Part:BBa_K4757062
_ TPA sensing XylS-K38R-L224Q/Pm expressing mKate2
Contents
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
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 4).
Values are presented as mean +/-SD. No statistical analysis was performed.
4.2 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).
5.3 Ps1/Ps2 XylS-MT TPA and MBA co-induction
To further test the influence of the Ps1/Ps2 promoter system on XylS-mt, the co-induction was tested with previously determined MBA and TPA concentrations. Three TPA concentrations were tested with one of four MBA concentrations. Fold change and normalized fluorescence were calculated (Figure 13). At an MBA concentration of 0.0025 mM, a significant TPA dependent fold change could be measured (1.29 +/- 0.056, p < 0.001). Higher MBA concentrations (0.0075 mM MBA, 0.015 mM MBA) showed an overall decreased fold change. Decrease after TPA induction is due to referencing errors caused by TPA precipitation. The expression strength shows an overall decreased fluorescence intensity at low MBA concentrations, despite co-induction with TPA (Figure 13, right)
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 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. (Figure 3).
Engnineering journey
Mutation -> weak expression/no expression after mutation -> enhancing with RBS -> still weak/no expression -> co-induction -> significant changes could be detected
co-induction showed so low exprssion -> pEM7 promoter was used to substitute Ps1/Ps2 -> high expression but leaky
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 212
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 934
- 1000COMPATIBLE WITH RFC[1000]
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