Experiments

Restriction double digest

• Restriction digest was performed with 20 units SacI-HF and 20 units PstI-HF (New England Biolabs (NEB))
• For preparative digest at least 1 µg was digested in 50 µL reaction volume for 1 h up to 16 h at 37 °C

Ligation

• Ligation was done with T4 DNA Ligase (NEB)
• 3:1 vector to insert molar ratio was used with 50 ng vector
• Incubated overnight at 16 °C

Oligo annealing

• 100 µM of forward and 100 µM of reverse oligo were annealed in a 50 µL reaction with 1x rCutsmart buffer (NEB)
• Reaction was incubated at 95 °C for 5 min and then cooled down at a rate of 0.75 K/min down to room temperature (25 °C)

Golden gate assembly

• Golden gate assembly was done with SapI or BsaI-HFv2 (NEB)
• 100 ng vector with 1:3 molar ration insert or 10 µM annealed oligos
• 25 µL reaction volume: 2.5 µL T4 Ligase buffer, 0.5 µL T4 Ligase (NEB), 1.5 µL type IIS enzyme
• 30 cycles of 5 min 37 °C, 5 min 16 °C, afterwards heat inactivation at 65 °C for 10 min and stored at 4 °C until further use

NEBuilder Hifi-DNA assembly

• Assembly was performed in 20 µL reaction volume with 0.1 or 0.15 pmol vector with 1:2 molar ration insert for one fragment assembly, or 1:1 molar ration for 5 fragment assembly
• Reaction was incubated at 50 °C for at least 1 h up to 4 h and stored at 4 °C until further assembly

PCR based assembly

• 0.025 pmol fragments were added in a PCR reaction without primer and 12 cycles were performed with recommended annealing temperature and extension time
• Primer were added in recommended concentration and 25 further cycles were performed

Agarose Gel electrophoresis

• Gel electrophoresis was performed with 7.5 V/cm for 30 to 90 min until desired separation was achieved
• Following agarose gel concentrations (wt/v) were used: (for preparative gels the concentration was reduced by 0.2-0.5 %)
• >3000 bp, 0.75 % Agarose
• 2000-3000 bp 1 % Agarose
• 500-2000 bp 2 % Agarose
• 500 bp 3 % Agarose
• SYBR safe DNA stain (Thermofisher scientific) was added 1:10 000 after agarose was dissolved completely
• Bands were visualized with an UV tray

KLD treatment

• After site directed mutagenesis PCR (insertion, single point mutation, deletion), the PCR produced was treated with the KLD mix (NEB), simultaneously phosphorylating and ligating the PCR ends, and DpnI digestion of methylated, not PCR amplified plasmid
• Either 1 µL PCR mix or 100 ng of purified DNA were used for the reaction

PCR

• PCR was either performed with the 2x KOD-one PCR master mix blue (Merck) or Q5 Phusion Polymerase master mix (NEB)
• Reactions were performed in a total volume ranging from 20-200 µL, 10-50 ng template were used with the recommended primer concentrations of 0.3 µM for KOD-one Polymerase or 0.5 µM for Phusion polymerase
• Annealing temperatures were calculated with NEB Tm calculator for the Phusion polymerase or 3-5 °C beneath the melting temperature calculated with the nearest neighbor method (with 50 mM Na+ concentration and 0.5 µM oligonucleotide concentration) for the KOD-one polymerase. The Tm’s were chosen within the range recommended by the manufacturer
• Extension time was 40 s/kb for the Phusion polymerase or 15 s/kb for the KOD-one polymerase with 72 °C or 68 °C extension temperatures, respectively
• For PCR amplification after Hifi-DNA assembly touch down PCR was used. The starting annealing temperature was chosen 10 °C above the recommended temperature, after 2 cycles the temperature was decreased by 2 °C after reaching the final annealing temperature 25 further cycles were performed.

DNA purification

• DNA clean up after enzymatic manipulation of DNA or agarose gel electrophoresis was done with NucleoSpin Gel and PCR clean up kit (Machery&Nagel). The recommended protocol was followed with the optional wash step was always included and the recommended steps and eluted twice in 20 µL Tris-HCl (pH 7.5)

Plasmid purification

• Plasmids were purified from 2-5 mL dense overnight culture of either P. fluorescens or E. coli, using the plasmid prep GeneElute (Sigma aldrich) kit or the NucleoSpin plasmid kit (Machery&Nagel) the recommended protocol was followed with the additional wash step for the Nucleospin kit. The plasmid was eluted in 50 µL dH2O (nuclease free) or Tris-HCl (pH 7.5)

E. coli

• Plasmid transformation was done either in BL21 (C2530H, NEB) for protein expression or DH5alpha (C2987, C2988, C2984, NEB) for all cloning work, with manufacturers recommended protocol
• Heat shock time was increased by 20 s (total time of 50 s)
• 500-100 ng purified plasmid were used or 2-5 µL reaction after ligation, Hifi assembly, golden gate assembly, or KLD treatment

Pseudomonas fluorescens (DSMZ 50090)

• P. fluorescens was transformed by electroporation
• 200 mL cells were harvested in the early to mid log phase (OD 0.4-0.8) and washed twice with 200 mL autoclaved dH2O at 4 °C, twice with 100 mL autoclaved dH2O, and once with 20 mL 10 % glycerol (v/v) and resuspended in a final volume of 2 mL with 10 % glycerol
• Washed cells were aliquoted in 100 µL and flash frozen at - 80 °C
• Thawed cells were mixed with 2 µL DNA from previous cloning reaction or 50-100 ng purified plasmid, after electroporation 900 µL SOC medium was added
• Transformed cells were plated on 100 µg/mL containing strep plates after 60-90 min outgrowth
• Electroporation was done in 2 mm cuvettes with a field strengths of 2 500 V (12.5 kV/cm), 25 µF capacity, and 200 Ohm resistance)

Bacteria selection

• Transformants harboring plasmids with aadA gene (StrepR) were selected on Streptomycin containing LB-Agar plates, 25 ng/µL for E. coli and 100 ng/µL for P. fluorescens
• After 14-16 h of growth colonies were picked and transferred in LB liquid culture containing the same antibiotic concentration as the selection plates
• For P. fluorescens 100 ng/µL ampicillin was sometimes added to avoid any unwanted contamination

Induction

• Expression controlled by the XylS/Pm systems were induced with m-toluate with a standard inducer concentration of 0.75 mM
• Expression controlled by the XylS-K38R-L224Q/Pm was induced with varying concentrations of terephthalate
• Expression controlled by the AlkS/pAlkB system were induced with alkanes varying in length (hexane up to heptadecane), with varying concentrations

Protein expression

• Protein expression was done either in E. coli BL21 (fhuA2 [lon] ompT gal [dcm] ΔhsdS, NEB) or Pseudomonas fluorescens (DSM 50090)
• 50 mL to 200 mL DYT (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl) were inoculated 1:50 with an overnight culture and incubated at 32 °C for E. coli or 28 °C for P. fluorescens
• At an OD₆₀₀ of approx. 0.6 expression was induced with 0.75 mM m-toluate (for XylS/Pm expression system), 0.02 % (w/v) arabinose (pBAD24), or 1 mM IPTG (lac promoter)
• Expression cultures were grown overnight at 32 °C or 28 °C, respectively

Protein extraction from cells

• Cells were harvested by centrifugation (3750 xg, 1 h, 4 °C) in 50 mL reaction tubes and resuspended in 10 mL lysis buffer (50 mM Tricine-HCl (pH 7.8), 300 mM NaCl, 10 mM Imidazole, 1 % (vol/vol) Triton X-100, 1 mg/mL Lysozyme) or PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄) with 10 mM Imidazole (PBS/10mM Imidazole).
• Lysis was performed by sonification for 5 min at 30 % power with 10 s intervals (Sonic Ruptor 400)
• Debris was removed by centrifugation (3750 xg, 60 min, 4 °C)

Protein precipitation

• Cells were pelleted by centrifugation (3750 xg, 1 h, 4 °C) and supernatant transferred to sterile shot flask
• Proteins precipitation was done by adding ammonium sulfate for a final saturation of 90 % at 4 °C
• Precipitated proteins were collected by centrifugation (3750 xg, 90 min, 4 °C) and pellet was resuspended in 10 mL PBS/10 mM Imidazole)
• To reduce salt concentration resuspended proteins were dialysed against 3 L PBS/10 mM Imidazole for 4-12 h (regenerated cellulose dialysis tube, 12000 -14000 MWCO, Carl Roth)

Protein purification

• Protein purification was performed in batch mode with 1 mL Ni-NTA (RotiGarose-His/Ni HPBeads, Carl Roth; or HisPur Ni-NTA resin, Thermo scientific)
• Raw protein extract was incubated with Ni-NTA resin for 1 h - 12 h at 4 °C, unbound fraction was separated by centrifugation (500 xg, 2 min)
• Ni-NTA was washed 3-4 times with 5 bed volumes of wash buffer (50 mM Tricine-HCl (pH 7.8), 300 mM NaCl, 20 mM Imidazole) or PBS/20 mM Imidazole, until A₂₈₀ absorbance in the wash fraction approaches baseline
• Elution was performed twice with two bed volumes of elution buffer (50 mM Tricine-HCl (pH 7.8), 300 mM NaCl, 250 mM Imidazole) or PBS/250 mM Imidazole

Protein concentration

• Protein concentration was estimated using Beer’s law by measuring absorption at 280 nm an dividing it by the molar extinction coefficient of the measured protein
• For more accurate protein quantification bradford assay or DC protein assay was used with the manufacturers recommended protocols (BioRad)

SDS-Page

• Sample was prepared with 4x Laemmli SDS sample buffer (Thermo scientific) for a final volume of 50 µL with additionally 1 µL 2-Mercaptoethanol was
• Samples were incubated at 95 °C for 5 min and then stored on ice until SDS-PAGE
• Samples were loaded on 4-15% polyacrylamide gels (15% Mini-PROTEAN® TGX™ Precast Protein Gels, BioRad), Page ruler prestained protein ladder (10-180 kDa, Thermofisher) was used as a marker
• Gels were run at 90 V for 15-20 min until the bands condensed to one line, afterwards the voltage was increased to 150 V until the loading dye left the gel (approx. 90 min)
• Gel electrophoresis was performed in tris/glycine running buffer (25 mM Tris (CarlRoth), 192 mM glycine (CarlRoth), 0.1 % SDS)

Coomassie staining

• SDS-Page was taken out of Gel-Electrophoresis and suspended in dH2O for 5 min, wash step was repeated a second time
• Washed gel was incubated (under constant agitation) with approx. 50 mL Coomassie staining solution (40 % methanol (Sigma Aldrich), 10 % glacial acetic acid (Sigma Aldrich), 0.1 % coomassie brilliant blue (wt/v) (US biological life sciences) )for 30 min - 6 h. Afterwards staining solution was collected for future use (this was done until the staining strength noticeably decreased)
• After staining gel was washed in dH2O for 5 min and afterwards destained with dH2O or destaining solution (7.5 % glacial acetic acid (v/v) (Sigma Aldrich), 10 % ethanol (v/v) (Sigma Aldrich)) until background color was minimized (water, or destaining solution was regularly exchanged)

Western-Blot (semi dry)

• Alternatively to Coomassie staining SDS-PAGE was used for western blotting
• Gel was incubated for 15 min in towbin buffer (25 mM Tris, 192 mM glycine, 20 % (v/v) methanol)
• Membrane (Transfer membrane ROTI®NC 0.2, CarlRoth) was incubated in TBS-T (20 mM Tris, 150 mM NaCl, 0.1 % (w/v) Tween 20) for 15-20 min
• Two filter papers (Rotilabo®, 1.5 mm, CarlRoth) was soaked in towbin buffer
• Blotting was prepared in Trans-Blot® SD Semi dry blot (anode, paper, membrane, gel, paper, membrane, gel, paper) and rolled out to get rid of anny bubbles
• Blotting was performed for 60 min at 7.5 V (~0.4 amp)

Antibody staining

• Washed membrane (or fully destained membrane) was blocked 1 h in TBS with 5 % (wt/v) nonfat dry milk powder and afterwards incubated with primary anti-his6 antibody (1:5000, monoclonal mouse antibody (HIS.H8), Invitrogen) overnight at 4 °C
• Membrane was washed for five minutes six times with TBS, bevor incubating it 1:2000 with the secondary anti-mouse antibody (Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, HRP; Invitrogen) fo 1 h
• Membrane was washed four times and imaged with Pierce^Tm ECL Western blotting membrane (Thermo Scientific)

Enzyme activity assay

• Esterase activity was tested with p-nitrophenyl ester (pNP ester)
• 200 µL reaction volumes were measured in 96 well microtiter plate (Thermo scientific), containing 0.25 µg enzyme in PBS with 20 µL of pNP ester dissolved in acetonitrile (0.1, 0.5, 1, 5 mM of pNP-ester was tested)
• Measurements were performed in a Tecan spark (Thermo Fisher Scientific) at 28 °C, pH 7.4 , after an initial 5 s shake period (orbital, 3 mm amplitude, 180 rpm) OD₄₁₅ was measured every 3 min for 6 h

Cell growth

• Cell based assays were performed in 96 well microtiter plates (Thermofisher scientific)
• 5 mL pre LB medium (10 g/L tryptone, 5 g/L NaCl, 5 g/L yeast extract (CarlRoth) was inoculated 1:100 or 1:200 with a dense overnight culture and grown until the early exponential phase OD₆₀₀ approx. 0.1-0.15
• Microtiter plates were prepared with 100 µL growth media with desired additives (e.g. inductor). If a different growth media than LB was used, 170 µL growth media were prepared
• Microtiter plates were incubated in a Tecan spark (Thermo scientific)
• 28 °C, 300 s orbital shaking (3 mm amplitude, 180 rpm)
• OD₆₀₀ absorbance measurement every 10 min (25 flashes, 0 ms settle time)
• If cell growth was measured in different media than the standard cultivation media (LB broth) cells were centrifuged (3750 xg, 4 °C, 30 min) and washed twice with M9 minimal salts medium (Sigma aldrich) and 30 µL were used to inoculate the microtiter plate

Fluorescence measurements

• Fluorescence assays were performed in a Tecan spark (Thermoscientific) in combination with the above mentioned incubation conditions and absorbance measurements
• Fluorescence was measured with an excitation measurement at 588 nm (20 nm bandwidth) and an excitation of 633 nm (20 nm bandwidth), 30 flashes per well, 40 µs integration time and a Z-position of 30624 µm
• An endpoint measurement (>16 h after induction) was used to find the optimal gain for time resolved measurements

• Inserts containing pSEVA438 homologous regions, SapI recognition sites, LUZ7 T50 terminator (BBa_K4757058), SSraA-ILV degradation tag (BBa_K4757001), ribosomal binding sites (BBa_J61100 (referred to as RBS 1), BBa_J61101 (referred to as RBS 2), BBa_K4757003 (referred to as RBS 3)), and pEM7 promoter(BBa_K4462022) were obtained by DNA synthesis (gBlock, IDT, codon optimized for P. putida). The sequences were PCR amplified (fwd_SapI_T_mKate2_RBS1_P, rev_SapI_T_mKate2_RBS_P)
• The sequence containing the synRBS (BBa_K4757003) could not be synthesized. Instead the assembled plasmid containing insert_RBS1 was used for site directed mutagenesis PCR and subsequent KLD treatment
• The pSEVA438 plasmid, a kind gift from our wetlab advisor Victoria Sajtovich, was PCR amplified with rev_Pm_pSEVA438 (sRNA construct_gibbson), fwd_pSEVA438 (MCS)
• After Hifi DNA assembly (0.15 pmol vector, 0.3 pmol insert, 75 min, 50 °C) 2 µL were transformed in E. coli
• 2-5 colonies were transferred to a liquid overnight culture and plasmid prep performed, successful assembly was verified by sanger sequencing (Eurofins genomics)
• DNA sequences coding for the sRNA’s were ordered as single strand DNA oligos (ssDNA oligos) (Merck, IDT) and annealed to yield dsDNA with 5’ caa (upper strand) and 5’ aag (lower strand) overhangs
• Purified plasmid and annealed oligos were used for golden gate assembly and afterwards transformed in E. coli
• Colonies were screened with colony PCR (fwd_sRNA validation, rev_sRNA validation) and agarose gel for successful assembly
• Colonies showing positive colony PCR results were picked and after liquid culture cultivation the plasmid was purified and validated with sanger sequencing. Correctly assembled plasmids were transformed in P. fluorescens by electroporation
• P. fluroescens containing successful plasmids were used for growth and fluorescens assays (as described above) and referenced against wildtype P. fluorescens as well as transformed cells without the sRNA coding sequence
• Cell growth and fluorescens intensity was measured in 96-well format with varying m-toluate concentrations for 16-24 h

• DNA sequence coding for pAlkB were obtained by DNA synthesis (gBlock, IDT, codon optimized for P. putida) with homologous overhangs for Hifi-DNA
• pSEVA438 was PCR amplified with fwd_pSEVA48_forAlkBP_gibson, rev_pSEVA438_forAlkBP_gibson, and purified with NucleoSpin
• Insert and vector were assembled with Hifi-DNA assembly master mix and 2 µL transformed in E. coli
• Single colonies were picked, cultured, plasmid purified and validated with sequencing
• AlkS presence in the genome was checked with colony PCR (fwd_AlkS_colony PCR, rev_AlkS_colony PCR; fwd_AlkSrev_colony PCR, rev_AlkSrev_colony PCR)
• The sequence coding for AlkS was obtained in 3 fragments with gene synthesis (gBlock, ID, Tcodon optimized for P. putida)
• The synthesized fragments were assembled with Hifi-DNA assembly and 2 µL assembly mix amplified with touch down PCR (fwd_AlkS insert_gibson, rev_AlkS insert_gibson) with subsequent agarose clean up
• pSEVA438_pAlkB was PCR amplified with fwd_pSEVA438_for AlkS, rev_pSEVA438_for AlkS
• Insert and vector were assembled with Hifi DNA assembly and 2 µL transformed in E. coli
• After sequence validation the plasmid (pSEVA438_AlkSpAlkB) and mKate2 (obtained by gene synthesis, and PCR amplified with fwd_mKate2(RE) and rev_mKate2(RE)) was digested with 20 units SacI-Hf and PstI-Hf. The linearized vector was phosphorylated with AnP and both fragments were purified.
• Vector and inserts were ligated with 1:3 vector to insert ratio and 2 µL transformed in E. coli
• Sequence verified plasmid (pSEVA438_AlkS-pAlkB_mKate2) was transformed in P. fluorescens
• pSEVA438_AlkS-pAlkB_mKate2 was PCR amplified with “fwd_mKate2(RE)_RBS addon” and “rev_pSEVA438 (RBS addon)” and treated with KLD mix and subsequent transformation into E. coli to introduce BBa_J61100 directly in front of mKate2
• pSEVA438_AlkS-pAlkB_RBS1-mKate2 was PCR amplified with pEM7_addon_XylS/AlkS_fwd and pEM7_addon_AlkS_rev to replace the Ps1/Ps2 promoter system with the pEM7 promoter and subsequently treated with KLD enzyme mix
• 2 µL were transformed in E. coli and after sequence validation purified plasmid was transformed in P. fluorescens
• Cell growth and fluorescence intensity of the different constructs were measured in 96-well format with alkanes varying lengths and concentrations for 16-24 h

• Site directed mutagenesis was used to introduce substitutions for the mutations K38R and L224Q, primers were designed with NEBuilder (XylS_K38R_XylS_L224Q:1_FWD, XylS_K38R_XylS_L224Q:1_REV, XylS_K38R_XylS_L224Q:2_FWD, XylS_K38R_XylS_L224Q:2_REV) in two different PCR reactions (primer pair 1 and primer pair 2)
• The fragments were assembled with Hifi DNA assembly and transformed in E. coli
• Mutations were validated by oxford nanopore sequencing (whole plasmid sequencing, Eurofins genomics)
• mKate2 was cloned via restriction digest with SacI and PstI as described in project 2
• The RBS BBa_J61100 was introduced with add on PCR (pEM7_addon_XylS/AlkS_fwd, pEM7_addon_XylS_rev)
• Cell growth and fluorescence intensity of the different constructs were measured in 96-well format with varying concentrations of terephthalate and m-toluate for 16-24 h

• Gel.Exp_LCC, AlkB and RBS-SPpstu_FAST-PETase with C terminal His6 was obtained by DNA synthesis (gBlock, IDT, codon optimized for P. putida) and PCR amplified with GelExp_LCC_primer01, rev_GelExp_LCC(&FAST-PETase), AlkB_primer 01_fw_MM, AlkB_primer 01_rev_MM, fwd_SPptsu_FAST-PETase_fwd
• Inserts were cloned in the pSEVA438 vector with SacI, PstI digest as described in project 2
• The ligation mix was transformed in E. coli and after sequencing validation, purified plasmid was transformed in P. fluorescens
• Plasmids harboring Gel.Exp_LCC or AlkB were PCR amplified with fwd_GelExpLCC (RBS addon), fwd_AlkB (RBS addon), rev_pSEVA438 (RBS addon) to add RBS BBa_J61100 (except for SPpstu_FAST-PETase which was synthesized with the RBS) after KLD treatment plasmids were transformed in E. coli, sanger sequencing validated plasmids were transformed in P. fluorescens
• Plasmids harboring Gel.Exp_LCC, AlkB or SPpstu_FAST-PETase were PCR amplified to add BBa_K4757002, BBa_K4757003, BBa_K4757005, respectively (fwd_LCC-synRBS, rev_V_LCC-synRBS, fwd_AlkB-synRBS, rev_V_AlkB-synRBS, fwd_FAST-synRBS, rev_V_FAST-synRBS) after KLD treatment plasmids were transformed in E. coli, sanger sequencing validated plasmids were transformed in P. fluorescens
• The coding sequences for Gel.Exp_LCC, AlkB, SPpstu-FAST-PETase were also cloned via restriction digest in the pBAD24 vector, PCR amplified with SacI and PstI recognition sites (fwd_pBAD24-PstI, rev_pBAD24-SacI) and transformed in E. coli BL21
• 100-300 mL cultures of BL21 and P. fluorescens were used for protein expression and subsequent purification as described above
• Raw lysates and purified proteins were loaded onto SDS-PAGEs and the purified protein was also used for activity assays as described above

• pSEVA438 harboring XylS-R38K-L224Q was amplified with BsaI-mut_XylS_fwd and BsaI-mut_XylS_rev to introduce silent mutations for removal of a BsaI recognition site, PCR product was treated with KLD mix and afterwards purified
• Previously obtain plasmid with XylS-R38K-L224QΔBsaI was PCR amplified was amplified with pSEVA_fwd, pSEVA_rev and correct size was validated with agarose gel
• Assembled plasmid from Project 1 (sRNA project) without any sRNA coding sequences were amplified with fwd_SapI_T_mKate2_RBS1_P and rev_SapI_T_mKate2_RBS_P
• Hifi assembly of PCR amplified vector and insert and transformation in E. coli
• Correct plasmid was assessed via Nanopore sequencing and amplified with pSEVA_fwd and pSEVA_rev and sequence validated with oxford nanopore sequencing
• Synthesized pAlkB fragment (described in project 3) was amplified with pAlkB_fwd (operon) and pAlkB_rev (operon), purified PCR product was hifi assembled with synthesized RBS2-BsaI-T sequence (GeneWiz, Azenta Life Sciences)
• Touch down PCR was performed directly after hifi assembly and cleaned up with preparative agarose gel (pAlkB_SacI_fwd, RBS2-BsaI-T_rev)
• Purified pAlkB_RBS2-BsaI-T sequence was hifi assembled with PCR amplified (assembled) AlkS fragment with AlkS_fwd, AlkS_rev
• Assembly was amplified from reaction mixture with Touch down PCR and purified with preoperative agarose gel (pAlkB_SacI_fwd, AlkS_rev2)
• Alternatively the three fragments (pAlkB, RBS2-BsaI-T, AlkS) were assembled with PCR as described above
• Complete insert was sequences were validated with oxford nanopore sequencing
• The synthesized mKate2 sequence (described above) was PCR amplified with mKate2-BsaI_fwd, mKate2-BsaI_rev
• The purified fragment pAlkB_RBS2-BsaI-T_AlkS was also used for golden gate assembly of
• Hifi assembly was done with PCR amplified vector (pSEVA_fwd, pSEVA_rev) and insert and transformed in E. coli and P. fluorescens, as of now no successful transformation could be done