Latest revision as of 14:51, 12 October 2023

TPA sensing XylS-K38R-L224Q/Pm expressing mKate2

BBa_K4757062

1. Usage and biology

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

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

2. Results

XylS-mt was first tested with the native Ps1/Ps2 promoter system, with different inducer compositions of TPA and 3-methyl-benzoate (MBA). The Ps1/Ps2 promoter was substituted with the constitutive promoter pEM7 using add-on PCR. TPA and MBA were tested separately in serial dilutions experiments (figure 2), and in combination (figure 3).

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 1).

**Figure 1: Comparison of expression strength of wildtype and mutated (K38R, L224Q) XylS, at three different inducer concentrations.**
Values are presented as mean +/-SD. No statistical analysis was performed.

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 factor XylR, previously described to jointly activate expression from the Ps1 promoter with XylS in P. putida. However, co-induction showed no increase in expression strength (data not shown).

2.3 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 2, 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 2, A). The calculated dose response curve (Figure 2, (B)) shows inductor saturation at 0.1 mM. For induction of TPA, no inductor saturation was observed (Figure 2, D). The fluorescence intensity of the XylS-WT compared to the XylS-mt shows an overall decreased expression strength. (Figure 2, E)

**Figure 2: MBA andTPA dependent induction of the XylS-MT transcription factor controlling mKate2 expresion.**
**(A)** Fluorescence intensity measurements at 8h, 12 h, 16 h after induction with serial dilutions of MBA
**(B)** Dose response curve of expression strength for different MBA inducer concentrations
**(C)** Fluorescence intensity measurements at 8 h, 12 h, 16 h after induction with serial dilutions of TPA
**(D)** Dose response curve of expression strength for different TPA inducer concentrations
**(E)** Time resolved measurements of dose response curves after induction with TPA and MBA
Values are presented as mean +/-SD. For statistical analysis groups were compared to 0 mg/mL MBA or 0 mg/mL TPA at each time point. Reported significances were determined with ordinary One-way ANOVA with Dunn's method for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. To reduce complexity only significant test results are shown.

2.4 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 3). At an MBA concentration of 0.0025 mM, a significant TPA dependent fold change could be measured (1.29 +/- 0.056, p < 0.001). Higher MBA concentrations (0.0075 mM MBA, 0.015 mM MBA) showed an overall decreased fold change. Decrease after TPA induction is due to referencing errors caused by TPA precipitation. The expression strength shows an overall decreased fluorescence intensity at low MBA concentrations, despite co-induction with TPA (Figure 3, right)

**Figure 3: Expression strength with TPA and MBA co-induction**
Left: Fold change in expression of different MBA inducer concentrations after co-induction of TPA
Right: Expression strength measured in relative fluorescence of different MBA inducer concentrations after co-induction of varying TPA concentrations.
Values are presented as mean +/-SD. For statistical analysis groups were compared to 0 mg/mL MBA or 0 mg/mL TPA at each time point. Reported significances were determined with ordinary One-way ANOVA with Dunn's method for multiple comparisons. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. To reduce complexity only significant test results are shown.

3. Engineering

3.1 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 fluorescence reporter gene mKate2 was cloned with SacI and PstI into the MCS downstream of Pm sensitivity of XylS-mt towards was studied using the native Ps1/Ps2 promoter system. In a second iteration, to improve overall expression strength, a ribosomal binding site (BBa_J61101) was added with add-on PCR. To further improve expression strength the Ps1/Ps2 promoter system was substituted with the constitutively active pEM7 (BBa_K4278402) promoter

XylS_mew — **Figure 4: Overview of genetic construct for XylS-MT testing**

3.2 Part evolution

To optimize terephthalic acid sensing, multiple experimental cycles had to be done After introducing the two point mutations (K38R, L224Q) significantly decreased fluorescence was measured. Since P. fluorescens natively have high auto-fluorescence, no good measurements could be taken.
A ribosomal binding site from the Anderson library was introduced (BBa_J61100) to increase expression, increasing induced fluorescence above backgroun. To better understand the underlying mechanism of different inducer concentrations of MBA and TPA weretested (figure 2). Altough significant changes after TPA induction the fluorescence intensity is very low compared to MBA induction (figure 2, (E)). For better understanding of the Ps1/Ps2 promoter system, co-induction with MBA and TPA was tested. After co-induction with low levels of MBA (< 0.005 mM MBA) significant, TPA concentration dependent, fold changes could be calculated.

Further characterization of XylS-mt was done by substituting th Ps1/Ps2 expression system with the constitutiveyl active pEM7 promoter(BBa_K4278402, yielding the composite part BBa_K4757063

During characterization of the engineered XylS-R38K-L224Q transcription factor, multiple design iterations were conducted, leading to an increased understanding of the sensor, as well as the Ps1/Ps2 promoter, giving valuable insights for future iGEM teams.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 212
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 934
1000
COMPATIBLE WITH RFC[1000]

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