Part:BBa_K4757999
Synthetic expression cassette regulated by terepthalic acid and alkanes for PET and PE sensing
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Abstract
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 yearâs 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.
With our novel approach we not only engineered the first PET and PE combination sensor system but also established P. fluorescens as a novel chassis for bioremediation.
Contents
4. Assembly and part evolution. 4
5.1. PE-degradation sensor (AlkS-V760E/pAlkB) 8
5.2. PET-degrdation sensor (XylS-K38R-L224Q/Pm) 10
5.2.1. Ps1/Ps2 XylSmt (with MBA or TPA) 10
5.2.2. PS1/Ps2 XylSmut TPA and MBA co-induction. 11
5.3. sRNA mediated repression. 12
5.3.1. SgrS1.2/MicC1.2 repression. 12
1. Outlook, future perspective. 13
Sequence overview
Usage and Biology
- Plastic sensor
- Expression control
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. PETâs plastics degradation product, terephthalic acid, binds to the transcription factor XylS-K38R-L224Q (XylSmt), leading to the expression of a dynamic sRNA repression system.
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. 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.
- ps1/ps2 altes paper
- Pem7
- Alkr
- Mutation
- Xlys well researched in p.putita
- Used in igem mut new to igem
The positive feedback relies on an alkane-AlkS complex binding to the PalkB promoter to control gene expression. Alkanes are a byproduct of PE degradation. AlkS, originally found in Pseudomonas oleovorans, recognizes short- to mid-range alkanes up to C12. 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 et al. (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.
The AlkS/pAlkB expression system has previously been used by iGEM teams unsuccessfully. We aimed to improve the part.
- C17 no transporter
- Mutation
- AlkL - transporter
- Other igem teams didnât work
- Improving parts
- V760E
Assembly and part evolution
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
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.
AlkS - cloning
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/pAlkBâs 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.
Detergent testing
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), TweenÂŽ 80, and rhamnolipids with varying concentrations. H2O and DMSO could not solubilize longer length alkanes making them unsuitable for future experiments.
Rhamnolipids could readily solubilize alkanes but showed high absorption at OD600 and strong auto-fluorescence, making them unsuitable as well.
The best results could be achieved by first solubilizing the alkanes in pure ethanol (> 99.8 %) supplemented with 1 % TweenÂŽ 80 (v/v), and then diluting them 1:100 on the microtiter plates for a final TweenÂŽ 80 concentration of 0.01%. This allows for biological availability of the n-alkanes, as well as low enough TweenÂŽ 80 concentration so as not to inhibit cell growth.
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Figure 1: Biological available of n-dodecane 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 different replicates. |
XylS - cloning
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.
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.
sRNA - cloning
To clone the sRNA coding sequences scarless 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.
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.
RBS comparison
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Figure 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. |
Before testing the sRNAâs 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 P. fluorescens (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).
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.
sRNA comparison
During the design process of the sRNA molecules, free binding energy and secondary structures for each construct were calculated in-silico to ensure efficient mRNA binding and degradation, by hfq recruitment.
###table of binding energy, picture of secondary structures not yet created###
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 E. coli. 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).
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Figure 3: 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 ### test comparing induced to un-induced constructs, and un-induced constructs to constructs without sRNA coding sequences. |
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.
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).
Final operon assembly
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 P. fluorescens nor E. coli DH5 alpha.
Results
PE-degradation sensor (AlkS-V760E/pAlkB)
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.
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.
Different n-alkanes, emulsified in TweenÂŽ 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.
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Figure 4: 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) TweenÂŽ 80 at two different concentrations. |
Serial dilution experiments of n-dodecane emulsified in TweenÂŽ 80 were performed and fluorescence intensity measured over 24 h. Fig. ### [A] shows normalized expression 6 h, 12 h and 20 h after induction with different concentrations, ranging from 2 mg/L up to 2000 mg/L. At 12 h significant increases could be measured with inducer concentrations > 200 mg/L, at 20 h concentrations as low as 20 mg/L were sufficient to measure a significant change in fluorescence (RFU##, RFU##, p-value). The fluorescence measurements at 20 h was used to further analyze the dose response curve (fig##,B), showing a stagnation in expression strength at concentrations above 2000 mg/L.
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Figure 5: 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) TweenÂŽ 80. [B] Dose response curve of serial dilutions of n-dodecane and expression strength 20 h after induction. |
PET-degrdation sensor (XylS-K38R-L224Q/Pm)
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 (Fig##), and in combination.
Ps1/Ps2 XylSmt (with MBA or TPA)
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) (fig##). 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##).
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 ##).
Comparing the fluorescence intensity of the wiltype XylS with the mutated one shows an overall decreased expression strength.
(hier nch nen plaot machen 20230824 endpoint sheet 3 um zu zeigen das mut schlechter aktiviert als WT).
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Figure 6: MBA and TPA dependant induction of the XylSmt transcription factor controlling mKate2 expression.
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PS1/Ps2 XylSmut TPA and MBA co-induction
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>###). The expression strength shows an overall decreased fluorescence intensity at low MBA concentrations, despite co-induction with TPA (Figure 7, B ).
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Figure 7 Expression strength with TPA and MBA co-induction
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pEM7 XylSmut
Alternatively, to the Ps1/Ps2 promoter system, pEM7 was tested as a constitutively active promoter, which was previously used for the expression of AlkSV760E.
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 < 0.01####).
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Figure 8 Expression strength of pEM7-XylSmt at different TPA concentrations. 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. |
sRNA mediated repression
SgrS1.2/MicC1.2 repression
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.
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).
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.
The inducer concentration dependant 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 need to be calculated##)
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Figure 9 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 triplicates were analyzed.
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##delta in repressions strength 0-6h 6-12h, mean abnahme alle 30 min mit new stdev, bar blot
Future perspectives
The composite part makes important contributions for the iGEM registy in form of two novel transcription factors sensing PET and PE, as well as newly characterized sRNAâs 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.
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 P. fluorescens related problem solving.
References
https://pubs.acs.org/doi/full/10.1021/acssynbio.2c00620
Expression cassette consinsting of XylS-K38R-L224Q/Pm promoter, activated by terepthalic acid, and AlkS-V760E/pAlkB activated by n-dodecane.
Gene expression controlled by the pAlkB promoter can be degraded with sRNA interference, expressed with Pm promoter. test 123
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4202
Illegal EcoRI site found at 4298 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4202
Illegal EcoRI site found at 4298
Illegal NotI site found at 1625 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4202
Illegal EcoRI site found at 4298
Illegal BglII site found at 212 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4202
Illegal EcoRI site found at 4298 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4202
Illegal EcoRI site found at 4298
Illegal NgoMIV site found at 934
Illegal NgoMIV site found at 2716
Illegal NgoMIV site found at 3187 - 1000COMPATIBLE WITH RFC[1000]
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