGATAGGGAATTTCTAGAAGCGGCCGCGAAT | Plasmid linearisation |
| Ptet-FP | GATAGAGATTGACATCCCTA | Insert-1 grad. Amplify FP |
| I1-amp-RP | GAGCGCCATTGAAATTACGTCGTCCAAGCTTAGTCGTG | Insert-1 amplify RP |
| I2-amp-FP | ATGGCACGACTAAGCTTGGACG | Insert-2 amplify |
| I2-amp-FP | CCATTTCCTCCTGCATGATC AAA ATG CAA GTG AGA AC | Insert-2 amplify |
| I3-amp-FP | TTATTAGTTCTCACTTGCATTTTGATC ATG CAG G | Insert-3 amplify |
| T7-O(P)-RP | AGCGGCCGCTACTAGTATTCAAAAAACCCCTCAAGACCCGTTT | Insert-3 amplify |
For PCM Characterization:
| Primers | Sequence | Used For |
|---|---|---|
| PL(Tet-R)-RP | TCGTCCAAGCTTAGTCGTG | GFP of tetR 3761 bp amplification |
| GFP-amp-FP(tetR) | ATGGCACGACTAAGCTTGGA ATG AGC AAA GGA GAA GAA CTT TTC ACT GG | GFP 757 bp for tetR amp. |
| PL(Antisense)-RP | CGA CGG TTT CGC TTT CAC TG | GFP of anti 5308 bp amplification |
| GFP-amp-FP(Antisense) | TACAGTGAAAGCGAAACCGTATG AGC AAA GGA GAA GAA CTT TTC ACT GG | GFP 757 bp for anti amp. |
| GFP-amp-RP | CCTTGCCCTTTTTTGCCGGA CTA TTT GTA GAG CTC ATC CAT G | GFP 757 bp for tetR and anti amp. |
| PL(hisTerm)-amp-FP | TCCGGCAAAAAAGGGCAAGG | For both GFP backbone amplification |
Antisense at different length:
| Primers | Sequence | Used For |
|---|---|---|
| PL-Anti-FP | TCC GGC AAA AAA GGG CAA GGT GTC ACC | To linearize the PSB1C3SA backbone of M20. |
| PL-Anti-RP | AAGTAAATTGTGAGCGCTCACAATTCCACACATTATACGAGCCG | To linearize the PSB1C3SA backbone of M20. |
| Anti-FP | CCTTGCCCTTTTTTGCCGGAATGTCACTTTCGCTTTGGC | Common FP to amplify variable bp of CCA from PCM. With overhangs to attach |
| Anti-RP1 | AGCGCTCACAATTTACTTTAACTCATCCTGCAATCGGGC | To amplify first 51 bp of CCA from PCM. With overhangs to attach to M20 PSB1C3SA |
| Anti-RP2 | AGCGCTCACAATTTACTTCCAATCGAGGACAAAACGG | To amplify first 150 bp of CCA from PCM. With overhangs to attach to M20 PSB1C3SA |
| Anti-RP3 | AGCGCTCACAATTTACTTTCGTCACTGCCGCTTGTG | To amplify first 277 bp of CCA from PCM. With overhangs to attach to M20 PSB1C3SA linearised backbone. |
| Anti-RP4 | AGCGCTCACAATTTACTTGTTAGAACGATAGGTCGGTTCTG | To amplify first 393 bp (selected for low hairpin, etc reasons) of CCA from PCM. With overhangs to attach to M20 PSB1C3SA |
| Anti-RP4 | AGCGCTCACAATTTACTTGTTAGAACGATAGGTCGGTTCTG | To amplify first 393 bp (selected for low hairpin, etc reasons) of CCA from PCM. With overhangs to attach to M20 PSB1C3SA |
| Anti-RP5 | AGCGCTCACAATTTACTTCGTTTACGTTAGAACGATAGGTCGGTTCTG | To amplify all 400 bp of CCA from our assembled PCM. |
MEV Primers
Primers for Assembly:
| Primers | Sequence |
|---|---|
| GPPS_FP | TTTCACACAGGAAAGATGGAATTTGAC |
| GPPS_RP | CATTTTCTCCTCTTTAATTTA ATTCTGACGAAATGCCACGTAG |
| LIS_FP | TAAATTAAAGAGGAGAAA ATGAGCATCAACATTAACATGCCG |
| LIS_RP | TATAAACGCAGAAAGGCCCACCCG |
Primers for Cloning:
| Primers | Sequence |
|---|---|
| PL-FP | TGGGCCTTTCTGCGTTTATA TCC GGC AAA AAA GGG CAA GGT GTC ACC |
| PL-RP | TCCATCTTTCCTGTGTGAAAAAGTAAATTGTGAGCGCTCACAATTCCACACATTATACGAGCCG |
Chemicals
| Chemical Name | Used For |
|---|---|
| Dry AHL(N-(3-Oxododecanoyl)-L-homoserine lactone) | For induction checking in PCM |
| Anhydro tetracycline | To check how tetR binding affects upon induction |
| IPTG | To check ptac promoter affecting upon induction |