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Synthetic expression cassette regulated by terepthalic acid and alkanes for PET and PE sensing <!DOCTYPE html> New Page

1. Abstract

With the global plastic waste crisis engulfing the planet, bioremediation of synthetic polymers has become an intense field of research. In vivo sensor systems capable of monitoring plastic degradation are necessary for establishing promising symbiotic bacterial degradation approaches. In this year's iGEM project, we introduce two novel transcription factors for sensing poly-ethylene-terephthalate (PET) and low-density polyethylene (PE) degradation. These are combined with a novel sRNA repressor system creating a synthetic operon, predicted to be capable of controlling the bacterium's growth behavior in a bioremediation co-culture.

In service of this goal, our team successfully established Pseudomonas fluorescens as a novel chassis for plastic bioremediation while engineering the first tandem in vitro PET and PE sensor system.

Contents

1. Abstract

2. Sequence overview

3. Usage and Biology

4. Assembly and part evolution

4.1. AlkS - cloning

4.1.1. Detergent testing

4.2. XylS - cloning

4.2.1 XylS-WT TPA sensitivity testing

4.2.2 XylS-mt induction with XylR activation

4.3. sRNA - cloning

4.3.1. RBS comparison

4.3.2. sRNA comparison

4.3.3 Comparison of experimental data with previously calculated properties

4.4. Final operon assembly

5. Results

5.1. PE-degradation Sensor (AlkS-V760E/pAlkB)

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

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

5.2.2. Ps1/Ps2 XylS-MT TPA and MBA co-induction

5.2.3. pEM7 XylS-MT

5.3. sRNA mediated repression

5.3.1. SgrS1.2/MicC1.2 repression

6. Future perspectives

7. References

2. Sequence overview

3. Usage and Biology

With an annual production of over 4000000 tons of plastic waste, the global recycling industry is grappling with an enormous challenge (Geyer et al., 2017). Chemical and mechanical recycling methods are not sustainable and require immense infrastructure to tackle this issue. However, in recent years enzymatic plastic degradation has become a viable option. Many plastic depolymerising enzymes have been discovered and engineered for physiological temperatures and pH levels (Lu et al., 2022). Expression of these enzymes in bacterial mono-cultures has already been tested as a viable bioremediation option (Sharma, 2018). However, recent findings reveal immense undiscovered potential in symbiotic co-culturing of different bacterial strains. The division of labor was found to reduce the individual metabolic burden and lead to increased degradation rates and growth (Bao et al., 2023). iGEM team Heidelberg 2023 leverages this new approach to create a co-culture of two strains of Pseudomonas fluorescens to efficiently break down mixed plastic waste consisting of PE and PET.

To stabilize this co-culture, we created a synthetic operon with two functions. It can sense PE and PET degradation and control the growth behavior of the co-culture by overexpressing or repressing different genes of interest (GOI), such as growth factors. Our biosensor consists of a positive and a negative feedback loop each capable of recognizing a plastic degradation product. To analyze the performance of the operon, the red fluorescent protein mKate2 was used as a reporter gene. Operon activity was measured as the amount of fluorescence of the culture normalized to optical density at 600 nm.

The PET degradation product terephthalic acid (TPA) is monitored by the XylS-K38R-L224Q (XylS-mt) transcription factor (Li et al., 2022). Li et al. (2022) discovered two point mutations K38R and L224Q makes XylS sensitive to TPA in concentrations as low as 10 mM in E. coli. 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 a dynamic sRNA repressor, 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.

The positive feedback senses PE-degradation products by relying on the alkane sensor AlkS-V760E. Upon activation with alkanes, AlkS-V760E binds the pAlkB promoter inducing gene expression (Tournier et al., 2020). Alkanes are a byproduct of PE degradation through the alkane-monooxygenase (AlkB) (Pinto et al., 2022). AlkS, originally found in Pseudomonas oleovorans, recognizes short- to mid-range alkanes up to C12 (Yuste et at., 1998). The small range of alkane recognition poses a problem as the exact mechanism of PE depolymerization is unknown, therefore the length of the resulting alkanes unknown. Chen et al. (2023) discovered the point mutation, V760E, which is capable of recognizing alkanes as long as C17, covering the alkane length range used as educts for AlkB. However, AlkS-V760E loses some sensitivity towards the shorter alkanes. While the mutation theoretically allows for a larger range of alkanes, the alkane transporter in P. fluorescens, AlkL, only transports alkanes up to C16 (Wu et al., 2015).

For the negative feedback switch, regulatory small RNA (sRNA) molecules were used as an alternative to protein based repressors, which pose high metabolic burden on the host cell and can't be easily expanded for repression of genome encoded genes (Na et al., 2013). Synthetic small regulatory RNA molecules regulate expression by utilizing mRNA interference and degradation (Kelly et al., 2013), ubiquitous in all organisms (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; Mller 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 sRNA repressor binds to the RBS, which inhibits the expression of the GOI.

Assembly and part evolution

For cloning of all the constructs, the pSEVA438 plasmid vector was used with the pBBR1 origin, 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) and the blank subtracted. The results were compared to the appropriate negative controls.

AlkS - cloning

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.

4.1.1. 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 pure ethanol (> 99.8 %) supplemented with 1 % Tween® 80 (v/v) and diluting them 1:100 on the microtiter plates for a final Tween® 80 concentration of 0.01%. This allows for biological availability of the n-alkanes without Tween® 80 concentration interfering with cell growth.

Figure 1: 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. Error bars were calculated with the standard deviation of three replicates.

4.2 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 (see section 5.2). 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.

4.2.1 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 2)

Figure 2 Comparison of expression strength of wildtype and mutated (K38R, L224Q) XylS, at three different inducer concentrations.

4.2.2 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 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).

4.3 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 (BBa_K4757031-BBa_K4757057).

4.3.1 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 3).

Figure 3: 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. Error was calculated with standard deviation.

Constructs with BBa_J61100 (RBS 1) showed minimal fold change in fluorescence levels (0.73-fold change). The second RBS from the Anderson library (BBa_J61101, RBS 2) had a distinct increase in fold change compared to BBa_J61100 (18.15 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-fold change).

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).

4.3.2 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 5)

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. 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. 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).

All constructs were tested with endpoint measurements in early stationary phase to see the maximum repression capability (Figure 4). Seed regions with target 3 showed the weakest repression rates. Seed regions with target 1 showed the strongest repression regardless of the RBS used.

(A)

(B)

(C)

Figure 4: 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. Measurements were taken with three technical replicates each. Significance was calculated with one sided two sample t-test comparing induced to un-induced constructs, and un-induced constructs to constructs without sRNA coding sequences.

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 are independent from the CDS.

4.3.3 comparison of experimental data with previously calculated properties

Figure 5: Dot plot of the in vivo measured relative repression strength against the in silico calculated free binding energy.

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

Figure 5 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).

4.4. 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 LU/ t50 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.

5. Results

5.1. PE-degradation Sensor (AlkS-V760E/pAlkB)

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 6). Only n-dodecane showed a change in fluorescence intensity and was used for further testing of the induction of AlkS at different concentrations.

Figure 6: AlkS expression strength with different n-alkane.

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.

Serial dilution experiments of n-dodecane emulsified in Tween� 80 were performed and fluorescence intensity measured over 20 h (Figure 5, 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.

Figure 7: Induction of AlkS/pAlkB expression system with different concentrations of n-dodecane.

(A) 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.

(B) Dose response curve of serial dilutions of n-dodecane and expression strength 20 h after induction.

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

The TPA sensing transcription factor XylS-K38R-L224Q (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 pEM7 using add-on PCR. TPA and MBA were tested separately in serial dilutions experiments (Figure 8), and in combination (Figure 9).

5.2.1. 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 8, 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 8, A). The calculated dose response curve (Figure 8, (B)) shows inductor saturation at 0.1 mM. For induction of TPA, no inductor saturation was observed (Figure 8, D). The fluorescence intensity of the XylS-WT compared to the XylS-mt shows an overall decreased expression strength. (Figure 8, E)

Figure 8: MBA and TPA dependent induction of the XylS-mt transcription factor controlling mKate2 expression.

(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

Significance was calculated from 3 biological replicates with one-way ANOVA.

5.2.2. 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 9). At an MBA concentration of 0.0025 mM, a significant TPA dependent fold change could be measured (1.3-fold change with 0.01 mM TPA, 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 9, B).

Figure 9 Expression strength with TPA and MBA co-induction

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

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

5.2.3. 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 normalized fluorescence intensity of the Ps1/Ps2 compared to the pEM7 led to overall higher expression strengths (measured in RFU/OD600), with significant changes above induction of 0.005 mM MBA (p<0.001) or 1 mM TPA (p<0.05).

Figure 10 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.

5.3. sRNA mediated repression

5.3.1. 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 11). Both sRNA constructs showed an overall continuous repression strength over time after induction. (Figure 11 A, B) with the highest repression after 20 h of 0.65 and 0.61 for SgrS and MicC, respectively.

The inducer concentration dependent repression strength was calculated at the time points 10 h and 15 h (Figure 11), which showed a linear increase in repression strength with a saturation above 100 mM MBA concentration .

Figure 11 Repression strength and dose response curve of SgrS1.2 and MicC1.2 at different time points 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 at the time points 5 h, 10 h, 15 h (B) Dose response curve of different MBA concentrations at 10 h and 15 h after induction (C) Repression strength of MicC1.2 with serial dilutions at the time points 5 h, 10 h, 15 h (D) Dose response curve of different MBA concentrations at 10 h and 15 h after induction

6. Future perspectives

The composite part makes important contributions for the iGEM registry in form of two novel transcription factors sensing PET and PE, as well as newly characterized sRNA's with different repression strength for use in different systems. Next to our three main contributions, we also introduced a bi-directional terminator (LUZ7 T50, BBa_K4757058), which is capable of efficiently terminating translation from both directions, and two existing ribosomal binding sites. These RBSs were compared to a synthetic designed RBS (BBa_J61100, BBa_J61101, BBa_K4757003). Conducting our experiments in Pseudomonas fluorescens further allowed us to establish a novel chassis organism, which has intriguing bioremediation capabilities.

We think our operon as a composite part has a valuable place for future bacteria-based plastic degradation, as well as enabling future teams to use the basic parts for plastic degradation or P. fluorescens related problem solving.

7. References

Bao, T., Qian, Y., Xin, Y., Collins, J. J., & Lu, T. (2023). Engineering microbial division of labor for plastic upcycling. Nature communications, 14(1), 5712. https://doi.org/10.1038/s41467-023-40777-x

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

Gallegos, M. T., Marqu�s, S., & Ramos, J. L. (1996). Expression of the tol plasmid xylS gene in pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and Alpha 54-dependent tandem promoters according to the compound used for Growth. Journal of Bacteriology, 178(8), 2356�2361. https://doi.org/10.1128/jb.178.8.2356-2361.1996

Gawin, A., Valla, S., & Brautaset, T. (2017). The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering. Microbial biotechnology, 10(4), 702�718. https://doi.org/10.1111/1751-7915.12701

Geyer, R., Jambeck, J. R., & Law, K. L. (2017). Production, use, and fate of all plastics ever made. Science advances, 3(7), e1700782. https://doi.org/10.1126/sciadv.1700782

Gottesman S. (2004). The small RNA regulators of Escherichia coli: roles and mechanisms*. Annual review of microbiology, 58, 303�328. https://doi.org/10.1146/annurev.micro.58.030603.123841

Kelly, C. L., Harris, A. W. K., Steel, H., Hancock, E. J., Heap, J. T., & Papachristodoulou, A. (2018). Synthetic negative feedback circuits using engineered small RNAs. Nucleic acids research, 46(18), 9875�9889. https://doi.org/10.1093/nar/gky828

Li, J., Nina, M. R. H., Zhang, X., & Bai, Y. (2022). Engineering Transcription Factor XylS for Sensing Phthalic Acid and Terephthalic Acid: An Application for Enzyme Evolution. ACS synthetic biology, 11(3), 1106�1113. https://doi.org/10.1021/acssynbio.1c00275

Lu, H., Diaz, D. J., Czarnecki, N. J., Zhu, C., Kim, W., Shroff, R., Acosta, D. J., Alexander, B. R., Cole, H. O., Zhang, Y., Lynd, N. A., Ellington, A. D., & Alper, H. S. (2022). Machine learning-aided engineering of hydrolases for PET depolymerization. Nature, 604(7907), 662�667. https://doi.org/10.1038/s41586-022-04599-z

Modi, S. R., Camacho, D. M., Kohanski, M. A., Walker, G. C., & Collins, J. J. (2011). Functional characterization of bacterial sRNAs using a network biology approach. Proceedings of the National Academy of Sciences of the United States of America, 108(37), 15522�15527. https://doi.org/10.1073/pnas.1104318108

Müller, T., Franch, T., H�jrup, P., Keene, D. R., B�chinger, H. P., Brennan, R. G., & Valentin-Hansen, P. (2002). Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. Molecular cell, 9(1), 23�30. https://doi.org/10.1016/s1097-2765(01)00436-1

Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., & Lee, S. Y. (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature biotechnology, 31(2), 170�174. https://doi.org/10.1038/nbt.2461

Sharma, S.R. (2018). Bioremediation of Polythenes and Plastics: A Microbial Approach. In: Prasad, R., Aranda, E. (eds) Approaches in Bioremediation. Nanotechnology in the Life Sciences. Springer, Cham. https://doi.org/10.1007/978-3-030-02369-0_6

Storz, G., Vogel, J., & Wassarman, K. M. (2011). Regulation by small RNAs in bacteria: expanding frontiers. Molecular cell, 43(6), 880�891. https://doi.org/10.1016/j.molcel.2011.08.022

Tournier, V., Topham, C. M., Gilles, A., David, B., Folgoas, C., Moya-Leclair, E., Kamionka, E., Desrousseaux, M. L., Texier, H., Gavalda, S., Cot, M., Gu�mard, E., Dalibey, M., Nomme, J., Cioci, G., Barbe, S., Chateau, M., Andr�, I., Duquesne, S., & Marty, A. (2020). An engineered PET depolymerase to break down and recycle plastic bottles. Nature, 580(7802), 216�219. https://doi.org/10.1038/s41586-020-2149-4

Wu, P., Wang, Z., Zhu, Q., Xie, Z., Mei, Y., Liang, Y., & Chen, Z. (2021). Stress preadaptation and overexpression of rpoS and hfq genes increase stress resistance of Pseudomonas fluorescens ATCC13525. Microbiological research, 250, 126804. https://doi.org/10.1016/j.micres.2021.126804

Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor for in situ detection of bio-synthesized alkanes. Scientific reports, 5, 10907. https://doi.org/10.1038/srep10907

Sequence and Features

Assembly Compatibility:

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Illegal EcoRI site found at 4298
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Illegal BglII site found at 212

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Illegal EcoRI site found at 4298

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Illegal NgoMIV site found at 934
Illegal NgoMIV site found at 2716
Illegal NgoMIV site found at 3187

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-   <h1><a>Abstract</a></h1>
+   <h1 id="Abstract">1. Abstract</h1>
-   <p>Enzymatic bioremediation of plastics has become more viable by engineering different depolymerizing enzymes. However, there is still a lack of sensor systems to regulate gene expression based on changes in plastic concentrations. In this year’s iGEM project we have introduced to novel transcription factors, XylS-K38R-L224Q capable of terephthalic acid sensing and AlkS-V760E, capable of sensing biologically relevant short polyethylene molecules. For low-burden gene expression 27 different sRNA molecules for mRNA interference were characterized.</p>
+   <p>With the global plastic waste crisis engulfing the planet, bioremediation of synthetic polymers has become an intense field of research. <i>In vivo</i> sensor systems capable of monitoring plastic degradation are necessary for establishing promising symbiotic bacterial degradation approaches. In this year's iGEM project, we introduce two novel transcription factors for sensing poly-ethylene-terephthalate (PET) and low-density polyethylene (PE) degradation. These are combined with a novel sRNA repressor system creating a synthetic operon, predicted to be capable of controlling the bacterium's growth behavior in a bioremediation co-culture.</p>
+  <p>In service of this goal, our team successfully established <i>Pseudomonas fluorescens</i> as a novel chassis for plastic bioremediation while engineering the first tandem in vitro PET and PE sensor system.</p>
+    <p>&nbsp;</p>
+  <p></p>
+  <p>Contents</p>
+  <p><a href="#Abstract">1. Abstract </a></p>
+  <p><a href="#Sequence_overview">2. Sequence overview</a></p>
+  <p><a href="#Usage_and_Biology">3. Usage and Biology</a></p>
+  <p><a href="#Assembly_and_part_evolution">4. Assembly and part evolution</a></p>
+  <p><a href="#AlkS_-_cloning">4.1. AlkS - cloning</a></p>
+  <p><a href="#Detergent_testing">4.1.1. Detergent testing</a></p>
+  <p><a href="#XylS_-_cloning">4.2. XylS - cloning</a></p>
+  <p><a href="#XylS-WT_TPA_sensitivity_testing">4.2.1 XylS-WT TPA sensitivity testing</a></p>
+  <p><a href="#XylS-mt_induction_with_XylR_activation">4.2.2 XylS-mt induction with XylR activation</a></p>
+  <p><a href="#sRNA_-_cloning">4.3. sRNA - cloning</a></p>
+  <p><a href="#RBS_comparison">4.3.1. RBS comparison</a></p>
+  <p><a href="#sRNA_comparison">4.3.2. sRNA comparison</a></p>
+  <p><a href="#4.3.3">4.3.3 Comparison of experimental data with previously calculated properties</a></p>
+  <p><a href="#4.4.">4.4. Final operon assembly</a></p>
+  <p><a href="#Results">5. Results</a></p>
+  <p><a href="#5.1.">5.1. PE-degradation Sensor (AlkS-V760E/pAlkB)</a></p>
+  <p><a href="#5.2.">5.2. PET-degradation sensor (XylS-K38R-L224Q/Pm)</a></p>
+  <p><a href="#5.2.1.">5.2.1. Ps1/Ps2 XylS-mt (with MBA or TPA)</a></p>
+  <p><a href="#5.2.2.">5.2.2. Ps1/Ps2 XylS-MT TPA and MBA co-induction</a></p>
+  <p><a href="#5.2.3.">5.2.3. pEM7 XylS-MT</a></p>
+  <p><a href="#5.3.">5.3. sRNA mediated repression</a></p>
+  <p><a href="#5.3.1.">5.3.1. SgrS1.2/MicC1.2 repression</a></p>
+  <p><a href="#6.">6. Future perspectives</a></p>
+  <p><a href="#7.">7. References</a></p>
+  <p><strong>&nbsp;</strong></p>
    <p>&nbsp;</p>
-   <p>With our novel approach we not only engineered the first PET and PE combination sensor system but also established <i>P. fluorescens</i> as a novel chassis for bioremediation.</p>
+   <h1 id="Sequence_overview"><a>2. Sequence overview</a></h1>
    <p>&nbsp;</p>
+  <h1 id="Usage_and_Biology"><a>3. Usage and Biology</a></h1>
+  <p>With an annual production of over 4000000 tons of plastic waste, the global recycling industry is grappling with an enormous challenge (Geyer <i>et al.,</i> 2017). Chemical and mechanical recycling methods are not sustainable&nbsp; and require immense infrastructure to tackle this issue. However, in recent years enzymatic plastic degradation has become a viable option. Many plastic depolymerising enzymes have been discovered and engineered for physiological temperatures and pH levels (Lu <i>et al.,</i> 2022). Expression of these enzymes in bacterial mono-cultures has already been tested as a viable bioremediation option (Sharma, 2018). However, recent findings reveal immense undiscovered potential in symbiotic co-culturing of different bacterial strains. The division of labor was found to reduce the individual metabolic burden and lead to increased degradation rates and growth (Bao <i>et al.,</i> 2023). iGEM team Heidelberg 2023 leverages this new approach to create a co-culture of two strains of <i>Pseudomonas fluorescens</i> to efficiently break down mixed plastic waste consisting of PE and PET.</p>
    <p>&nbsp;</p>
+  <p>To stabilize this co-culture, we created a synthetic operon with two functions. It can sense PE and PET degradation and control the growth behavior of the co-culture by overexpressing or repressing different genes of interest (GOI), such as growth factors. Our biosensor consists of a positive and a negative feedback loop each capable of recognizing a plastic degradation product. To analyze the performance of the operon, the red fluorescent protein mKate2 was used as a reporter gene. Operon activity was measured as the amount of fluorescence of the culture normalized to optical density at 600 nm.</p>
    <p>&nbsp;</p>
-   <p>Contents</p>
+   <p>The PET degradation product terephthalic acid (TPA) is monitored by the XylS-K38R-L224Q (XylS-mt) transcription factor (Li <i>et al.,</i> 2022). Li <i>et al.</i> (2022) discovered two point mutations K38R and L224Q makes XylS sensitive to TPA in concentrations as low as 10 mM in <i>E. coli</i>. Upon activation with TPA or the well described XylS inducer 3-methyl-benzoate (MBA), XylS-mt dimerizes and binds the Pm promoter (Gawin <i>et al.,</i> 2017). Pm activation results in the expression of a dynamic sRNA repressor, blocking the translation of the GOI. A negative feedback loop is established, downregulating the GOI activity at high PET depolymerization rates.</p>
-  <p><a href="#_Toc147753944">1.&nbsp;&nbsp;&nbsp;&nbsp; Abstract 1</a></p>
-  <p><a href="#_Toc147753945">2.&nbsp;&nbsp;&nbsp;&nbsp; Sequence overview.. 1</a></p>
-  <p><a href="#_Toc147753946">3.&nbsp;&nbsp;&nbsp;&nbsp; Usage and Biology. 2</a></p>
-  <p><a href="#_Toc147753947">4.&nbsp;&nbsp;&nbsp;&nbsp; Assembly and part evolution. 3</a></p>
-  <p><a href="#_Toc147753948">4.1.&nbsp;&nbsp;&nbsp;&nbsp; AlkS - cloning. 3</a></p>
-  <p><a href="#_Toc147753949">4.1.1. Detergent testing. 3</a></p>
-  <p><a href="#_Toc147753950">4.2.&nbsp;&nbsp;&nbsp;&nbsp; XylS - cloning. 4</a></p>
-  <p><a href="#_Toc147753951">4.3.&nbsp;&nbsp;&nbsp;&nbsp; sRNA - cloning. 4</a></p>
-  <p><a href="#_Toc147753952">4.3.1. RBS comparison. 4</a></p>
-  <p><a href="#_Toc147753953">4.3.2. sRNA comparison. 5</a></p>
-  <p><a href="#_Toc147753954">4.4.&nbsp;&nbsp;&nbsp;&nbsp; Final operon assembly. 7</a></p>
-  <p><a href="#_Toc147753955">5.&nbsp;&nbsp;&nbsp;&nbsp; Results. 7</a></p>
-  <p><a href="#_Toc147753956">5.1.&nbsp;&nbsp;&nbsp;&nbsp; PE-degradation Sensor (AlkS-V760E/pAlkB) 7</a></p>
-  <p><a href="#_Toc147753957">5.2.&nbsp;&nbsp;&nbsp;&nbsp; PET-degrdation sensor (XylS-K38R-L224Q/Pm) 9</a></p>
-  <p><a href="#_Toc147753958">5.2.1. Ps1/Ps2 XylSmt (with MBA or TPA) 9</a></p>
-  <p><a href="#_Toc147753959">5.2.2. PS1/Ps2 XylS-MT TPA and MBA co-induction. 10</a></p>
-  <p><a href="#_Toc147753960">5.2.3. pEM7 XylS-MT. 10</a></p>
-  <p><a href="#_Toc147753961">5.3.&nbsp;&nbsp;&nbsp;&nbsp; sRNA mediated repression. 11</a></p>
-  <p><a href="#_Toc147753962">5.3.1. SgrS1.2/MicC1.2 repression. 11</a></p>
-  <p><a href="#_Toc147753963">1.&nbsp;&nbsp;&nbsp;&nbsp; Future perspectives. 12</a></p>
-  <p><a href="#_Toc147753964">2.&nbsp;&nbsp;&nbsp;&nbsp; References. 12</a></p>
    <p>&nbsp;</p>
+  <p>The expression of XylS-mt itself is regulated through the Ps1/Ps2 promoter (Gallegos <i>et al</i>., 1996; Gawin <i>et al.,</i> 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 <i>et al</i>., 1996). This is the first time a TPA sensor is characterized in <i>P. fluorescens</i> and in the iGEM parts registry.</p>
    <p>&nbsp;</p>
-   <h1><a>Sequence overview</a></h1>
+   <p>The positive feedback senses PE-degradation products by relying on the alkane sensor AlkS-V760E. Upon activation with alkanes, AlkS-V760E binds the pAlkB promoter inducing gene expression (Tournier <i>et al.</i>, 2020). Alkanes are a byproduct of PE degradation through the alkane-monooxygenase (AlkB) (Pinto <i>et al</i>., 2022). AlkS, originally found in <i>Pseudomonas oleovorans</i>, recognizes short- to mid-range alkanes up to C12 (Yuste <i>et at.,</i> 1998). The small range of alkane recognition poses a problem as the exact mechanism of PE depolymerization is unknown, therefore the length of the resulting alkanes unknown. Chen et al. (2023) discovered the point mutation, V760E, which is capable of recognizing alkanes as long as C17, covering the alkane length range used as educts for AlkB. However, AlkS-V760E loses some sensitivity towards the shorter alkanes. While the mutation theoretically allows for a larger range of alkanes, the alkane transporter in <i>P. fluorescens</i>, AlkL, only transports alkanes up to C16 (Wu <i>et al.,</i> 2015).</p>
    <p>&nbsp;</p>
-   <h1><a>Usage and Biology</a></h1>
+   <p>For the negative feedback switch, regulatory small RNA (sRNA) molecules were used as an alternative to protein based repressors, which pose high metabolic burden on the host cell and can't be easily expanded for repression of genome encoded genes (Na <i>et al</i>., 2013). Synthetic small regulatory RNA molecules regulate expression by utilizing mRNA interference and degradation (Kelly <i>et al.</i>, 2013), ubiquitous in all organisms (Modi <i>et al.<i>, 2011). Prokaryotic Organisms natively regulate gene expression through small RNAs (sRNAs) and RNA chaperon protein hfq mediated sRNA-mRNA binding and degradation (Na <i>et al.</i>, 2013; Gottesman, 2004; Storz <i>et al</i>., 2011; Modi <i>et al</i>., 2011; Mller <i>et al.</i>, 2002), which is also present in <i>Pseudomonas</i> species (Trouillon <i>et al.</i>, 2022; Wu <i>et al.</i>, 2021) and confirmed in our sub-strain ATCC 50090 by BLAST. The sRNA repressor binds to the RBS, which inhibits the expression of the GOI.</p>
-  <ul>
-    <li>Plastic sensor</li>
-    <li>Expression control</li>
-  </ul>
    <p>&nbsp;</p>
-   <p>With biosensors applications expanding, the ReMixHD iGEM team Heidelberg 2023 aimed to create a plastic biosensor capable of controlling gene expression. The biosensor consists of a positive and a negative feedback loop each capable of recognizing a plastic degradation product. PET’s plastics degradation product, terephthalic acid, binds to the transcription factor XylS-K38R-L224Q (XylSmt), leading to the expression of a dynamic sRNA repression system.</p>
+  <h1 id="Assembly_and_part_evolution"><a>Assembly and part evolution</a></h1>
+   <p>For cloning of all the constructs, the pSEVA438 plasmid vector was used with the pBBR1 origin, 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) and the blank subtracted. The results were compared to the appropriate negative controls.</p>
    <p>&nbsp;</p>
-   <p>The point mutations K38R and L224Q enable XylS to sense TPA, a function that the WT XylS does not possess. The mutation was characterized in E. coli in an endpoint measurement to be sensitive to TPA concentrations as low as ___ mM<a>.</a>&nbsp; The Pm promoter can be induced using benzoate and 3-methyl-benzoate (MBA). The latter being the preferred inducer as it is non-metabolic in nature. The XylS expression system is well established in P. putida and has been used by iGEM in the past. However, it has not been used in P. fluorescens and the point mutation allowing for TPA sensing is also new.</p>
+   <h2 id="AlkS_-_cloning"><a>AlkS - cloning</a></h2>
-  <ul>
+   <p>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.</p>
-    <li>
+  <p>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.</p>
-      <a>ps1/ps2 altes paper</a>
-    </li>
-    <li>Pem7</li>
-    <li>Alkr&nbsp;</li>
-    <li>Mutation</li>
-    <li>Xlys well researched in p.putita</li>
-    <li>Used in igem mut new to igem</li>
-  </ul>
-  <p>The positive feedback relies on an alkane-<a>AlkS</a>&nbsp; complex binding to the PalkB promoter to control gene expression. Alkanes are a byproduct of PE degradation. AlkS, originally found in <i>Pseudomonas oleovorans</i>, recognizes short- to mid-range alkanes up to C12.<a href="#_ftn1"></a> The small range of alkane recognition poses a problem as the exact mechanism of PE degradation is unknown, therefore the length of the resulting alkanes unknown. Chen <i>et al.</i> (2023) discovered the point mutation, V760E, which is capable of recognizing alkanes as long as C17. However, AlkS-V760 loses some sensitivity towards the shorter alkanes. While the mutation theoretically allows for a larger range of alkanes, the alkane transporter in P. fluorescens, AlkL, only transports alkanes up to C16.</p>
-  <p>The AlkS/pAlkB expression system has previously been used by iGEM teams unsuccessfully. We aimed to improve the part.</p>
-  <ul>
-    <li>C17 no transporter</li>
-    <li>Mutation</li>
-    <li>AlkL - transporter</li>
-    <li>Other igem teams didn’t work</li>
-    <li>Improving parts</li>
-    <li>V760E</li>
-    <li>&nbsp;</li>
-  </ul>
-  <h1><a>Assembly and part evolution</a></h1>
-  <p>All parts were assembled in the pSEVA438 plasmid vector. The experiments 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 for 16-24 h</p>
-  <p>To ensure comparison between the different technical and biological replicates, the fluorescence of each well was normalized with the cell count (referenced to OD600) and appropriate negative controls and blanks were subtracted or used for comparison. &nbsp;</p>
-  <p>&nbsp;</p>
-  <h2><a>AlkS - cloning</a></h2>
-   <p>The plasmid pSEVA438 was used as a basis for integration of the AlkS/pAlkB expression system. The sequences were obtained by gene synthesis and cloned with Gibson assembly into the vector, replacing XylS/pAlkB, with their respective regulatory parts encoded on the plasmid. To further analyze AlkS-V760E/pAlkB’s behavior the Ps1/Ps2 promoter system, regulating AlkS expression, was replaced by add-on PCR with the constitutively active pEM7 promoter. As a reporter gene mKate2 was used and cloned with SacI, PstI restriction digest in the multiple cloning site downstream of pAlkB. To further increase fluorescence a synthetic RBS (BBa_J61100) was added upstream of the coding sequence with substitution PCR.</p>
-  <h3><a>Detergent testing</a></h3>
-  <p>Before characterizing the transcription factor, preliminary tests were conducted to optimize solubility of different length n-alkanes (hexane, heptane, dodecane, heptadecane) and biological availability. Solubility was tested in H2O, di-methyl sulfoxide (DMSO), Tween® 80, and rhamnolipids with varying concentrations. H2O and DMSO could not solubilize longer length alkanes making them unsuitable for future experiments.</p>
-  <p>Rhamnolipids could readily solubilize alkanes but showed high absorption at OD600 and strong auto-fluorescence, making them unsuitable as well.</p>
-  <p>The best results could be achieved by first solubilizing the alkanes in pure ethanol (&gt; 99.8 %) supplemented with 1 % Tween® 80 (v/v), and then diluting them 1:100 on the microtiter plates for a final Tween® 80 concentration of 0.01%. This allows for biological availability of the n-alkanes, as well as low enough Tween® 80 concentration so as not to inhibit cell growth.</p>
    <p>&nbsp;</p>
+  <h3 id="Detergent_testing"><a>4.1.1. Detergent testing</a></h3>
+  <p>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&#174; 80, and rhamnolipids. Long chain alkanes could not be brought into solution using H2O and DMSO, making them unsuitable for future experiments.</p>
+  <p>While rhamnolipids could readily solubilize alkanes, they showed high absorption at OD600 and strong auto-fluorescence, making them unsuitable.
+  <p>The best results were achieved by first solubilizing the alkanes in pure ethanol (&gt; 99.8 %) supplemented with 1 % Tween&#174; 80 (v/v) and diluting them 1:100 on the microtiter plates for a final Tween&#174; 80 concentration of 0.01%. This allows for biological availability of the n-alkanes without Tween&#174; 80 concentration interfering with cell growth.</p>
+    <p>&nbsp;</p>
    <table>
      <tr>
-       <td></td>
+       <td>
+        <p><img style="width: 50%;" src="https://static.igem.wiki/teams/4757/wiki/alks-induction-of-pem7-alks-palkb-mkat2-with-n-dodecane-after-20-h.svg" alt="image002"></p>
+      </td>
      </tr>
      <tr>
        <td>
-         <p><strong>Figure</strong> <strong>1</strong><strong>: Biological available of n-dodecane in different detergents</strong>.</p>
+         <p><strong><i>Figure 1: Biological availability of n-dodecane solubilized in different detergents</i></strong><i>.</i></p>
-         <p>Biological availability was measured by fluorescence increase of mKate2 regulated by the n-dodecane sensing transcription factor AlkS-V760E. Error bars were calculated with the standard deviation of three replicates.</p>
+         <p><i>Biological availability was measured by fluorescence increase of mKate2 regulated by the n-dodecane sensing transcription factor AlkS-V760E. Error bars were calculated with the standard deviation of three replicates.</i></p>
        </td>
      </tr>
    </table>
    <p>&nbsp;</p>
-   <h2><a>XylS - cloning</a></h2>
+   <h2 id="XylS_-_cloning"><a>4.2 XylS - cloning</a></h2>
-   <p>Since the XylS/Pm expression system is natively found on the pSEVA438 plasmid, the two amino acid substitutions were introduced with two primer pairs with the needed single base substitutions. Analogous to AlkSV760E the Ps1/Ps2 promoter system was substituted with pEM7 to further test the functionality in different scenarios. The CDS for mKate2 was also cloned with SacI and PstI restriction digest, and substitution PCR was used to introduce the RBS BBa_J61100.</p>
+   <p>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 (see section 5.2). 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 <a href="https://parts.igem.org/Part:BBa_J61100">RBS BBa_J61100</a>.</p>
-   <p>The wildtype XylS/Pm expression system is natively on pSEVA438. To get the two amino acid substitutions, site directed mutagenesis was used with two primer pairs and subsequent Gibson assembly.</p>
+  <h3 id="XylS-WT_TPA_sensitivity_testing"><a>4.2.1 XylS-WT TPA sensitivity testing</a></h3>
+   <p>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 2)</p>
    <p>&nbsp;</p>
-   <h2><a>sRNA - cloning</a></h2>
+   <p><img style="width: 50%;" src="https://static.igem.wiki/teams/4757/wiki/induction-strength-comparison-xylswt-vs-xylsmt.svg" alt="image004"></p>
-   <p>To clone the sRNA coding sequences <a>scarless</a>&nbsp; behind the Pm promoter, as well as mKate2 behind the constitutively active promoter pEM7, three different gene sequences were designed, each with a different RBS, synthesis and cloned into the linearized pSEVA438 vector with Gibson assembly.</p>
+   <p><strong><i>Figure 2 Comparison of expression strength of wildtype and mutated (K38R, L224Q) XylS, at three different inducer concentrations.</strong></i></p>
-  <p>The sRNA coding sequences were ordered as primer pairs that, after annealing, had 3 bp overhangs on each site. The 27 annealed primer oligos were assembled into the new vector with golden gate assembly (with SapI), and after cloning validated with sanger sequencing.</p>
-  <h3><a>RBS comparison</a></h3>
    <p>&nbsp;</p>
+  <h3 id="XylS-mt_induction_with_XylR_activation"><a>4.2.2 XylS-mt induction with XylR activation</a></h3>
+  <p>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 XylR, previously described to jointly activate expression from the Ps1 promoter with XylS in <i>P. putida</i>. However, co-induction showed no increase in expression strength (data not shown).</p>
+  <p>&nbsp;</p>
+  <h2 id="sRNA_-_cloning"><a>4.3 sRNA - cloning</a></h2>
+  <p>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 (BBa_K4757031-BBa_K4757057).&nbsp;&nbsp;</p>
+  <h3 id="RBS_comparison"><a>4.3.1 RBS comparison</a></h3>
+  <p>To find optimal expression levels of mKate2 and establish new ribosomal binding sites for <i>P. fluorescens</i>, 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 <i>P. fluorescens</i> was calculated (Figure 3).</p>
    <p>&nbsp;</p>
    <table>
      <tr>
-       <td></td>
+       <td>
+        <p><i>&nbsp;</i></p>
+      </td>
      </tr>
      <tr>
        <td>
-         <p><a>Figure</a> 2: 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. Error was calculated with standard deviation.</p>
+         <p><img width="258" height="373" src="https://static.igem.wiki/teams/4757/wiki/fold-change-fluorescence-intensity-with-testsed-rbs.svg" alt="image006"></p>
+        <p><strong><i> Figure 3: 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 <i>P. fluorescens<i>. Measurements were taken in the early stationary phase. Error was calculated with standard deviation.<strong></i></p>
        </td>
      </tr>
    </table>
    <p>&nbsp;</p>
-   <p>Before testing the sRNA’s construct, three different strength ribosomal binding sites were characterized by measuring the mKate2 fluorescence expressed by pEM7 promoter and calculating the fold change over the auto-fluorescence of <i>P. fluorescens</i> (Figure 2). Constructs with BBa_J61100 (RBS 1) showed minimal fold change in fluorescence levels (0.73-fold change). The second RBS from the Anderson library (BBa_J61101, RBS 2) had a distinct increase in fold change compared to BBa_J61100 (18.15 compared to 0.73). The synthetic RBS (RBS 3) designed by the Salis-lab calculator (calculated for maximal expression strength) showed the strongest fluorescence (48.56-fold change).</p>
+   <p>Constructs with BBa_J61100 (RBS 1) showed minimal fold change in fluorescence levels (0.73-fold change). The second RBS from the Anderson library (BBa_J61101, RBS 2) had a distinct increase in fold change compared to BBa_J61100 (18.15 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-fold change).</p>
    <p>&nbsp;</p>
-   <p>Although RBS 3 showed the highest expression strength the RBS 2 (BBa_J61101) was chosen as a strong, non mRNA specific RBS for our final operon.</p>
+   <p>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).</p>
    <p>&nbsp;</p>
-  <h3><a>sRNA comparison</a></h3>
-  <p>During the design process of the sRNA molecules, free binding energy and secondary structures for each construct were calculated <i>in-silico</i> to ensure efficient mRNA binding and degradation, by hfq recruitment.</p>
-  <p>###table of binding energy, picture of secondary structures not yet created###</p>
    <p>&nbsp;</p>
+  <h3 id="sRNA_comparison"><a>4.3.2 sRNA comparison</a></h3>
+  <p>Before ordering the different sRNA constructs, <i>in silico</i> analysis of the free binding energy of sRNA-mRNA hybridization was calculated and compared to literature to ensure efficient repression. (figure 5)</p>
    <p>&nbsp;</p>
-   <p>Three different scaffolds, previously used for synthetic sRNA repression, as well as three different binding sites were chosen, resulting in 27 different sRNA constructs being tested. The scaffolds SgrS and MicC were chosen since both have been used by previous iGEM teams but lack characterization in different bacteria than <i>E. coli</i>. Additionally, an engineered version of SgrS (SgrS-S CUUU 6 nts stem (SgrSmt)), optimized for repression in <i>E. coli</i> DH5 alpha, was chosen. 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).</p>
+   <p>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. As they lack characterization in bacteria other than <i>E. coli</i>, we could establish sRNAs in the novel chassis <i>P. fluorescens</i>. Additionally, an engineered version of SgrS (SgrS-S CUUU 6 nts stem (SgrSmt)), optimized for repression in <i>E. coli</i> DH5 alpha, was chosen. 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).</p>
-   <p>&nbsp;</p>
+   <p>All constructs were tested with endpoint measurements in early stationary phase to see the maximum repression capability (Figure 4). Seed regions with target 3 showed the weakest repression rates. Seed regions with target 1 showed the strongest repression regardless of the RBS used.</p>
    <p>&nbsp;</p>
    <table>
@@ Line 132: / Line 126: @@
        <td>
          <p>(A)</p>
+        <p><img width="606" height="298" src="https://static.igem.wiki/teams/4757/wiki/repression-strength-against-rbs-1.svg" alt="image008"></p>
        </td>
      </tr>
@@ Line 137: / Line 132: @@
        <td>
          <p>(B)</p>
+        <p><img width="606" height="335" src="https://static.igem.wiki/teams/4757/wiki/repression-strength-against-rbs-2.svg" alt="image010"></p>
        </td>
      </tr>
@@ Line 142: / Line 138: @@
        <td>
          <p>(C)</p>
+        <p><img width="606" height="305" src="https://static.igem.wiki/teams/4757/wiki/repression-strength-against-rbs-3.svg" alt="image012"></p>
        </td>
      </tr>
      <tr>
        <td>
-         <p><a><strong>Figure</strong></a> <strong>3</strong><strong>: Repression strength of all tested sRNA constructs.</strong></p>
+         <p><strong><i>Figure 4: Repression strength of all tested sRNA constructs. </i></strong></p>
-         <p>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. Measurements were taken with three technical replicates each. Significance was calculated with ### test comparing induced to un-induced constructs, and un-induced constructs to constructs without sRNA coding sequences.</p>
+         <p><i>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. Measurements were taken with three technical replicates each. Significance was calculated with one sided two sample t-test comparing induced to un-induced constructs, and un-induced constructs to constructs without sRNA coding sequences.</i></p>
        </td>
      </tr>
    </table>
    <p>&nbsp;</p>
-   <p>All constructs were tested with endpoint measurements in early stationary phase to see the maximum repression capability (Figure 2). For all scaffolds the seed region targeting only the coding sequence showed the weakest repression, and the seed region only targeting the RBS showed the strongest repression, independently which RBS was used.</p>
+   <p>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 are independent from the CDS.</p>
    <p>&nbsp;</p>
-   <p>Constructs with the seeds region targeting only the RBS BBa_J61101 with SgrS and MicC as scaffolds, were used for the final operon, as they showed the highest repression strength, as well as being independent from the coding sequence (CDS).</p>
+   <h2 id="4.3.3"><a>4.3.3 comparison of experimental data with previously calculated properties</a></h2>
+  <p><img width="378" height="321" src="https://static.igem.wiki/teams/4757/wiki/srna-effect-of-free-binding-energy-on-repression-dot-plot.svg" alt="image014">&nbsp;</p>
+  <p><strong><i>Figure 5: Dot plot of the in vivo measured relative repression strength against the in silico calculated free binding energy.</i></strong> &nbsp;&nbsp;</p>
    <p>&nbsp;</p>
-   <h2><a>Final operon assembly</a></h2>
+  <p>After conducting repression experiments with all sRNA constructs, possible correlation between the relative repression strength and calculated free binding energy was calculated.</p>
+  <p>Figure 5 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).</p>
+   <h2 id="4.4."><a>4.4. Final operon assembly</a></h2>
    <p>&nbsp;</p>
-   <p>To combine all the previously tested parts into one operon, the XylS-K38R-L224Q on the pSEVA438 plasmid was used as a basis. Sequences coding for pAlkB and AlkS were PCR amplified to add homologous overhangs, a new sequence containing the regulatory parts BBa_J61101 (RBS 2) BsaI restriction sites and LUZ7 T50 terminator (BBa_K4757058) was synthesized with homologous overhangs and all inserts were assembled with Gibson assembly. Golden gate assembly (with BsaI restriction enzyme) was used for cloning of mKate2 behind RBS 2. Insert and vector sequences were verified with sequencing but could be successfully combined and transformed into neither <i>P. fluorescens</i> nor <i>E. coli</i> DH5 alpha.</p>
+   <p>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 LU/ t50 terminator (BBa_K4757058), was synthesized with homologous sequences (IDT). All three insert fragments (pAlkB, RBS2-BsaI-LUZ7 T50) were assembled using Gibson assembly.</p>
+  <p>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 <i>P. fluorescens</i> or <i>E. coli</i> DH5 alpha.</p>
+  <h1 id="Results"><a>5. Results</a></h1>
+  <h2 id="5.1."><a>5.1. PE-degradation Sensor (AlkS-V760E/</a>pAlkB)</h2>
+  <p>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.</p>
    <p>&nbsp;</p>
-  <h1><a>Results</a></h1>
+   <p>Different n-alkanes emulsified in Tween&#174; 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 6). Only n-dodecane showed a change in fluorescence intensity and was used for further testing of the induction of AlkS at different concentrations.</p>
-  <h2><a>PE-degradation Sensor (AlkS-V760E/pAlkB)</a></h2>
-  <p>For one of our plastic sensing transcription factors, we used the engineered AlkS-V760E variant capable of sensing biologically relevant polyethylene degradation products, which can be hydroxylated and introduced into the fatty acid cycle.</p>
-  <p>&nbsp;</p>
-  <p>In its final configuration, the AlkS-V760E transcription factor is constitutively expressed by the pEM7 promoter and the AlkS-V760E/pAlkB expression strength is measured with mKate2 fluorescence as a reporter gene.</p>
-  <p>&nbsp;</p>
-   <p>Different n-alkanes, emulsified in Tween® 80 (0.1 % (v/v)) were tested for their inducer capabilities at different concentrations (100 mg/L, 200 mg/L), with time-resolved fluorescence measurements (Figure 4). As only n-dodecanes showed a change in fluorescence intensity, this alkane was used for further testing of the transcription factors dependency on different inducer concentrations.</p>
    <p>&nbsp;</p>
    <table>
      <tr>
-       <td></td>
+       <td>
+        <p><img width="598" height="395" src="https://static.igem.wiki/teams/4757/wiki/alks-sensitivity-of-pem7-alksv760e-towards-n-alkanes.svg" alt="image016"></p>
+      </td>
      </tr>
      <tr>
        <td>
-         <p><a><strong>Figure</strong></a> <strong>4</strong><strong>: AlkS expression strength with different n-alkane.</strong></p>
+         <p><strong><i>Figure 6: AlkS expression strength with different n-alkane. </i></strong></p>
-         <p>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.</p>
+         <p><i>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.</i></p>
        </td>
      </tr>
    </table>
    <p>&nbsp;</p>
-   <p>Serial dilution experiments of n-dodecane emulsified in Tween® 80 were performed and fluorescence intensity measured over 20 h (<strong>Figure 5</strong> (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. &nbsp;At 12 h significant increases could be measured with inducer concentrations &gt; 200 mg/L (1800 RFU compared to 800 RFU, p&lt;0.01####), at 20 h concentrations as low as 20 mg/L were sufficient to measure a significant change in fluorescence (11000 RFU,1000 RFU, p&lt;0.001). The fluorescence measurements at 20 h was used to further analyze the dose response curve (<strong>Figure 5</strong>, B), showing a stagnation in expression strength at concentrations above 2000 mg/L.</p>
+   <p>Serial dilution experiments of n-dodecane emulsified in Tween� 80 were performed and fluorescence intensity measured over 20 h (Figure 5, 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&lt;0.01). At 20 h concentrations as low as 20 mg/L were sufficient to measure a significant change in fluorescence (p&lt;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.</p>
    <p>&nbsp;</p>
    <table>
      <tr>
-       <td></td>
+       <td>
+        <p><img width="535" height="187" src="https://static.igem.wiki/teams/4757/wiki/alks-sensitivtiy-of-pem7-alksv760e-towards-n-dodecane-pem7.svg"> </p>
+      </td>
      </tr>
      <tr>
        <td>
-         <p><a><strong>Figure</strong></a> <strong>5</strong><strong>: Induction of AlkS/pAlkB expression system with different concentrations of n-dodecane.</strong></p>
+         <p><strong><i>Figure 7: Induction of AlkS/pAlkB expression system with different concentrations of n-dodecane.</i></strong></p>
-         <p>[A] 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.</p>
+         <p><i>(A) 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&#174; 80.</i></p>
-         <p>[B] Dose response curve of serial dilutions of n-dodecane and expression strength 20 h after induction.</p>
+         <p><i>(B) Dose response curve of serial dilutions of n-dodecane and expression strength 20 h after induction.</i></p>
        </td>
      </tr>
    </table>
    <p>&nbsp;</p>
-   <h2>&nbsp;<a>PET-degrdation sensor (XylS-K38R-L224Q/Pm)</a></h2>
+   <h2 id="5.2."><a>5.2. PET-degradation sensor (XylS-K38R-L224Q/Pm)</a></h2>
-   <p>The TPA sensing transcription factor XylS-K38R-L224Q (XylSmt) was first tested with the native Ps1/Ps2 promoter system, with different inducer compositions of TPA and m-toluate (MBA). TPA and MBA were tested separately in serial dilutions experiments (<strong>Figure 6</strong>), and in combination (<strong>Figure 7</strong>).</p>
+   <p>The TPA sensing transcription factor XylS-K38R-L224Q (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 pEM7 using add-on PCR. TPA and MBA were tested separately in serial dilutions experiments (Figure 8), and in combination (Figure 9).</p>
    <p>&nbsp;</p>
-   <h3><a>Ps1/Ps2 XylSmt (with MBA or TPA)</a></h3>
+   <h3 id="5.2.1."><a>5.2.1. Ps1/Ps2 XylS-mt (with MBA or TPA)</a></h3>
-  <p>&nbsp;</p>
+   <p>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&lt;0.01) (Figure 8, 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&lt;0.001) (Figure 8, A). The calculated dose response curve (Figure 8, (B)) shows inductor saturation at 0.1 mM. For induction of TPA, no inductor saturation was observed (Figure 8, D). The fluorescence intensity of the XylS-WT compared to the XylS-mt shows an overall decreased expression strength. (Figure 8, E)</p>
-   <p>Serial dilution experiments of only TPA showed significant fluorescence increases above un-induced controls, for concentrations above ## mM (time point # h, # mM, RFU#1, RFU#1, p-value) (<strong>Figure 6</strong>).&nbsp; The same experiments were performed with MBA as an inducer, showing an overall stronger expression strength as well as significant changes 0.01 mM MBA (8 h after induction, RFU#1, RFU#2, p-value##).</p>
-  <p>The calculated dose response curve (Figure 6, (B)) shows inductor saturation at 0.1 mM with no significant fluorescence increase above (##RFU at 0.1 mM, ##RFU at 0.75 mM, p-value ##).</p>
-  <p>Comparing the fluorescence intensity of the wildtype XylS with the mutated one shows an overall decreased expression strength.</p>
-  <p>&nbsp;</p>
-  <p>(<strong>hier nch nen plaot machen 20230824 endpoint sheet 3 um zu zeigen das mut schlechter aktiviert als WT)</strong>.</p>
    <p>&nbsp;</p>
    <table>
      <tr>
-       <td></td>
+       <td>
+        <p><img width="606" height="255" src="https://static.igem.wiki/teams/4757/wiki/xyls-overview-graphc-a-e.svg" alt="image020"></p>
+      </td>
      </tr>
      <tr>
        <td>
-         <p><a><strong>Figure</strong></a> <strong>6</strong><strong>: MBA and TPA dependent induction of the XylSmt transcription factor controlling mKate2 expression</strong>.</p>
+         <p><strong><i>Figure 8: MBA and TPA dependent induction of the XylS-mt transcription factor controlling mKate2 expression</i></strong><i>.</i></p>
          <ul>
-           <li>Fluorescence intensity measurements at 8h, 12 h, 16 h after induction with serial dilutions of MBA</li>
+           <li>(A) Fluorescence intensity measurements at 8h, 12 h, 16 h after induction with serial dilutions of MBA</i></li>
-           <li>Dose response curve of expression strength for different MBA inducer concentrations</li>
+           <li>(B) Dose response curve of expression strength for different MBA inducer concentrations</i></li>
-           <li>Fluorescence intensity measurements at 8 h, 12 h, 16 h after induction with serial dilutions of TPA</li>
+           <li>(C) Fluorescence intensity measurements at 8 h, 12 h, 16 h after induction with serial dilutions of TPA</i></li>
-           <li>Dose response curve of expression strength for different TPA inducer concentrations</li>
+           <li>(D) Dose response curve of expression strength for different TPA inducer concentrations</i></li>
-         </ul>
+          <li>(E) Time resolved measurements of dose response curves after induction with TPA and MBA</i></li>
+         <p><i>Significance was calculated from 3 biological replicates with one-way ANOVA.</i></p>
        </td>
      </tr>
    </table>
    <p>&nbsp;</p>
-   <h3><a>PS1/Ps2 XylS-MT TPA and MBA co-induction</a></h3>
+   <h3 id="5.2.2."><a>5.2.2. Ps1/Ps2 XylS-MT TPA and MBA co-induction</a></h3>
-   <p>To further test the influence of the Ps1/Ps2 promoter system on XylSmt, the co-induction was tested with different, previously determined MBA and TPA concentrations. Three TPA concentrations were tested for four different MBA concentrations. Fold change and normalized fluorescence were calculated (Figure 7). At MBA concentrations of 0.0025 mM significant TPA dependent fold changes could be measured (1.3-fold change with 0.01 mM TPA, p-value). Higher MBA concentrations (0.0075 mM MBA, 0.015 mM MBA showed no TPA dependent change in expression (p&gt;0.05). The expression strength shows an overall decreased fluorescence intensity at low MBA concentrations, despite co-induction with TPA (Figure 7, <a>B</a>&nbsp;&nbsp;).</p>
+   <p>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 9). At an MBA concentration of 0.0025 mM, a significant TPA dependent fold change could be measured (1.3-fold change with 0.01 mM TPA, p&lt;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 9, B).</p>
    <p>&nbsp;</p>
    <table>
      <tr>
-       <td></td>
+       <td>
+        <p><img width="602" height="228" src="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"</p>
+      </td>
      </tr>
      <tr>
        <td>
-         <p><a><strong>Figure</strong></a> <strong>7</strong> <strong>Expression strength with TPA and MBA co-induction</strong></p>
+         <p><strong><i>Figure 9 Expression strength with TPA and MBA co-induction</i></strong></p>
          <ul>
-           <li>Fold change in expression of different MBA inducer concentrations after co-induction of TPA</li>
+           <li><i>Fold change in expression of different MBA inducer concentrations after co-induction of TPA</i></li>
-           <li>Expression strength measured in relative fluorescence of different MBA inducer concentration after co-induction of varying TPA concentrations.</li>
+           <li><i>Expression strength measured in relative fluorescence of different MBA inducer concentration after co-induction of varying TPA concentrations.</i></li>
          </ul>
        </td>
@@ Line 241: / Line 245: @@
    </table>
    <p>&nbsp;</p>
-   <h3><a>pEM7 XylS</a>-MT</h3>
+   <h3 id="5.2.3."><a>5.2.3. pEM7 XylS-MT</a></h3>
-   <p>Alternatively, to the Ps1/Ps2 promoter system, pEM7 was tested as a constitutively active promoter, which was previously used for the expression of AlkSV760E.</p>
+   <p>Alternative to the Ps1/Ps2 promoter system, the constitutively active pEM7 promoter was tested, which was previously used for the expression of AlkSV760E. The normalized fluorescence intensity of the Ps1/Ps2 compared to the pEM7 led to overall higher expression strengths (measured in RFU/OD600), with significant changes above induction of 0.005 mM MBA (p&lt;0.001) or 1 mM TPA (p&lt;0.05).</p>
-  <p>Comparing the normalized fluorescence intensity (Figure 8), the substitution of Ps1/Ps2 with pEM7 led to overall higher expression strengths with decreased sensitivity towards TPA and MBA. For high TPA concentrations (above 1 mM) significant changes in fluorescence intensity could be measured (14000 RFU to 11000 RFU, p &lt; 0.05####).</p>
+  <p>&nbsp;</p>
    <p>&nbsp;</p>
    <table>
      <tr>
-       <td></td>
+       <td>
+        <p><img width="483" height="255" src="https://static.igem.wiki/teams/4757/wiki/xyls-expressopm-stremgth-of-pem7-xylsmt-upon-induction-with-tpa-or-mba.svg"
+      </td>
      </tr>
      <tr>
        <td>
-         <p><a><strong>Figure</strong></a> <strong>8</strong> <strong>Expression strength of pEM7-XylSmt at different TPA concentrations.</strong></p>
+         <p><strong><i>Figure 10 Expression strength of pEM7-XylS-mt at different TPA concentrations.</i></strong></p>
-         <p>TPA dependent expression strength was measured by fluorescence intensity of the pEM7-XylSmt. Measurement was done with three replicates and error bars calculated with the standard deviation.</p>
+         <p><i>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.</i></p>
        </td>
      </tr>
    </table>
    <p>&nbsp;</p>
-   <h2><a>sRNA mediated repression</a></h2>
+   <h2 id="5.3."><a>5.3. sRNA mediated repression</a></h2>
-   <h3><a>SgrS1.2/MicC1.2 repression</a></h3>
+   <h3 id="5.3.1."><a>5.3.1. SgrS1.2/MicC1.2 repression</a></h3>
-   <p>After preliminary testing of 27 different sRNA molecules, the scaffolds SgrS and MicC were chosen with the RBS 1 (BBa_J661101) target region to further characterize the repression characteristics.</p>
+   <p>The scaffolds SgrS and MicC with the RBS 2 (BBa_J661101) target region were used for further characterization of the repression characteristics.</p>
-  <p>&nbsp;</p>
-  <p>The sRNA expression was controlled by the m-toluate inducible XylS/Pm promoter system. By targeting the constitutively expressed mKate2, repression strength was calculated with decrease in fluorescence intensity (Figure 9).</p>
-  <p>Both sRNA constructs showed increasing repression strength with proceeding time after induction. (Figure 9 A, B), with the highest repression after 20 h of 0.65 and 0.61 for SgrS and MicC respectively.</p>
-  <p>The inducer concentration dependent repression strength was calculated at the time points 10 h and 15 h (Figure 9), showing a linear increase in repression strength up to a MBA concentration of 100 mM, above which increasing inductor concentrations, showed no significant changes in repression strength. (Repression 0.09 mM, repression 0.75 mM, pValue##sig test needs to be calculated##)</p>
    <p>&nbsp;</p>
+  <p>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 11). Both sRNA constructs showed an overall continuous repression strength over time after induction. (Figure 11 A, B) with the highest repression after 20 h of 0.65 and 0.61 for SgrS and MicC, respectively.</p>
+  <p>The inducer concentration dependent repression strength was calculated at the time points 10 h and 15 h (Figure 11), which showed a linear increase in repression strength with a saturation above 100 mM MBA concentration&nbsp;.</p>
    <table>
      <tr>
-       <td></td>
+       <td>
-       <td></td>
+        <p><img width="335" height="313" src="https://static.igem.wiki/teams/4757/wiki/srna-repression-sgrs-log2-5-h-10-h-15-h-barplot.svg"</p>
+      </td>
+       <td>
+        <p><img width="255" height="318" src="https://static.igem.wiki/teams/4757/wiki/srna-repression-micc-log2-5-h-10-h-15-h-barplot.svg" alt="image028"></p>
+      </td>
      </tr>
      <tr>
-       <td></td>
+       <td>
-       <td></td>
+        <p><img width="323" height="236" src="https://static.igem.wiki/teams/4757/wiki/dose-response-curve-sgrs-10-h-and-15-h-after-induction-mt-dilution.svg" alt="image030"></p>
+      </td>
+       <td>
+        <p><img width="316" height="383" src="https://static.igem.wiki/teams/4757/wiki/dose-response-curve-micc-10-h-and-15-h-after-induction-mt.svg" alt="image032"></p>
+      </td>
      </tr>
      <tr>
        <td colspan="2">
-         <p><a><strong>Figure</strong></a> <strong>9</strong> <strong>Repression strength and dose response curve of SgrS1.2 and MicC1.2 at different time points with MBA serial dilutions.</strong> 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 triplicates were analyzed.</p>
+         <p><strong><i>Figure 11 Repression strength and dose response curve of SgrS1.2 and MicC1.2 at different time points with MBA serial dilutions.</i></strong> <i>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.</i></p>
          <ul>
-           <li>Repression strength of SgrS with serial dilutions at the time points 5 h, 10 h, 15 h</li>
+           <p>(A) Repression strength of SgrS with serial dilutions at the time points 5 h, 10 h, 15 h
-          <li>Dose response curve of different MBA concentrations at 10 h and 15 h after induction</li>
+            (B) Dose response curve of different MBA concentrations at 10 h and 15 h after induction
-          <li>Repression strength of MicC1.2 with serial dilutions at the time points 5 h, 10 h, 15 h</li>
+            (C) Repression strength of MicC1.2 with serial dilutions at the time points 5 h, 10 h, 15 h
-          <li>Dose response curve of different MBA concentrations at 10 h and 15 h after induction</li>
+            (D) Dose response curve of different MBA concentrations at 10 h and 15 h after induction</p>
          </ul>
        </td>
      </tr>
    </table>
-   <p>##delta in repressions strength 0-6h 6-12h, mean abnahme alle 30 min mit new stdev, bar blot</p>
+   <p>&nbsp;</p>
    <p>&nbsp;</p>
    <p><strong>&nbsp;</strong></p>
-   <h1><a>Future perspectives</a></h1>
+   <h1 id="6."><a>6. Future perspectives</a></h1>
+  <p>The composite part makes important contributions for the iGEM registry in form of two novel transcription factors sensing PET and PE, as well as newly characterized sRNA's with different repression strength for use in different systems.&nbsp; Next to our three main contributions, we also introduced a bi-directional terminator (LUZ7 T50, BBa_K4757058), which is capable of efficiently terminating translation from both directions, and two existing ribosomal binding sites. These RBSs were compared to a synthetic designed RBS (BBa_J61100, BBa_J61101, BBa_K4757003). Conducting our experiments in <i>Pseudomonas fluorescens</i> further allowed us to establish a novel chassis organism, which has intriguing bioremediation capabilities.</p>
+  <p>&nbsp;</p>
+  <p>We think our operon as a composite part has a valuable place for future bacteria-based plastic degradation, as well as enabling future teams to use the basic parts for plastic degradation or <i>P. fluorescens</i> related problem solving.</p>
+  <p>&nbsp;</p>
+  <p>&nbsp;</p>
+  <h1 id="7."><a>7. References</a></h1>
+  <p>Bao, T., Qian, Y., Xin, Y., Collins, J. J., & Lu, T. (2023). Engineering microbial division of labor for plastic upcycling. <i>Nature communications</i>, <i>14</i>(1), 5712. <a href="https://doi.org/10.1038/s41467-023-40777-x">https://doi.org/10.1038/s41467-023-40777-x</a></p>
+  <p>&nbsp;</p>
+  <p>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. <a href="https://doi.org/10.1021/acssynbio.2c00620">https://doi.org/10.1021/acssynbio.2c00620</a></p>
+  <p>Gallegos, M. T., Marqu�s, S., & Ramos, J. L. (1996). Expression of the tol plasmid xylS gene in pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and Alpha 54-dependent tandem promoters according to the compound used for Growth. <i>Journal of Bacteriology</i>, <i>178</i>(8), 2356�2361. https://doi.org/10.1128/jb.178.8.2356-2361.1996</p>
+  <p>Gawin, A., Valla, S., & Brautaset, T. (2017). The XylS/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering. <i>Microbial biotechnology</i>, <i>10</i>(4), 702�718. <a href="https://doi.org/10.1111/1751-7915.12701">https://doi.org/10.1111/1751-7915.12701</a></p>
+  <p>&nbsp;</p>
+  <p>Geyer, R., Jambeck, J. R., & Law, K. L. (2017). Production, use, and fate of all plastics ever made. <i>Science advances</i>, <i>3</i>(7), e1700782. <a href="https://doi.org/10.1126/sciadv.1700782">https://doi.org/10.1126/sciadv.1700782</a></p>
+  <p>&nbsp;</p>
+  <p>Gottesman S. (2004). The small RNA regulators of Escherichia coli: roles and mechanisms*. Annual review of microbiology, 58, 303�328. <a href="https://doi.org/10.1146/annurev.micro.58.030603.123841">https://doi.org/10.1146/annurev.micro.58.030603.123841</a></p>
+  <p>&nbsp;</p>
+  <p>Kelly, C. L., Harris, A. W. K., Steel, H., Hancock, E. J., Heap, J. T., & Papachristodoulou, A. (2018). Synthetic negative feedback circuits using engineered small RNAs. Nucleic acids research, 46(18), 9875�9889. <a href="https://doi.org/10.1093/nar/gky828">https://doi.org/10.1093/nar/gky828</a></p>
+  <p>&nbsp;</p>
+  <p>Li, J., Nina, M. R. H., Zhang, X., & Bai, Y. (2022). Engineering Transcription Factor XylS for Sensing Phthalic Acid and Terephthalic Acid: An Application for Enzyme Evolution. <i>ACS synthetic biology</i>, <i>11</i>(3), 1106�1113. <a href="https://doi.org/10.1021/acssynbio.1c00275">https://doi.org/10.1021/acssynbio.1c00275</a></p>
+  <p>&nbsp;</p>
+  <p>Lu, H., Diaz, D. J., Czarnecki, N. J., Zhu, C., Kim, W., Shroff, R., Acosta, D. J., Alexander, B. R., Cole, H. O., Zhang, Y., Lynd, N. A., Ellington, A. D., & Alper, H. S. (2022). Machine learning-aided engineering of hydrolases for PET depolymerization. <i>Nature</i>, <i>604</i>(7907), 662�667. <a href="https://doi.org/10.1038/s41586-022-04599-z">https://doi.org/10.1038/s41586-022-04599-z</a></p>
+  <p>&nbsp;</p>
+  <p>Modi, S. R., Camacho, D. M., Kohanski, M. A., Walker, G. C., & Collins, J. J. (2011). Functional characterization of bacterial sRNAs using a network biology approach. Proceedings of the National Academy of Sciences of the United States of America, 108(37), 15522�15527. <a href="https://doi.org/10.1073/pnas.1104318108">https://doi.org/10.1073/pnas.1104318108</a></p>
+  <p>&nbsp;</p>
+  <p>Müller, T., Franch, T., H�jrup, P., Keene, D. R., B�chinger, H. P., Brennan, R. G., & Valentin-Hansen, P. (2002). Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. Molecular cell, 9(1), 23�30. <a href="https://doi.org/10.1016/s1097-2765(01)00436-1">https://doi.org/10.1016/s1097-2765(01)00436-1</a></p>
+  <p>&nbsp;</p>
+  <p>Na, D., Yoo, S. M., Chung, H., Park, H., Park, J. H., & Lee, S. Y. (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature biotechnology, 31(2), 170�174. <a href="https://doi.org/10.1038/nbt.2461">https://doi.org/10.1038/nbt.2461</a></p>
+  <p>Sharma, S.R. (2018). Bioremediation of Polythenes and Plastics: A Microbial Approach. In: Prasad, R., Aranda, E. (eds) Approaches in Bioremediation. Nanotechnology in the Life Sciences. Springer, Cham. <a href="https://doi.org/10.1007/978-3-030-02369-0_6">https://doi.org/10.1007/978-3-030-02369-0_6</a></p>
+  <p>Storz, G., Vogel, J., & Wassarman, K. M. (2011). Regulation by small RNAs in bacteria: expanding frontiers. Molecular cell, 43(6), 880�891. <a href="https://doi.org/10.1016/j.molcel.2011.08.022">https://doi.org/10.1016/j.molcel.2011.08.022</a></p>
    <p>&nbsp;</p>
-   <p>The composite part makes important contributions for the iGEM registry in form of two novel transcription factors sensing PET and PE, as well as newly characterized sRNA’s with different repression strength for use in different systems. Next to our three main contributions we also introduced a bi-directional terminator (LUZ7 T50, BBa_K4757058), characterized two existing ribosomal binding sites and compared them to a new synthetic designed RBS (BBa_J61100, BBa_J61101, BBa_K4757003). Conducting our experiments in Pseudomonas fluorescence, further allowed as establish a novel chassis organism as well.</p>
+   <p>Tournier, V., Topham, C. M., Gilles, A., David, B., Folgoas, C., Moya-Leclair, E., Kamionka, E., Desrousseaux, M. L., Texier, H., Gavalda, S., Cot, M., Gu�mard, E., Dalibey, M., Nomme, J., Cioci, G., Barbe, S., Chateau, M., Andr�, I., Duquesne, S., & Marty, A. (2020). An engineered PET depolymerase to break down and recycle plastic bottles. Nature, 580(7802), 216�219. <a href="https://doi.org/10.1038/s41586-020-2149-4">https://doi.org/10.1038/s41586-020-2149-4</a></p>
    <p>&nbsp;</p>
-   <p>We think our operon as a composite part has a valuable place for future bacteria based plastic degradation, as well as enabling future teams to use the basic parts for plastic or <i>P. fluorescens</i> related problem solving.</p>
+   <p>Wu, P., Wang, Z., Zhu, Q., Xie, Z., Mei, Y., Liang, Y., & Chen, Z. (2021). Stress preadaptation and overexpression of rpoS and hfq genes increase stress resistance of Pseudomonas fluorescens ATCC13525. Microbiological research, 250, 126804. <a href="https://doi.org/10.1016/j.micres.2021.126804">https://doi.org/10.1016/j.micres.2021.126804</a></p>
-  <h1><a>References</a></h1>
    <p>&nbsp;</p>
-   <p><a href="#_ftnref1"></a> <a href="https://pubs.acs.org/doi/full/10.1021/acssynbio.2c00620">https://pubs.acs.org/doi/full/10.1021/acssynbio.2c00620</a></p>
+   <p>Wu, W., Zhang, L., Yao, L., Tan, X., Liu, X., & Lu, X. (2015). Genetically assembled fluorescent biosensor for in situ detection of bio-synthesized alkanes. Scientific reports, 5, 10907. <a href="https://doi.org/10.1038/srep10907">https://doi.org/10.1038/srep10907</a></p>
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-<!-- Add more about the biology of this part here
-===Usage and Biology===
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Difference between revisions of "Part:BBa K4757999"

Revision as of 03:40, 11 October 2023

1. Abstract

5.1. PE-degradation Sensor (AlkS-V760E/pAlkB)