iGEM 2023 — Team heidelberg

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RemixHD

# Notebook

![](https://static.igem.wiki/teams/4757/wiki/banner-over.png)

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RemixHD

# Engineering

**Synthetic biology has become one of the promising technologies around the globe. The intersection between biology and engineering aims to
construct biological systems for a helpful purpose. The framework of engineering cycles is crucial to succeed since they facilitate the systematic development and optimization of biological systems to execute designated functions.
Navigating through our project, we encountered numerous challenges that demanded innovative solutions and a rethinking of our designs.
Here, we illustrate our iterations within the engineering design cycles.**

![](https://static.igem.wiki/teams/4757/wiki/banner-over.png)

#### Sections

[Abstract](#Abstract)
[Wetlab](#Sec1)
[Self-Feedback Loop](#Sec11)
[Construction of AlkS fragment](#Sec12)
[Cloning of AlkS/pAlkB](#Sec13)
[Drylab](#Sec2)
[Digital Twin](#Sec21)
[Dynamic Flux Balance Analysis](#Sec22)
[Human Practices](#Sec3)
[GPS Tracking](#Sec31)
[References](#Sec6)

### Wetlab
### Drylab
### Human Practices

### Self-Feedback Loop

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-glu-hbirne.svg	)

#### Idea

We decided to regulate the operon via a negative feedback loop dependent on the terephthalic acid concentration.
XylS/pS1/pS2 expression system is a naturally occurring expression system that we wanted to use.

#### Results & Learnings

* Evaluation of the expression system
  pS1/pS2/XylS-mt in the presence of TPA as an inducer resulted in significant mKate2 fluorescence read-out.
* A combination of TPA and 3-methyl benzoate (MBA) was tested to boost XylS-mt expression.
  At high MBA concentrations, TPA dependency was abolished.
* Xylene and toluene were tested as they are inducers of the XlyS/XylR operon found in related Pseudomonas species *P. putida* (González-Pérez *et
  al., 2004*). The two inducers had no effect on XlyS-mt expression.

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-gehirn.svg)

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-kolben.svg)

#### Fix

The pS1/pS2 expression system was exchanged with the constitutive promoter pEM7 with a
Shine-Dalgarno sequence to increase basal XylS-mt expression. mKate2 fluorescence read-out overall
was higher compared to the initial system. However, the expression was less sensitive to TPA induction.

### Construction of AlkS fragment

#### Idea

As we needed the gene for AlkS, we wanted to order the fragment.

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-glu-hbirne.svg	)

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-gehirn.svg)

#### Results & Learnings

Due to the high complexity of the fragment, a synthesis was not possible. We learned that the size
of a construct matters.

#### Fix

We order it as three fragments and one backbone. We assembled the three fragments without the
backbone, PCR amplified, and purified. The fragment sequence was tested with an analytical agarose
gel and sequencing. The single AlkS fragment and backbone were then successfully added.

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-kolben.svg)

### Cloning of AlkS/pAlkB

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-glu-hbirne.svg	)

#### Idea

To improve the solubility and uptake of long-chain alkanes, we used a biosurfactant. (Chen et al., 2023) used rhamnolipids to successfully suspend the alkanes
and induce the AlkS/pAlkB expression system in *E. coli*.

#### Results & Learnings

The alkanes were resuspended in varying rhamnolipid concentrations. Cell growth and fluorescence
was measured over a few hour period. No usable results were generated due to the red coloring of
the rhamnolipids, which masked the fluorescence emitted by mKate2. (Chen et
al., 2023) worked in *E. coli* with the fluorescent reporter protein GFP and did not
have an issue with overlapping color.

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-gehirn.svg)

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-kolben.svg)

#### Fix

Two different biosurfactants, Tween® 80 and DMSO, were tested to suspend the alkanes and
proper fluorescence measurements were taken.

### Digital Twin development - finding the right model

#### Idea

Having settled for the idea of creating a digital twin for our organism, we researched existing
models for *P. fluorescens* and found one that promised a high accuracy. Using this model we
wanted to predict how well the colony would grow in specific conditions and when the resources
would be depleted.

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-glu-hbirne.svg)

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-gehirn.svg)

#### Results & Learnings

We ran different simulations and found out that using this model, the results only indicate how
much the colony grows at one point, when the initial medium concentration is present.

#### Fix

As we needed to know the proportions of the medium and colony at all times, this steady-state
modeling was not suitable for us. By using dynamic flux balance analysis, this problem was solved
later on.

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-computer.svg)

### Optimizing Dynamic Flux Balance Analysis

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-glu-hbirne.svg	)

#### Idea

Having established the dynamic flux balance analysis, we attempted a second round of calculating
the growth and medium composition.

#### Results & Learnings

This time, the analysis produced results where the colony starts small and grows over time, while
consuming energy from the medium. However, comparing the *in silico* results to the
experimentally measured data showed significant differences.

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-gehirn.svg)

![](https://static.igem.wiki/teams/4757/wiki/engineering-cycle-computer.svg)

#### Fix

By applying a correction factor *k = 0.04* the model produced reliable results over
multiple iterations during the dFBA.

### GPS Tracking

Explore the integration of an engineering cycle in our GPS Tracking project by visiting our [Sustainable Development Page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D#gps-container).

References

Chen, D., Xu, S., Li, S., Tao, S., Li, L., Chen, S., & Wu, L. (2023). Directly
Evolved AlkS-Based Biosensor Platform for Monitoring and High-Throughput Screening of Alkane
Production. ACS synthetic biology, 12(3), 832–841. <https://doi.org/10.1021/acssynbio.2c00620>

González-Pérez, M. M., Ramos, J. L., & Marqués, S. (2004). Cellular XylS levels are
a function of transcription of xylS from two independent promoters and the differential efficiency of
translation of the two mRNAs. Journal of bacteriology, 186(6), 1898–1901. <https://doi.org/10.1128/JB.186.6.1898-1901.2003>

[Abstract](#Abstract)
[Header 1](#Sec1)
[Header 2](#Sec2)
[Header 3](#Sec3)
[Header 4](#Sec4)
[Header 5](#Sec5)
[References](#Sec6)

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RemixHD

# Experiments

![](https://static.igem.wiki/teams/4757/wiki/banner-over.png)

#### Sections

[Abstract](#Abstract)
[The crisis](#Sec1)
[What is ReMixHD?](#Sec2)
[Bioinformatics](#Sec3)
[Why P. fluorescens](#Sec4)
[Our contribution](#Sec5)

DNA cloning method

##### 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)

Transformation

##### 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

Protein expression and purification

##### 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 PierceTm 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 based assays

##### 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

Project 1 sRNA

* 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

Sequence

| Sequence (5' -> 3') | description |
| --- | --- |
| gcggatacaggagtgcaaaaaatggctatctctagtaaggcctaccccttaggctttatgcaa  cGAAGAGCagatcgacagctgactgatGCTCTTCa  ttctgggagatcctggcaaacgcattcaaaaagaaaggggagcattagctccccttcttctttacaggttcatcaaacctttcaccatcggcagttcact  GACCAGGATCGCAAACTCCTGGCCATAGTTTTCATCGTTAGC  GCGGTGCCCGAGCTTGGATGGCAGATCGCAGTACCGAGCGACCGCGACCTCATGCTGTTCCACATACGTTTCTTTATCCGCCTCCTTGATGCGTTCGAGGCGGCGGTCCACATAATACACACCCGGCATTTTCAGATTTTTTGCTGGTTTCTTGCTGCGGTACGTGGTCTTCAGGTTACAAATCAGGTGCCCCCCGCCCACGAGTTTCAGTGCCATGTCCGCCCGGCCTTCCAAACCGCCATCGGCCGGATACAGCGTTTCGGTGGATGCTTCCCAGCCGAGCGTCTTCTTCTGCATGACTGGACCATTGGAGGGGAAGTTCACACCCCGAATTTTGACGTTGTAGATCAAGCAGCCGTCTTGCAGCGAGGTGTCCTGCGTCGCCGTCAGCACCCCACCGTCCTCGTACGTCGTGACGCGCTCCCAGGTGAAGCCTTCGGGAAAGCTCTGCTTGAAAAAATCAGGGATACCCTGCGTGTGATTGATGAACGTCTTGGAACCATACATAAAGGACGTCGCCAGAATATCGAAGGCAAATGGCAAGGGACCCCCTTCCACTGCCTTAATACGCATGGTCTGGGTGCCCTCATATGGCTTCCCCTCCCCCTCGGAGGTGCACTTAAAGTGATGATTGTTGACCGTACCTTCCATATACAATTTCATATGCATATTCTCCTTAATGAGTTCGCTGACCAT  CATCTAGTTTGTCCCCTCTTTCTCTAGA ggtttagttcctcacctt gtcgtattatactatgccgatatactatgccgatgattaattgtcaac  agagtcgacctgcaggcatgcaagcttgcggccgcgtcgtgactgggaaaac | insert\_RBS1 |
| gcggatacaggagtgcaaaaaatggctatctctagtaaggcctaccccttaggctttatgcaa  cGAAGAGCagatcgacagctgactgatGCTCTTCa  ttctgggagatcctggcaaacgcattcaaaaagaaaggggagcattagctccccttcttctttacaggttcatcaaacctttcaccatcggcagttcact  GACCAGGATCGCAAACTCCTGGCCATAGTTTTCATCGTTAGC  GCGGTGCCCGAGCTTGGATGGCAGATCGCAGTACCGAGCGACCGCGACCTCATGCTGTTCCACATACGTTTCTTTATCCGCCTCCTTGATGCGTTCGAGGCGGCGGTCCACATAATACACACCCGGCATTTTCAGATTTTTTGCTGGTTTCTTGCTGCGGTACGTGGTCTTCAGGTTACAAATCAGGTGCCCCCCGCCCACGAGTTTCAGTGCCATGTCCGCCCGGCCTTCCAAACCGCCATCGGCCGGATACAGCGTTTCGGTGGATGCTTCCCAGCCGAGCGTCTTCTTCTGCATGACTGGACCATTGGAGGGGAAGTTCACACCCCGAATTTTGACGTTGTAGATCAAGCAGCCGTCTTGCAGCGAGGTGTCCTGCGTCGCCGTCAGCACCCCACCGTCCTCGTACGTCGTGACGCGCTCCCAGGTGAAGCCTTCGGGAAAGCTCTGCTTGAAAAAATCAGGGATACCCTGCGTGTGATTGATGAACGTCTTGGAACCATACATAAAGGACGTCGCCAGAATATCGAAGGCAAATGGCAAGGGACCCCCTTCCACTGCCTTAATACGCATGGTCTGGGTGCCCTCATATGGCTTCCCCTCCCCCTCGGAGGTGCACTTAAAGTGATGATTGTTGACCGTACCTTCCATATACAATTTCATATGCATATTCTCCTTAATGAGTTCGCTGACCAT  CATCTAGTTTGTCCCCTCTTTCTCTAGA ggtttagttcctcacctt gtcgtattatactatgccgatatactatgccgatgattaattgtcaac  agagtcgacctgcaggcatgcaagcttgcggccgcgtcgtgactgggaaaac | insert\_RBS2 |
| gcggatacaggagtgcaaaaaatggctatctctagtaaggcctaccccttaggctttatgcaa  cGAAGAGCagatcgacagctgactgatGCTCTTCa  ttctgggagatcctggcaaacgcattcaaaaagaaaggggagcattagctccccttcttctttacaggttcatcaaacctttcaccatcggcagttcact  GACCAGGATCGCAAACTCCTGGCCATAGTTTTCATCGTTAGC  GCGGTGCCCGAGCTTGGATGGCAGATCGCAGTACCGAGCGACCGCGACCTCATGCTGTTCCACATACGTTTCTTTATCCGCCTCCTTGATGCGTTCGAGGCGGCGGTCCACATAATACACACCCGGCATTTTCAGATTTTTTGCTGGTTTCTTGCTGCGGTACGTGGTCTTCAGGTTACAAATCAGGTGCCCCCCGCCCACGAGTTTCAGTGCCATGTCCGCCCGGCCTTCCAAACCGCCATCGGCCGGATACAGCGTTTCGGTGGATGCTTCCCAGCCGAGCGTCTTCTTCTGCATGACTGGACCATTGGAGGGGAAGTTCACACCCCGAATTTTGACGTTGTAGATCAAGCAGCCGTCTTGCAGCGAGGTGTCCTGCGTCGCCGTCAGCACCCCACCGTCCTCGTACGTCGTGACGCGCTCCCAGGTGAAGCCTTCGGGAAAGCTCTGCTTGAAAAAATCAGGGATACCCTGCGTGTGATTGATGAACGTCTTGGAACCATACATAAAGGACGTCGCCAGAATATCGAAGGCAAATGGCAAGGGACCCCCTTCCACTGCCTTAATACGCATGGTCTGGGTGCCCTCATATGGCTTCCCCTCCCCCTCGGAGGTGCACTTAAAGTGATGATTGTTGACCGTACCTTCCATATACAATTTCATATGCATATTCTCCTTAATGAGTTCGCTGACCAT  AAATACCTCCTTACCTTTCAATTGACGCTGCTAACGCAGCCGG ggtttagttcctcacctt gtcgtattatactatgccgatatactatgccgatgattaattgtcaac  agagtcgacctgcaggcatgcaagcttgcggccgcgtcgtgactgggaaaac | Insert\_RBS 3 |
| ttgcataaagcctaaggggtaggccttactagag | rev\_Pm\_pSEVA438 (sRNA construct\_gibson) |
| agagtcgacctgcaggcatgcaagc | fwd\_pSEVA438\_MCS (for mKate gibbson) |
| gcggatacaggagtgcaaaaaatggctatctc | fwd\_SapI\_T\_mKate2\_RBS1\_P |
| gttttcccagtcacgacgcggccg | rev\_SapI\_T\_mKate2\_RBS\_P |
| agaagcggatacaggagtgcaaaaaatg | fwd\_sRNA validation |
| tgaactgccgatggtgaaaggtttgatg | rev\_sRNA validation |
| CTGCTAACGCAGCCGGggtttagttcctcaccttgtcgtattatactatgcc | fwd\_pEM7\_addon2 (binding region is in lower case) |
| CGTCAATTGAAAGGTAAGGAGGTATTTatggtcagcgaactcattaaggagaatatgc | rev\_mKate2\_addon2 |

Project 2 AlkS/pAlkB

* 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

Sequence

| Sequence (5' -> 3') | description |
| --- | --- |
| aaacaataataatggagtcatgaccatgcctaggc | fwd\_pSEVA48\_forAlkBP\_gibson |
| tatccctatcaaaccggacactgcactttatgc | rev\_pSEVA438\_forAlkBP\_gibson |
| GATTTCCCGGTCGCTAAGGTCGGAG | fwd\_AlkS\_colony PCR |
| CTTGACAGACTAAGCGCTGATAGATCTC | rev\_AlkS\_colony PCR |
| GCGATTCACGACGTTCAGCGTTGC | fwd\_AlkSrev\_colony PCR |
| CAATAACGACTTCCCGGTGGCAAAAGTCG | rev\_AlkSrev\_colony PCR |
| ttcgactgagcctttcgttttatttgatgc | fwd\_AlkS insert\_gibson |
| ggcgcctttctacatcacaccaagc | rev\_AlkS insert\_gibson |
| agttcacggttctcttattttaatgtgggctgc | fwd\_pSEVA438\_for AlkS |
| aggcatcaaataaaacgaaaggctcagtcg | rev\_pSEVA438\_for AlkS |
| ATCCgagctcATGGTCAGCGAAC | fwd\_mKate2(RE) |
| gatgtactGGATctgcagGCGGTG | rev\_mKate2(RE) |
| ATGGTCAGCGAACTCATTAAGGAGAATATGC | fwd\_mKate2(RE)\_RBS addon |
| CATCTAGTTTGTCCCCTCTTTCTCTAGAcgcggcctaggcatggtcatgac | rev\_pSEVA438 (RBS addon) (binding region in lower case) |
| ATGCCGATATACTATGCCGATGATTAATTGTCAACcataaagtgcagtgtcc | pEM7\_addon\_XylS/AlkS\_fwd (binding region in lower case) |
| AGTTAATACGACAAGGTGAGGAACGGATCCAAGGAcgtgaactatgaaaattattatc | pEM7\_addon\_AlkS\_rev (binding region in lower case) |
| tgcagccagcataaccagcataaagtgcagtgtccggtttgatagggata  atggctaatattattcagataactacaccaaggagtagttgaagcaggcttaaggtgagtgcagttttaccatgtcagatgtgagagtagatggcctgattttccctgttgggacaggaattaggttgtgtggtgctcgcagagacttattggacaataggagaacaacaaa  cagaaacaataataatggagtcatgaccatgcctaggccgcggccgcgcg | pAlkB fragment with homologous regions to vector |
| ttcgactgagcctttcgttttatttgatgcctttaattaaacgttcgtaa ttaaataataccctgacgctccgcctcgatggttgcctgggtgcgattcacgacgttcagcgttgcaaagattttccgcatgtgccatttaatagcatcctcggtcacgtgcatattcgttgcaatttgcttgttgctacacccctctttgacgagccgcaagaccgcaatttgtttccgggtgagcagtgcgtccgctttattctccgccgacttaccaatttcgacgatacgcggcagcgagaaggccccgatcgcctggtccaggttgaccgcggtgaacgcctcgcaggaggcggggatgatgcgttcaatcaggcacacctcgtccaacacggtttgaaaacattgcaactgcttagcaatctcctcggcatagacaatgttcaattgtgccttcttgagatcgccagcacctgcttgcgcgcctgccaaacacaacagcccacggacctccaactgcccggcattgattttgcgagcctgttggattgccaggagagcccgatgggcagcggaatggaaattccggtcacgcgagaggacgagggattgcacgaggagcagccgcgccttgagcggcgccgagtgctgcccggagaagattttatggtcttccaaggtcttcagattgttgatgccggtgataccttggcagacgaggcgctgataaatttcaatctgggacatgacttcgagccgaggcagatttttctccacagcatgggcttttgcccattccaggatctcaatggacccattgaggtccgaacgaccgagccgccatgcgctgaccgcacggcagacggagaagaacacgtcggtgacgccatggttggagatgaattccaaaatcttgctcagcttttcttccgacgtatccaggcaacg caactcataatgcagttccagctccaacgcatcaaacatt | AlkS fragment 1 |
| caactcataatgcagttccagctccaacgcatcaaacatt ttcgaggtgaattcgctctccatcatctgggcacgcgagtccgtccgagcttgggtaataatttgacgcgcccaccccatcttaccgcttgccaacgcttgttgaaagcgagcgacgtagagccaaccaaaggcgaagttctgcttagcaaatttgttcacagcctgggcctgcgccagcaccttttccagttccgcaaaacggtattcggaagcaaagataaaagccaaacaggtgagagcagcgcccttacccactgcgttcgagtcaccaaagaggctaatccacttattgcacaactcctcgctcgacagcatctcgtccttggtagctttcccgatcgccaggaccagttgcagccattctttttcctgccatttattctttttgtcgctctgcgaggacaggtccttgatcaacttctcagcccgagcaccttgttgggagaaatacaacacccaagcatagctgatcaacacaatgggtttttggtgccaggcttgtttgggcagttccaacagccattgacggagagcatcgatttcgccctgccggaaggacaggtccaaaatgatccgttccgacatgctcacagcccagcgacagtcgttggcacggaggctaatccggatagcgtactggtactcaccccggcgccagtgccagaaggctgcacgcttcaacaggtacgaccgtttggccgggttttccgtccaggtaatctcccgcaaaaagttccggagcacaggatggagggtaaattgcgccggttccccggagacgtgacgcaacagcatatagttggtggagaggtatttaatacacgagacaccattcacgcacttgaagacataattatattggtccggggtcacaaagtcgagcaaggacgaatttgccaagaacacacgataacgttc cgggatcgcttcgaagatttcgtcccgaaaataattgtcg | AlkS fragment 2 |
| cgggatcgcttcgaagatttcgtcccgaaaataattgtcg acctccaccacggcggaaatgtgtttcgcaggcaattcgcgcttcaagagaaagacgaccagtgccggccaaccctccacttcttggacgagcgtttcaatttgttcttcaggcacacccagcacggactcagcctccgccaatgcgaccgcttcctcagcggagaaggcgaggtctttctccgtgtactcccgcatcgcgccagcgagtttcagttgggagaagcctttgatggtattccccgcgacggcaaaacggatgttttttggcgtgttcagcatgaactccatgagcgcgtgcaacagaggcaaatccagatcatggttaatgttatccaggcacaccaacgtttcgatctcattattcgacgtgctttgccacaacgacgatgccagatcacggagcagagcgggcttgctgaccccctcgcgcacccgggagaacttgaccatttcaaaggtttccagctgttcaatgatctccgcgcagatatcgaactcggagtaggaggacgcccggaggctcaaccagactgccggccgaccagccgtccggtgacggagccactcgaaagccaacgcgaccgtcttaccatacccaggtggagcacggtagagacacacccgcggcgctgcaccgtcagcgatgctgagccgggggcggtaaatgcagctgtgcactttagcgctcaccaaggtggtgatttgatcggcaccgacttttgccaccgggaagtcgttattgataataattttcat agttcacggttctcttattttaatgtgggctgcttggtgtgatgtagaaaggcgcc | AlkS fragment 3 |

Project 3 XylS

* 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

Sequence

| Sequence (5' -> 3') | description |
| --- | --- |
| TCGCCTGCCCaggGGCGGGCGCC | XylS\_K38R\_XylS\_L224Q:1\_FWD (heterologous bases are lower case) |
| GATATTCCGTTTctgATTCTCCTCA | XylS\_K38R\_XylS\_L224Q:1\_REV (heterologous bases are lower case) |
| TGAGGAGAATcagAAACGGAATATC | XylS\_K38R\_XylS\_L224Q:2\_FWD (heterologous bases are lower case) |
| GGCGCCCGCCcctGGGCAGGCGA | XylS\_K38R\_XylS\_L224Q:2\_REV (heterologous bases are lower case) |
| ATGCCGATATACTATGCCGATGATTAATTGTCAACcataaagtgcagtgtcc | pEM7\_addon\_XylS/AlkS\_fwd (binding region in lower case) |
| AGTTAATACGACAAGGTGAGGAACGGATCCAAGGAcgtgaactatggatttttgc | pEM7\_addon\_XylS\_rev (binding region in lower case) |

Project 4 LCC/AlkB

* 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

Sequence

| Sequence (5' -> 3') | description |
| --- | --- |
| ATCCGAGCTC ATGATGAAGGGTAATAAAATCCTCTACATCTTGGGGACGGGGATCTTCGTCGGGAGTTCCTGCCTCTTCTCGTCGCTGTTTGTCGCTGCT  CAATCCAATCCATACCAGCGGGGTCCAAATCCTACGCGTTCCGCGCTGACGGCAGACGGGCCGTTCTCCGTCGCTACCTATACCGTCTCCCGTCTCTCCGTGTCCGGTTTCGGCGGCGGTGTCATCTACTATCCCACGGGTACCAGCCTGACCTTCGGGGGGATTGCCATGTCGCCAGGCTATACGGCAGATGCCAGCTCCTTGGCCTGGCTCGGCCGTCGTCTCGCTTCCCACGGTTTCGTCGTCCTCGTCATCAACACGAACTCGCGTTTTGATTATCCTGATTCGCGGGCGTCGCAGCTGAGCGCTGCGCTGAACTATTTGCGGACGTCGTCCCCGTCCGCAGTGCGTGCACGTCTCGACGCAAATCGTTTGGCCGTCGCAGGGCACAGCATGGGTGGCGGGGGTACGTTGCGGATCGCAGAGCAGAATCCCTCGCTCAAAGCGGCAGTCCCGCTCACGCCGTGGCACACCGACAAGACCTTCAATACGTCCGTCCCAGTCCTCATCGTCGGCGCTGAGGCCGATACGGTGGCCCCTGTGTCCCAGCACGCAATTCCATTTTACCAGAACCTGCCCTCCACCACGCCTAAAGTCTATGTCGAACTGGACAACGCTTCGCATTTTGCCCCTAATAGCAACAATGCAGCAATTTCCGTGTACACGATTTCGTGGATGAAACTGTGGGTCGACAACGATACGCGGTATCGGCAGTTCCTCTGCAATGTCAATGACCCCGCTCTGAGCGACTTTCGCACGAATAACCGGCATTGTCAGCTCGAG  CACCATCACCATCACCAC TGATGACTGCAGATCC | Synthesized Gel.Exp.\_LCC fragment (BBa\_K4757017, BBa\_K4757016) |
| ATCCGAGCTC ATGTTTGAGAATTTCTCGCCCAGCACGATGCTGGCGATTAAAAAGTACGCTTATTGGCTCTGGCTCTTGCTGGCATTGTCCATGCCCTTTAACTATTGGATGGCCCAGGATTCGGCTCATCCAGCCTTTTGGGCGTTTTCCCTGGTGATCGCGGTGTTCGGGATTGGTCCCCTGCTCGATATGCTCTTTGGCCGTGACCCCGCTAATCCCGACGAGGAAACCCAGACGCCTCAGTTGCTCGGGCAGGGCTATTACGTGCTGCTGACCTTGGCAACGGTGCCGGTGCTCATCGGGACGCTGGTGTGGGCAGCTGGGGTGTTCGTGGCATTTCAAGAGTGGGGCTGGTTGGGTCGCCTCGGTTGGATCCTCAGCATGGGGACCGTCATGGGCGCAGTGGGTATTGTCGTCGCTCACGAACTGATTCACAAGGACTCCGCGCTGGAGCAAGCAGCGGGCGGCATCCTCCTGGCCGCAGTCTGCTACGCGGGGTTTAAGGTCGAACACGTCCGCGGTCACCACGTCCATGTGTCCACCCCCGAGGACGCATCCTCCGCTCGTTTTGGCCAATCGGTCTACCAGTTTCTGCCCCACGCTTACAAGTATAACTTTTTGAATGCTTGGCGTCTCGAGGCGGTCCGCTTGCGGAAGAAGGGCTTGCCAGTCTTTGGCTGGCAGAACGAGTTGATCTGGTGGTATCTGCTGTCCCTGGCTTTGCTCGTGGGTTTCGGTTGGGCCTTCGGGTGGCTGGGGATGGTGTTCTTTTTGGGCCAGGCGTTCGTCGCTGTCACCTTGTTGGAGATCATCAATTACGTGGAGCACTATGGCTTGCACCGGCGCAAGGGTGAGGACGGTCGTTATGAACGGACGAATCACACCCACAGCTGGAACTCCAACTTTGTCTTCACGAACTTGGTCCTCTTTCATTTGCAACGCCACTCGGACCACCACGCCTTTGCCAAACGGCCCTACCAGGTGCTCCGGCACTACGACGATAGCCCGCAGATGCCCAGCGGGTATGCAGGCATGGTGGTCTTGGCTCTGATTCCTCCTCTCTGGCGGGCCGTGATGGATCCGAAGGTCCGGGCGTACTACGCCGGCGAGGAGTTTCAACTGACCGCCGAACAATCCGAGCGTCCAGCGGCTTCC  CACCACCACCATCACCAT TGATGACTGCAGATCC | Synthesized AlkB fragment |
| ATCCGAGCTC TCTAGAGAAAGAGGGGACAAACTAGATG  atgagccacatcctgcgagccgccgtattggcggcgatgctgttgccgttgccgtccatggcc  CAGACGAACCCGTATGCCCGCGGTCCGAATCCCACGGCAGCCTCCTTGGAGGCTTCGGCGGGTCCGTTCACCGTGCGCTCCTTTACCGACTCGCGTCCATCCGGCTATGGCGCTGGCACCGTGTACTACCCGACCAATGCCGGTGGTACGGTGGGTGCCATTGCTATCGTCCCTGGGTACACCGCCCGCCAAAGCTCCATCAAGTGGTGGGGTCCGCGGCTGGCCAGCCATGGGTTCGTGGTGATCACCATCGATACGAACTCCACGCTCGATCAGCCGGAAAGCCGGAGTTCACAGCAGATGGCCGCATTGCGTCAAGTCGCCTCGCTCAACGGGACGAGCAGCTCCCCTATTTATGGTAAAGTGGACACCGCCCGGATGGGCGTCATGGGGTGGTCCATGGGTGGCGGGGGGTCCCTCATCAGCGCGGCCAACAACCCTTCCTTGAAAGCCGCGGCACCACAAGCGCCTTGGGAGTCGTCCACCAACTTCTCCAGCGTCACGGTCCCGACCCTCATCTTCGCGTGTGAAAACGATAGCATCGCGCCTGTGAACTCCTCGGCGTTGCCGATCTACGATAGCATGTCGCAAAACGCGAAGCAGTTCCTGGAGATCAAGGGCGGGTCGCATTCGTGTGCTAACTCGGGTAATAGCAATCAGGCGTTGATCGGTAAAAAAGGCGTCGCCTGGATGAAACGGTTCATGGATAACGACACGCGGTATTCCACGTTTGCATGCGAGAACCCAAACAGCACCGCGGTGAGCGATTTCCGTACCGCAAACTGCTCGCTCGAA  CACCACCACCATCACCAC TGATGACTGCAGATCC | Synthesized RBS\_SPpstu-FAST-PETase fragment |
| ATCCGAGCTCATGATGAAGGGTAATAAAATCC | GelExp\_LCC\_primer01 |
| GGATCTGCAGTCATCAGTGGTGATGG | rev\_GelExp\_LCC(&FAST-PETase) |
| ATCCGAGCTCATGTTTGAGAATTTCTCG | AlkB\_primer 01\_fw\_MM |
| GGATCTGCAGTCATCAATGGTGATGGTG | AlkB\_primer 01\_rev\_MM |
| ATCCGAGCTCTCTAGAGAAAGAGGG | SPptsu\_FAST-PETase\_fwd |
| GGATCTGCAGTCATCAGTGGTGATGG | fwd\_GelExpLCC (RBS addon) |
| ATGTTTGAGAATTTCTCGCCCAGCACG | fwd\_AlkB (RBS addon) |
| CATCTAGTTTGTCCCCTCTTTCTCTAGAcgcggcctaggcatggtcatgac | rev\_pSEVA438 (RBS addon) (binding region in lower case) |
| GAGGTTTTAAatgatgaagggtaataaaatcctctacatcttggg | fwd\_LCC-synRBS (binding region in lower case) |
| CTTAAAGCATGTGCGCAcctaggcatggtcatgactccattattattgtttc | rev\_V\_LCC-synRBS (binding region in lower case) |
| AAGAACTACAAAAGAAAAGGTTAAGGAGGTTATTTatgtttgagaatttctcgcccagcacg | fwd\_AlkB-synRBS (binding region in lower case) |
| TTCTTCCGAAAGGActaggcatggtcatgactccattattattgtttc | rev\_V\_AlkB-synRBS (binding region in lower case) |
| GGCCTATAGTCCAGAACTAAGGAGGTATTTTTatgagccacatcctgcgagcc | fwd\_FAST-synRBS (binding region in lower case) |
| CTGTGGCCGTctaggcatggtcatgactccattattattgtttc | rev\_V\_FAST-synRBS (binding region in lower case) |
| ATACTGCAGagcttggctgttttggcggatgag | fwd\_pBAD24-PstI (binding region in lower case) |
| ATCCGAGCTCattcctcctgctagcccaaaaaaacggg | rev\_pBAD24-SacI (binding region in lower case) |

Final operon cloning

* 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

Sequence

| Sequence (5' -> 3') | description |
| --- | --- |
| TAATCCAGGCGAGATTAC | BsaI-mut\_fwd |
| GAGACGTGTTATCATCTGCAAATAATACTC | BsaI-mut\_rev |
| agagtcgacctgcaggcatgcaagc | pSEVA438\_MCS (for mKate gibson) |
| ttgcataaagcctaaggggtaggccttactagag | rev\_Pm\_pSEVA438 (sRNA construct gibson) |
| gcggatacaggagtgcaaaaaatggctatctc | fwd\_pSEVA438\_SapI\_T\_mKate2\_RBS |
| ATAGTTCACGCTCCTTGGATCCGTTCCTCACCTTGTCGTATTAAC | pSEVA\_fwd |
| TATTAGCCATGAGCTCAGTGAACTGCCGATGGTG | pSEVA\_rev |
| CACTGAGCTCatggctaatattattcagataactacacc | pAlkB\_fwd |
| GCCTAGGCATGGTCATGAC | pAlkB\_rev |
| cactgagctcatggctaatattattcag | pAlkB\_SacI\_fwd |
| gcaacgctgaacgtcg | RBS2-BsaI-T\_rev |
| TGCGATTCACGACGTTCAG | AlkS\_fwd |
| GGATCCAAGGAGcgtgaactatgaaaattattatcaataacgac | AlkS\_rev |
| ggatccaaggagcgtgaac | AlkS\_rev2 |
| CAGAAACAATAATAATGGAGTCATGACCATGCCTAGGCCGCGGCCGCGCG  TCTAGAGAAAGACAGGACCCACTAGATG AGAGACCAGATCGACAGTACTCTGATGGTCTCGCGACGTC TTCTGGGAGATCCTGGCAAACGCATTCAAAAAGAAAGGGGAGCATTAGCTCCCCTTCTTCTTTACAGGTTCATCAAACCTTTCACCATCGGCAGTTCACT GCTAGC TTAAATAATACCCTGACGCTCCGCCTCGATGGTTGCCTGGGTGCGATTCACGACGTTCAGCGTTGC | RBS2-BsaI-T |
| cactgagct c atggctaatattattcagataactacaccaaggagtagttgaagcaggcttaaggtgagtgcagttttaccatgtcagatgtgagagtagatggcctgattttccctgttgggacaggaattaggttgtgtggtgctcgcagagacttattggacaataggagaacaacaaa  CAGAAACAATAATAATGGAGTCATGACCATGCCTAGGCCGCGGCCGCGCG  TCTAGAGAAAGACAGGACCCACTAGATG  AGAGACCAGATCGACAGTACTCTGATGGTCTCGCGACGTCTTCTGGGAGATCCTGGCAAACGCATTCAAAAAGAAAGGGGAGCATTAGCTCCCCTTCTTCTTTACAGGTTCATCAAACCTTTCACCATCGGCAGTTCACTGCTAGCTTAAATAATACCCTGACGCTCCGCCTCGATGGTTGCCTGGGTGCGATTCA  CGACGTTCAGCGTTGCaaagattttccgcatgtgccatttaatagcatcctcggtcacgtgcatattcgttgcaatttgcttgttgctacacccctctttgacgagccgcaagaccgcaatttgtttccgggtgagcagtgcgtccgctttattctccgccgacttaccaatttcgacgatacgcggcagcgagaaggccccgatcgcctggtccaggttgaccgcggtgaacgcctcgcaggaggcggggatgatgcgttcaatcaggcacacctcgtccaacacggtttgaaaacattgcaactgcttagcaatctcctcggcatagacaatgttcaattgtgccttcttgagatcgccagcacctgcttgcgcgcctgccaaacacaacagcccacggacctccaactgcccggcattgattttgcgagcctgttggattgccaggagagcccgatgggcagcggaatggaaattccggtcacgcgagaggacgagggattgcacgaggagcagccgcgccttgagcggcgccgagtgctgcccggagaagattttatggtcttccaaggtcttcagattgttgatgccggtgataccttggcagacgaggcgctgataaatttcaatctgggacatgacttcgagccgaggcagatttttctccacagcatgggcttttgcccattccaggatctcaatggacccattgaggtccgaacgaccgagccgccatgcgctgaccgcacggcagacggagaagaacacgtcggtgacgccatggttggagatgaattccaaaatcttgctcagcttttcttccgacgtatccaggcaacgcaactcataatgcagttccagctccaacgcatcaaacattttcgaggtgaattcgctctccatcatctgggcacgcgagtccgtccgagcttgggtaataatttgacgcgcccaccccatcttaccgcttgccaacgcttgttgaaagcgagcgacgtagagccaaccaaaggcgaagttctgcttagcaaatttgttcacagcctgggcctgcgccagcaccttttccagttccgcaaaacggtattcggaagcaaagataaaagccaaacaggtgagagcagcgcccttacccactgcgttcgagtcaccaaagaggctaatccacttattgcacaactcctcgctcgacagcatctcgtccttggtagctttcccgatcgccaggaccagttgcagccattctttttcctgccatttattctttttgtcgctctgcgaggacaggtccttgatcaacttctcagcccgagcaccttgttgggagaaatacaacacccaagcatagctgatcaacacaatgggtttttggtgccaggcttgtttgggcagttccaacagccattgacggagagcatcgatttcgccctgccggaaggacaggtccaaaatgatccgttccgacatgctcacagcccagcgacagtcgttggcacggaggctaatccggatagcgtactggtactcaccccggcgccagtgccagaaggctgcacgcttcaacaggtacgaccgtttggccgggttttccgtccaggtaatctcccgcaaaaagttccggagcacaggatggagggtaaattgcgccggttccccggagacgtgacgcaacagcatatagttggtggagaggtatttaatacacgagacaccattcacgcacttgaagacataattatattggtccggggtcacaaagtcgagcaaggacgaatttgccaagaacacacgataacgttccgggatcgcttcgaagatttcgtcccgaaaataattgtcgacctccaccacggcggaaatgtgtttcgcaggcaattcgcgcttcaagagaaagacgaccagtgccggccaaccctccacttcttggacgagcgtttcaatttgttcttcaggcacacccagcacggactcagcctccgccaatgcgaccgcttcctcagcggagaaggcgaggtctttctccgtgtactcccgcatcgcgccagcgagtttcagttgggagaagcctttgatggtattccccgcgacggcaaaacggatgttttttggcgtgttcagcatgaactccatgagcgcgtgcaacagaggcaaatccagatcatggttaatgttatccaggcacaccaacgtttcgatctcattattcgacgtgctttgccacaacgacgatgccagatcacggagcagagcgggcttgctgaccccctcgcgcacccgggagaacttgaccatttcaaaggtttccagctgttcaatgatctccgcgcagatatcgaactcggagtaggaggacgcccggaggctcaaccagactgccggccgaccagccgtccggtgacggagccactcgaaagccaacgcgaccgtcttaccatacccaggtggagcacggtagagacacacccgcggcgctgcaccgtcagcgatgctgagccgggggcggtaaatgcagctgtgcactttagcgctcaccaaggtggtgatttgatcggcaccgacttttgccaccgggaagtcgttattgataataattttcatagttcacgctccttggatcc |

[DNA cloning method](#Abstract)
[Transformation](#Sec1)
[Protein expression and purification](#Sec2)
[Cell based assays](#Sec3)
[Project 1 sRNA](#Sec4)
[Project 2 AlkS/pAlkB](#Sec5)
[Project 3 XylS](#Sec6)
[Project 4 LCC/AlkB](#Sec6)
[Final operon cloning](#Sec6)

{% endblock %}

{% extends "layout.html" %}
{% block page\_content %}

RemixHD

# Results

**ReMixHD’s goal is to demonstrate a proof of concept for mixed plastic bioremediation using smart co-culture
of
*Pseudomonas fluorescens*. By engineering a helper strain capable of depolymerizing polyethylene and
polyethylene
terephthalate and a main strain utilizing the intermediates as a carbon source, a recombinant product can be
synthesized. The bacteria co-culture will be regulated by a custom-engineered operon capable of controlling
the
helper strains growth using an operon dependent on plastic degradation. For sensing the PE-breakdown
product,
alkanes, the alkane sensor, AlkS-V760E/PalkB, can be used to express a P. fluorescens growth factor
predicted by
our dry lab team. Using a novel engineered XylS-K38R-L224Q/Pm expression system, the PET-breakdown product,
terephthalic acid, is recognized and activates the sRNA repressor system, downregulating the growth of the
helper strain.
The main strain is capable of using terephthalic acid as a carbon source. Once the terephthalic acid levels
are high enough to maintain maximum main strain growth, the operon dynamically represses the helper strain
growth,
enabling synthesis of the recombinant product.**
![4-operon-zugeschnitten.svg](https://static.igem.wiki/teams/4757/wiki/4-operon-zugeschnitten.svg)

Overview of genetic construct of the final operon composite part

![](https://static.igem.wiki/teams/4757/wiki/banner-over.png)

#### Sections

[Abstract](#Abstract)
[Background Research](#Research-results)
[PE-degradation Sensor (AlkS-V760E/pAlkB)](#Sec1)
[AlkS-cloning](#Sec11)
[AlkS - results](#Sec12)
[PET-degradation sensor (XylS-K38R-L224Q/Pm)](#Sec2)
[XylS-cloning](#Sec21)
[XylS - results](#Sec22)
[sRNA mediated repression](#Sec3)
[sRNA - cloning](#Sec31)
[sRNA - results](#Sec32)
[Final operon assembly](#Sec4)
[Plastic degrading enzymes](#Sec5)
[References](#Sec6)

##### Modal title

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### 1. Background Research

Background Research

##### XylS

The PET degradation product terephthalic acid (TPA) is monitored by the XylS-K38R-L224Q
(*XylS-mt*)
transcription factor. Li *et al.* discovered two point mutations K38R and L224Q makes XylS
sensitive to TPA in
concentrations as low as 10 µM in *E. coli* (Li *et al.*, 2022). Upon activation with TPA or
the well
described XylS inducer 3-methyl-benzoate (MBA), XylS-mt dimerizes and binds the *Pm* promoter
(Gawin et al., 2017). *Pm* activation results in the expression of
small regulatory RNAs (sRNAs),
capable of blocking
the translation of the GOI. A negative feedback loop is established, downregulating the GOI activity at
high PET
depolymerization rates.

The expression of XylS-mt itself is regulated through the *Ps1/Ps2* promoter (Gallegos et al., 1996; Gawin et al.,
2017). In the absence of TPA, a low baseline of XylS-mt is present in the cell
through
constitutive low expression from the *Ps2* promoter. However, upon XylS-mt activation the
transcription factor
also binds the *Ps1* promoter leading to high levels of induction (Gallegos et
al., 1996).
This is the
first time a TPA sensor is characterized in *P. fluorescens* and in the iGEM parts registry.

##### sRNA

We introduced a synthetic sRNAs repressor system in Pseudomonas fluorescens to dynamically adjust gene
expression dependent on changing plastic concentrations. While extensively characterized and capable of
tightly regulating expression, protein-based repressors pose a high metabolic burden on the host cell
and cannot be easily expanded for repression of genome-encoded genes (Na et al.,
2013). Regulatory small RNA (sRNA) molecules are a viable option for an expandable and
low-burden repression system as they can dynamically and reversibly control gene expression.
Previous research has explored the use of synthetic small regulatory RNA molecules (Kelly et al., 2018).

mRNA interference and degradation ubiquitous in all organisms can achieve repression
(Modi et al., 2011). Prokaryotic Organisms natively regulate gene expression
through small RNAs (sRNAs)
and RNA chaperon protein hfq mediated sRNA-mRNA binding and degradation (Na et al.,
2013; Gottesman, 2004; Storz et al., 2011; Modi et al., 2011; Møller et al., 2002), which is
also present in Pseudomonas species (Trouillon et al., 2022; Wu et al.,
2021) and confirmed in our sub-strain ATCC 50090 by BLAST. The sRNAs have two main regions: the
scaffold and seed regions. The scaffold region forms the secondary structure, recruiting hfq and aiding
in the RNA degradation (Na et al., 2013; Møller et al., 2002). The seed or
target region is homologous to the mRNA and needed for sRNA-mRNA binding (Kelly et
al., 2018).
For our experimental design, three different sRNA scaffolds were tested that had previously been used
for synthetic sRNAs and three seed regions in the 5’UTR or 5’ beginning of the CDS. The chosen scaffold
included one previously used iGEM part (MicC, [Part:BBa
K1124005](http://parts.igem.org/Part%3ABBa_K1124005), (Na et al., 2013), an engineered scaffold, and its WT
version (SgrS, SgrSmt (SgrS-S CUUU 6 nts stem)) (Na et al., 2013, Noh et al.,
2019).

Along with testing different sRNA scaffolds, multiple RBS were tested as no standardized RBS is
available for *P. fluorescens*. Three synthetic RBS, including two previously used in iGEM and
taken from the Anderson library (RBS1 [Part:BBa
J61100](http://parts.igem.org/Part%3ABBa_J61100); RBS2 [Part:BBa J61101](http://parts.igem.org/Part%3ABBa_J61101), and one designed
using the Salis lab RBS calculator, were tested. The RBS designed by the Salis lab RBS calculator was
designed to maximize the expression of mKate2 mRNA with a predicted translation initiation rate of
516487.12 (Salis, 2023).
To test the gene expression, a C-terminal degradation tag was linked to the reporter gene mKate2. The
degradation tag was shown to work in *Pseudomonas putida*  (Halvorsen et al.,
2022). The degradation tag reduced the halflife of the constitutive expression mKate2, which
was expressed using the promoter pEM7. This enabled us to observe the dynamic repression resulting from
the sRNAs.

##### AlkS

An alkane sensor using the AlkS/pAlkB expression system creates a positive feedback loop promoting the
expression of our GOI dependent on the PE degradation products n-alkanes. During depolymerization, PE’s
carbon backbone fragments into n-alkanes, which are further metabolized by alkane monooxygenase AlkB
(Pinto et al., 2022). The length of the n-alkanes resulting from the PE
depolymerization is unknown, as the mechanism is yet to be determined. However, the length of the alkane
chains is presumably below C17 as long-chain alkanes cannot pass through the cell membrane via
diffusion, and there is no known transporter in *P. fluorescens* for alkanes longer than C16. The
alkane, along with the transcription factor AlkS originally found in *P. oleovorans*, builds a
complex leading to the activation of the AlkB promotor and the expression of the GOI (Yuste et at., 1998). The alkane sensing abilities of AlkS are well-researched.
WT AlkS is only sensitive to short alkanes and is not sensitive to chain lengths between C12 and C16
(Wu et al., 2015). Dongdong Chen et al. identified AlkS with the mutation
V760E, which can recognize alkanes with carbon chain lengths up to C17 (Chen et
al., 2023). AlkS and the alkane bind to the AlkB promoter, leading to the expression of the GOI
(Yuste et al., 1998). The constitutive promoter pEM7 or pS2 expressed
AlkSmt, and pAlkB expressed mKate 2 in *P. fluorescens*. Alkanes needed to be introduced to the
cell to test the alkane-responsive transcriptional regulatory system. As alkanes are not soluble in
water, a biosurfactant is necessary to make them soluble. Multiple biosurfactants were tested, including
rhamnolipids, tween 80, and DMSO (Chen et al., 2023). The growth rate of the
cells and the fluorescence were then measured and used to determine the expression rate of AlkS and
pAlkB.

##### LCC/PETase/AlkB

The growing amount of plastic polymer pollution, like polyethylene terephthalate (PET) and low-density
polyethylene (LDPE), poses a formidable challenge that scientists are trying to solve bioremediation
using enzymatic degradation. Enzymes have been identified in nature capable of breaking down these
polymers.

PET is composed of alternating terephthalic acid and ethylene glycol molecules connected by ester bonds.
Esterases, such as WT-Petase from *Ideonella sakaiensis* or leaf-branch compost cutinase (LCC)
discovered through metagenomic analysis, can cleave the esters and, when secreted, lead to PET
degradation (Yoshida et al., 2012; Tournier et al., 2020). However,
optimizing these enzymes for *P. fluorescens* poses distinct challenges. Most PETases reach peak
activity at an alkaline pH and temperatures above 50°C (Yoshida et al.,
2012). At the optimal culture conditions for *P. fluorescens* (pH 7 and 28°C), the PETase
activity severely decreases. Even with recent breakthroughs, temperature- and pH-optimized mutants such
as the fast-PETase and LCC are still suboptimal for use within the ReMix framework (Lu et al., 2022; Tournier et al., 2020).

For enzymatic plastic degradation to occur, they need to be exported out of the cell using export tags.
One tag of two tags tested hijacks the naturally occurring gelatinase export system found in *P.
fluorescence*. The second tag utilizes the signal peptide SPpstu from the maltotetraose-formin
amylase Pseudomonas stutzeri MO-19 (Lu et al., 2022).

PE has a pure carbon backbone structure, making enzymatic degradation difficult as it lacks easily
hydrolyzable groups. Alkane monooxygenase (AlkB) found in various Pseudomonas species plays a role in
breaking down PE (Pinto et al., 2022). Expression of AlkB in *E. coli*
enabled growth on LDPE. AlkB oxidizes medium-length n-alkanes in the periplasm into their corresponding
primary alcohols, subsequently ferrying them across the inner membrane. The 1-alkanols are oxidized
intracellularly into fatty acids, feeding into the cell's metabolism via the beta-oxidation cycle (Callaghan et al., 2006). The exact PE depolymerization mechanism is not fully
understood. It is unlikely that AlkB alone facilitates the depolymerization into n-alkanes as the enzyme
resides in the inner membrane, unreachable for any alkane-like structures above C16 (Grant et al., 2014). AlkB cannot oxidize n-alkanes longer than n-dodecane,
which supports this hypothesis. For iGEM, we want to demonstrate that *P. fluorescens* can
functionally express AlkB as an additional depolymerizing gene for our helper strain.

##### mKATE2

We replaced the GOI with a fluorescent reporter protein to test our expression systems. As *P.
fluorescens* is a naturally fluorescent organism, we must choose a reporter protein whose
excitation and emission wavelengths are distinct from the naturally emitted wavelengths. mKate2 has an
excitation wavelength of 588 nm and an emission wavelength of 633 nm (Shcherbo et
al., 2009). Another criterion was the fast maturation time as we designed our operon to be
dynamic. With a 20-minute maturation time, mKate2 fulfilled this criterion (Heppert
et al., 2016).

##### Main strain

The main strain's goal is to utilize the TPA as a carbon source and generate a recombinant product. The
set of genes for TPA uptake were introduced to our organism using the pBAMD1-2 plasmid containing the
tphII operon (Brandenberg et al., 2022). Brandenberg et
al. (2022) presented four genes found in *Comamonas sp.* E6m needed to convert TPA into
PCA, which can reach the central carbon metabolism via the ß-ketoadipate pathway. The mini-Tn5
transposon vector pBAMD1-2 integrates the operon randomly in the genome. Due to the nature of the
operon, TPA is a selection marker, and no further antibiotic resistance is necessary (Brandenberg et al., 2022).

### 2. PE-degradation Sensor (AlkS-V760E/pAlkB)

AlkS - cloning

For cloning of all the constructs, the pSEVA438 plasmid vector was used with the pBBR1 ori, which is
compatible with
a broad range of prokaryotic organisms. The plasmid carries the *XylS/Pm* expression cassette,
which was used as
a basis for the experiments. The growth assays were done in 96-well microtiter plates incubated at 28 °C
and
OD600 and fluorescence (588 nm excitation, 633 nm emission) measurements were taken every 10
min over a
time period of 16-24 h. The fluorescence of each well was normalized with cell count (referenced to
OD600).
The results were compared to the appropriate negative controls.

Sequences coding for AlkS and pAlkB were obtained by gene synthesis (IDT) and cloned via Gibson
assembly into the
plasmid vector. Transcription factor expression was regulated by the constitutive *pEM7* promoter,
replacing
the *XylS/Pm* system. The fluorescence reporter gene *mKate2* was cloned with SacI and PstI
into the MCS
downstream of *pAlkB*. To increase fluorescence intensity with clearer read-outs, a synthetic RBS
from the
Anderson library (BBa\_J61100) was added upstream of the coding sequence via substitution PCR.

![AlkS overview](https://static.igem.wiki/teams/4757/wiki/alks-overview-mew.svg)

***Figure 1: Overview of genetic construct for AlkS
testing***

##### Detergent testing

Before characterizing the transcription factor, preliminary tests were conducted to optimize the
solubility and
bioavailability of different length n-alkanes (hexane, heptane, dodecane, heptadecane). Solubility was
tested in
varying concentrations of H2O, dimethyl sulfoxide (DMSO), Tween® 80, and rhamnolipids. Long chain
alkanes could not be
brought into solution using H2O and DMSO, making them unsuitable for future experiments.
While rhamnolipids
could readily solubilize alkanes, they showed high absorption at OD600 and strong
auto-fluorescence, making
them unsuitable.

The best results were achieved by first solubilizing the alkanes in 1 % Tween80<®> in ethanol
absolute. To
allow for bioavailability this solution was suspended in H2O at 100-fold dilution.

![](https://static.igem.wiki/teams/4757/wiki/alks-induction-of-pem7-alks-palkb-mkat2-with-n-dodecane-after-20-h.svg)

**Figure 2: Biological availability of n-dodecane solubilized in
different detergents**

Biological availability was measured by fluorescence increase of mKate2 regulated by the
n-dodecane sensing
transcription factor AlkS-V760E. Values are presented as mean +/-SD. For statistical analysis groups
were
compared to AlkS 0 mg/mL+Tween80®. Reported significances were determined with ordinary One-way
ANOVA with
Dunn's method for multiple comparisons. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.001>/0.001>
0.0001. To
reduce complexity only significant test results are shown.

PE-degradation Sensor (AlkS-V760E/pAlkB) - Results

The final PE biosensor has the AlkS-V760E transcription factor constitutively expressed by the pEM7
promoter and
the AlkS-V760E/pAlkB expression strength is measured with mKate2 fluorescence as a reporter gene.

Different n-alkanes emulsified in Tween® 80 (0.1 % (v/v)) were tested as inducers of mKate 2 at
different
concentrations (100 mg/L, 200 mg/L) with time-resolved fluorescence measurements (figure 10). Only
n-dodecane
showed a change in fluorescence intensity and was used for further testing of the induction of AlkS at
different
concentrations.

![](https://static.igem.wiki/teams/4757/wiki/alks-sensitivity-of-pem7-alksv760e-towards-n-alkanes.svg)

**Figure 10: AlkS expression strength with different length n-alkanes.**

Time resolved fluorescence measurements of pEM7-AlkS/pAlkB controlled mKate2 expression. Induction
with
different length alkanes (hexane, heptane, dodecane, heptadecane) solubilized in 0.1 % (v/v)
Tween® 80 at
two different concentrations. Values are presented as mean +/- SD.

Serial dilution experiments of n-dodecane emulsified in Tween® 80 were performed and fluorescence
intensity
measured over 20 h (Figure 10, A). Expression strength was calculated 6 h, 12 h and 20 h after induction
with
different concentrations, ranging from 2 mg/L up to 2000 mg/L. Twelve hours after induction, significant
increases
could be measured with inducer concentrations smaller than 200 mg/L (p<0.01). At 20 h concentrations
as low as
20 mg/L were sufficient to measure a significant change in fluorescence (p<0.01). The fluorescence
measurements
at 20 h were used to further analyze the dose-response curve (Figure 5, B), showing inducer saturation
above 2000
mg/L n-dodecane.

![](https://static.igem.wiki/teams/4757/wiki/alks-sensitivtiy-of-pem7-alksv760e-towards-n-dodecane-pem7.svg)

**Figure 11: Induction of AlkS/pAlkB expression system with different concentrations of
n-dodecane.**

Left graph shows induction of pEM7-AlkS-V760E with n-dodecane constructions ranging from 2 mg/L up
to 2000
mg/L, supplemented with 0.01 % (v/v) Tween® 80.

Right graph shows the dose response curve of serial dilutions of n-dodecane and expression
strength 20 h
after induction.

Values are presented as mean +/-SD. For statistical analysis groups were compared to 0 mg/mL at
each time
point. Reported significances were determined with ordinary One-way ANOVA with Dunn’s method for
multiple
comparisons. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001. To reduce complexity only
significant test results are shown.

### 3. PET-degradation sensor (XylS-K38R-L224Q/Pm)

XylS - cloning

Since the XylS/Pm expression system is natively found on the pSEVA438 plasmid only the two point
mutations, K38R
and L224Q, needed to be introduced. Two primer pairs were used to add the single base pair
substitutions. The
sensitivity of XylS-mt towards was studied using the native Ps1/Ps2 promoter system but found to yield
low
expression levels in the TPA sensitive range. To mitigate this problem, the
Ps1/Ps2 promoter system was substituted with pEM7 to further test the functionality in different
scenarios. The
fluorescence reporter gene mKate2 was cloned with SacI and PstI into the MCS downstream of Pm, add-on
PCR was used
to introduce the Anderson library promoter RBS BBa\_J61100. (Figure 3).

![XylS_mew](https://static.igem.wiki/teams/4757/wiki/xyls-overview-mew.svg)

***Figure 3: Overview of genetic construct for XylS-MT
testing***

##### XylS-WT TPA sensitivity testing

The XylS-mt sensitivity towards TPA was compared to the XylS-WT sensitivity. XylS-WT showed no
sensitivity
towards TPA and good sensitivity towards MBA. When comparing the sensitivities of XylS-mt and XylS-WT to
MBA, the
introduced mutations seemed to cause a 60-70 % decrease in expression strength (figure 4).

![](https://static.igem.wiki/teams/4757/wiki/induction-strength-comparison-xylswt-vs-xylsmt.svg)

**Figure 4: Comparison of expression strength of
wildtype and
mutated (K38R, L224Q) XylS, at three different inducer concentrations.**

Values are presented as mean +/-SD. No statistical analysis was performed.

##### XylS-mt induction with XylR activation

Co-induction with varying concentrations of TPA and m-Xylene or TPA and Toluene (5 nM, 50 nM, 500 nM
m-Xylene or
Toluene mixed with 0 nM, 2.5 nM, 5 nM, 10 nM, 50 nM, 500 nM, or 1 mM TPA) was tested to improve the
induction of
XylS-mt and the expression of the GOI. Toluene and Xylene are inductors of the genomic transcription
factor XylR,
previously described to jointly activate expression from the *Ps1* promoter with XylS in  *P.
putida*.
However, co-induction showed no increase in expression strength (data not shown).

PET-degradation sensor (XylS-K38R-L224Q/Pm) - Results

XylS-mt was first tested with the native Ps1/Ps2 promoter system, with different inducer compositions
of TPA and
3-methyl-benzoate (MBA). The *Ps1/Ps2* promoter was substituted with the constitutive promoter
*pEM7*
using add-on PCR. TPA and MBA were tested separately in serial dilutions experiments (figure 11), and in
combination (figure 12).

##### Ps1/Ps2 XylS-mt (with MBA or TPA)

Serial dilution experiments of only TPA showed significantly increased fluorescence compared to the
uninduced
controls for concentrations above 1 mM at 8 h and 12 h after induction (p<0.01) (Figure 11, C). The
same
experiments performed with MBA as an inducer showed an overall stronger expression strength and
significant
changes in fluorescence after induction with 0.01 mM MBA (p<0.001) (Figure 11, A). The calculated
dose response
curve (Figure 11, (B)) shows inductor saturation at 0.1 mM. For induction of TPA, no inductor saturation
was
observed (Figure 11, D). The fluorescence intensity of the XylS-WT compared to the XylS-mt shows an
overall
decreased expression strength. (Figure 11, E)

![](https://static.igem.wiki/teams/4757/wiki/xyls-overview-graphc-a-e.svg)

**Figure 12: MBA andTPA dependent induction of the XylS-MT transcription factor controlling
mKate2
expresion.**

**(A)** Fluorescence intensity measurements at 8h, 12 h, 16 h after induction with
serial
dilutions of MBA

**(B)** Dose response curve of expression strength for different MBA inducer
concentrations

**(C)** Fluorescence intensity measurements at 8 h, 12 h, 16 h after induction with
serial
dilutions of TPA

**(D)** Dose response curve of expression strength for different TPA inducer
concentrations

**(E)** Time resolved measurements of dose response curves after induction with TPA
and MBA

Values are presented as mean +/-SD. For statistical analysis groups were compared to 0 mg/mL MBA
or 0 mg/mL
TPA at each time point. Reported significances were determined with ordinary One-way ANOVA with Dunn's
method
for multiple comparisons. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001. To reduce complexity
only significant test results are shown.

##### Ps1/Ps2 XylS-MT TPA and MBA co-induction

To further test the influence of the Ps1/Ps2 promoter system on XylS-mt, the co-induction was tested
with
previously determined MBA and TPA concentrations. Three TPA concentrations were tested with one of four
MBA
concentrations. Fold change and normalized fluorescence were calculated (Figure 13). At an MBA
concentration of
0.0025 mM, a significant TPA dependent fold change could be measured (1.29 +/- 0.056, p < 0.001). Higher
MBA concentrations (0.0075 mM MBA, 0.015 mM MBA) showed an overall decreased fold change. Decrease
after TPA induction is due to referencing errors caused by TPA precipitation. The expression strength
shows an overall decreased fluorescence intensity at low MBA concentrations, despite co-induction with
TPA (Figure 13, right)

![](https://static.igem.wiki/teams/4757/wiki/xyls-tpa-sensitivity-of-ps1ps2-xylsmt-upon-mba-activation-expression-strength-of-ps1ps2-upon-induction-with-tpa-and-mba.svg)

**Figure 13: Expression strength with TPA and MBA co-induction**

Left: Fold change in expression of different MBA inducer concentrations after co-induction of
TPA

Right: Expression strength measured in relative fluorescence of different MBA inducer
concentrations
after co-induction of varying TPA concentrations.

Values are presented as mean +/-SD. For statistical analysis groups were compared to 0 mg/mL
MBA or 0
mg/mL TPA at each time point. Reported significances were determined with ordinary One-way ANOVA
with Dunn's
method for multiple comparisons. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001. To reduce
complexity only significant test results are shown.

##### pEM7 XylS-MT

Alternative to the *Ps1/Ps2* promoter system, the constitutively active *pEM7* promoter
was
tested, which was previously used for the expression of AlkSV760E. The new promoter showed overall
higher
fluorescence intensities, compared to the previously tested *Ps1/Ps2* promoter system.
Significant
changes above induction could be measured with 0.005 mM MBA (p<0.001) or 1 mM TPA (p<0.05)
(figure 14).

![](https://static.igem.wiki/teams/4757/wiki/xyls-expressopm-stremgth-of-pem7-xylsmt-upon-induction-with-tpa-or-mba.svg)

**Figure 14: Expression strength of pEM7-XylS-mt at different TPA concentrations.**

TPA dependent expression strength was measured by fluorescence intensity of the pEM7-XylS-mt.
Measurement was done with three replicates and error bars calculated with the standard
deviation.Values are
presented as mean +/-SD. Reported significances were determined with ordinary One-way ANOVA with
Dunn’s
method for multiple comparisons. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001. To reduce
complexity only significant test results are shown.

### 4. sRNA mediated repression

sRNA - cloning

Three gene constructs were obtained by gene synthesis (IDT) each with a different ribosomal binding
site
(BBa\_J61100, BBa\_J61101, BBa\_K4757003). The construct also contains two SapI recognition sites, a
bi-directional
terminator (LUZ7 T50, BBa\_K4757058), mKate2 in reverse complement with degradation tag (BBa\_K4757000,
BBa\_K4757001), and the constitutive promoter pEM7. The sRNA coding oligo sequences were cloned scarless
behind the
Pm promoter with SapI golden gate assembly, yielding 27 different composite parts (By combining the seed
regions
BBa\_K4757021 - BBa\_K4757027 with the scaffolds BBa\_K4757031 - BBa\_K4757033 ).

![sRNA_mew](https://static.igem.wiki/teams/4757/wiki/srna-overview-02-mew.svg)

**Figure 5: Overview of genetic construct for sRNA testing.**

The sRNA repressor consists of one seed region (BBa\_K4757021 - BBa\_K4757027) combined with one
scaffold
(BBa\_K4757031 - BBa\_K4757033), repressing on, or near, the different ribosomal binding sites tested
(BBa\_J61100,
BBa\_J61101, BBa\_K4757002).

##### RBS comparison

To find optimal expression levels of mKate2 and establish new ribosomal binding sites for *P.
fluorescens*,
different ribosomal binding sites were tested. For the experiments, the repression of the fluorescence
intensity
of constitutively expressed mKate2 was measured and the fold change over the auto-fluorescence of *P.
fluorescens* was calculated (Figure 6).

![](https://static.igem.wiki/teams/4757/wiki/fold-change-fluorescence-intensity-with-testsed-rbs.svg)

***Figure 6: Calculated fold change of fluorescence intensity of mKate2 with different
ribosomal binding
sites**

(RBS 1: BBa\_J61100, RBS 2: BBa\_J61101, RBS 3: synthetic de-novo RBS) compared to wild-type P.
fluorescens.
Measurements were taken in the early stationary phase. Values are presented as mean +/-SD. For
statistical
analysis groups were compared to wild-type P. fluorescens Reported significances were determined
with ordinary
One-way ANOVA with Dunn's method for multiple comparisons. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001;
\*\*\*\*p < 0.0001. All differences in fold changes were significant (p < 0.001 for RBS 1 and 2). To
reduce complexity only edge cases are shown.*

Constructs with BBa\_J61100 (RBS 1) showed minimal fold-change in fluorescence levels (0.73 +/- 0.316).
The second
RBS from the Anderson library (BBa\_J61101, RBS 2) had a distinct increase in fold change compared to
BBa\_J61100
(18.15 +/- 3.21 compared to 0.73). The synthetic RBS (RBS 3) designed by the Salis-lab calculator
(calculated for
maximal expression strength for mKate2 mRNA) showed the strongest fluorescence (48.56 +/- 3.394).

Although RBS 3 showed highest expression strength, RBS 2 was used for the final operon as the
binding was
independent from the coding sequence (CDS).

##### sRNA comparison

Before ordering the different sRNA constructs, *in silico* analysis of the free binding energy of
sRNA-mRNA
hybridization was calculated and compared to literature to ensure efficient repression (figure 8).

The 27 different sRNA constructs were tested using three different scaffolds, previously used for
synthetic sRNA
repression, and three different binding sites. The scaffolds SgrS and MicC were chosen since they have
been used
by previous iGEM teams (e.g. Team Peking 2011, Team Edinburgh 2018, Team UT-Tokyo 2013) and have been
established
in the literature (No et al., 2019). As they lack characterization in bacteria other than *E.
coli* ,
we could establish sRNAs in the novel chassis *P. fluorescens* . Additionally, an engineered
version of SgrS
(SgrS-S CUUU 6 nts stem (SgrSmt)), optimized for repression in *E. coli* DH5 alpha, was chosen (Noh
et al., 2019). Seed regions (homologous to the mRNA) were chosen with 25 bp homology, targeting
either the RBS
(target 1), both the RBS (12 nt) and CDS (13 nt) (target 2), or the CDS starting with AUG (target 3).

(A)

![](https://static.igem.wiki/teams/4757/wiki/repression-strength-against-rbs-1.svg)

(B)

![](https://static.igem.wiki/teams/4757/wiki/repression-strength-against-rbs-2.svg)

(C)

![](https://static.igem.wiki/teams/4757/wiki/repression-strength-against-rbs-3.svg)

**Figure 7: Repression strength of all tested sRNA constructs.**

Bar plots showing the repression strength of the 27 tested sRNA constructs with measurements
taken in the
early stationary phase. The naming scheme is the scaffold name followed by the seed region 1.#,
2.#, or 3.#,
targeting 25 nt of the RBS, 13 nt of the RBS and 12 nt of the CDS, or 25 nt of the CDS starting
with AUG,
respectively. The second number indicates different ribosomal binding sites upstream of mKate2
CDS((A): #.1
BBa\_J61100; (B) #.2 BBa\_J61101; (C) #.3 BBa\_K4757003) Repression was calculated by dividing
fluorescence
intensity of the respective RBS-mKate2 constructs with constructs lacking the sRNA coding
sequence.Values are
presented as mean +/-SD. For statistical analysis groups were compared to negative control for
each sRNA.
Reported significances were determined with ordinary One-way ANOVA with Dunn's method for multiple
comparisons.
\*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001.

All constructs were tested with endpoint measurements in early stationary phase to see the maximum
repression
capability (figure 3). Seed regions with target 3 showed the weakest repression rates (0.367 +/- 0.118
mean
repression compared to 0.544 +/- 0.153 and 0.569 +/- 0.102, for target 1 and 2 respectively). Seed
regions with
target 1 showed the strongest repression regardless of the RBS used.

The final operon construct contained the seed region targeting only the RBS (target 1, RBS2:
BBa\_J61101) with the
SgrS and MicC scaffolds, as they showed the highest repression strength and the RBS was not designed for
a
specific mRNA sequence.

##### comparison of experimental data with previously calculated properties

After conducting repression experiments with all sRNA constructs, possible correlation between the
relative
repression strength and calculated free binding energy was calculated.

Figure 8 shows repression strength against the free binding energy. For all three tested targets no
correlation between binding energy could be found. Interestingly target 3 showed an overall increased
variance
(0.0678 mean error) compared to target 1 (0.042 mean error) and target 2 (0.0422 mean error).

![](https://static.igem.wiki/teams/4757/wiki/srna-effect-of-free-binding-energy-on-repression-dot-plot.svg)

**Figure 8: Dot plot of the in vivo measured relative repression strength against the in
silico calculated
free binding energy.**

The free binding energy was calculated with ViennaRNA. Experimental values are presented as
means +/- SD, no
significance was calculated.

sRNA mediated repression

##### SgrS1.2/MicC1.2 repression

The scaffolds SgrS and MicC with the RBS 2 (BBa\_J661101) target region were used for further
characterization
of the repression characteristics.

The sRNA expression was controlled by the MBA inducible XylS-WT/Pm promoter system. By targeting the
constitutively expressed mKate2, repression strength was calculated with decrease in fluorescence
intensity
(Figure 15). Both sRNA constructs showed an overall continuous repression strength over time after
induction.
(Figure 15 A, B) with the highest repression after 20 h of 0.65 +/- 0.033 and 0.61 +/- 0.034 for SgrS
and
MicC, respectively.

The inducer concentration dependent repression strength was calculated at the time points 10 h and
15 h
(Figure 15), which showed a linear increase in repression strength with a saturation above 100 mM MBA
concentration.

(A)

![](https://static.igem.wiki/teams/4757/wiki/dose-response-curve-micc-10-h-and-15-h-after-induction-mt.svg)

(B)

![](https://static.igem.wiki/teams/4757/wiki/srna-repression-sgrs-log2-5-h-10-h-15-h-barplot.svg)

(C)

![](https://static.igem.wiki/teams/4757/wiki/dose-response-curve-micc-10-h-and-15-h-after-induction-mt.svg)

(D)

![](https://static.igem.wiki/teams/4757/wiki/srna-repression-micc-log2-5-h-10-h-15-h-barplot.svg)

**Figure 15: Repression strength and dose response curve for SgrS1.2 and MicC1.2 at 5 h, 10 h,
15 h
with MBA serial dilutions.**

Repression strength was measured by dividing measured fluorescence intensity with fluorescence
intensity
of constructs without sRNA coding genes. Error bars were calculated with the standard deviation. Three
biological replicates were analyzed.

(A) Repression strength of SgrS with serial dilutions

(B) Repression strength of MicC1.2 with serial dilutions

(C) Dose-respsonse curve of different MBA concentrations

(D) Dose-response curve of different MBA concentrations

Values are presented as mean +/-SD. For statistical analysis groups were compared to 0 nM at each
time
point. Reported significances were determined with ordinary One-way ANOVA with Dunn's method for
multiple
comparisons. \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; \*\*\*\*p < 0.0001. To reduce complexity only the first
significant test results are shown.

### 5. Final operon assembly

Final operon assembly

The XylS-K38R-L224Q on the pSEVA438 plasmid was used as a basis for assembling the final operon. The
vector was
linearized by PCR, adding homologous overhangs for pAlkB and AlkS sequences. A new sequence containing
RBS 2
(BBa\_J61101), BsaI restriction sites, and the LUZ7T50 terminator (BBa\_K4757058), was synthesized with
homologous
sequences (IDT). All three insert fragments (pAlkB, RBS2-BsaI-LUZ7 T50) were assembled using Gibson
assembly.

Golden Gate assembly, with BsaI restriction enzyme, was used for inserting mKate2 behind RBS 2.
Insert and
vector sequences were verified with sequencing, but after multiple attempts with different molar ratios,
they
could neither be successfully combined nor transformed into either *P. fluorescens* or *E.
coli* DH5
alpha.

![final operon_mew](https://static.igem.wiki/teams/4757/wiki/operon-overview-mew.svg)

**Figure 9: Overview of genetic construct of the final operon composite part**

### 6. PET degrading enzymes

Cloning

Sequences coding for WT-PETase, FAST-PETase, LCC, and AlkB were synthesized by IDT and added into the
pSEVA438 vector
using SacI und PstI restriction digest. The enzymes (fused to His6-tag) were expressed in
*P.
fluorescens*.
Proteins were from raw cell lysate or, if excreted by SPpstu secretion tag, from culture media, were
purified with
Ni-NTA affinity chromatographie, and competetively eluted.
Time resolved, colorimetric assays with 0.25 µg enzyme and p-nitrophenyl-acetate (pNP-Ac) and
pNP-butyrate (pNP-But)
were done to asses enzyme activity.
Reaction was incubated at28 °C, pH7 over 90 mins, with OD415 measurements every three
minutes.

*AlkB* was successfully transformed into *P. fluorescens* and confirmed by Sanger sequencing,
however, as
of now the enzyme's expression and functionality could not be tested.
AlkB is a membrane bound protein with membrane specific cofactors making the isolation difficult.

The tested enzymes, WT-PETase, FAST-PETase, and LCC, all showed catalytic activity under previously
described
conditions (figure 1).
Based on these measurements Michaelis Menten kinetics could be calculated, with the FAST-PETase purified
from the
growth media showing a 4 fold, higher vmax compared to the other WT-PETase and LCC (Figure 2,
A).
The Km value was higher for the FAST-PETase from the medium compared to the
others (Figure 2, B). The initial rate of the extracellular FAST-PETase was fitted with a
Michaelis-Mentan curve
(Figure 3).

![OD_LCC_lysate](https://static.igem.wiki/teams/4757/wiki/od-time-lcc-lysate.svg)

***Figure 1: Time resolved, colorimetric measurements of tested Enzymes**

Optical density measurements (OD415) were taken every 3 min, after combining 0.25 µg Enzyme with
pNP-Ac or pNP-But.
Tested enzymes were His6-tag purified by IMAC, prior to measurements. Values are presented as mean
+/-
SD. No statistical analysis was performed.*

![Km-value_enzyme](https://static.igem.wiki/teams/4757/wiki/michaelis-constant.svg)

***Figure 2: Calculated enzyme kinetics based**

**(A)**Calculated vmaxvalues of tested enzymes

**(B)**Calculated Michaelis Menten constants (KM)

Enzyme kinetics were calculated with the substrates pNP-Ac and pNP-But via non-linear regression.
OD415 measurements were taken every 3 min.*

![](https://static.igem.wiki/teams/4757/wiki/fast-petase-activity-curve-fit.svg)

***Figure 3: Fitted Michaelis Menten kinetic for extracellular FAST-PETase***

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{% endblock %}