Previous Engineering Efforts

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XylR is an intensively studied regulatory protein mined from Pseudomonas putida[1]. It responds strongly to toluene, xylene and 4-chlro-toluene, while weakly to 3-methyl benzyl alcohol[1].

XylR activates the Pu promoter to express the "upper pathway" (xylMABC) when exposed to m-xylene (Fig. 1). It also activates the Ps1 promoter, thus to produce another transcriptional activator, called XylS, to turn on the expression of the downstream pathway (xylXYZLTEGFJGKIH, the meta-cleavage operon)[1][2](Fig. 1, Fig. 2). Notably, the entire regulatory network is also controlled by several global regulatory elements, such as IHF. This provides an explanation for the genetic-context-dependent performance of XylR-Pu pair; namely, when expressed in different bacterial species, the regulatory performance of XylR/Pu pair often fails[3]. Therefore, fine-tuning is probably necessary.

The XylR protein consists of four domains (Fig. 3): Domain A is the sensing domain, which will conduct a conformational change upon effector binding. Previous studies showed that Domain A inhibits the DNA binding affinity before conformational change[4][5]. Domain B is a linker domain; mutations in this domain will disrupt the functional coupling and spatial interactions between Domain A and C[6].

Domain C is the activation domain with ATPase activity that is crucial for XylR dimerization. A subdomain in domain C is assumed to account for the dimerization. Domain D is the DNA binding domain featured by helix-turn-helix motif whose DNA binding is sequence-specific.

XylR is capable of forming tetramer when Domain C binds with ATP [7]. Without ATP, the XylR dimers could only bind to two sequence-specific DNA sites, but won't initiate transcription even exposed to inducers. As a typical σ54-dependent transcriptional activator, with the binding of ATP, two dimers of XylR tend to further cooperatively tetramerize, thus to bend the promoter region DNA with the help of integrated host factor (IHF). As a result, the transcription will be launched by the interaction between the XylR tetramer and RNA Polymerase (RNAP). ATP hydrolysis provides energy for this process [8].

Since one of the criteria of our Sensor Mining for aromatics-sensing transcriptional regulators is "well-studied", it can be expected that some mutants of XylR with novel aromatics-sensing characteristics have been identified. Therefore, we set out to collect the information of XylR. As expected, random mutagenesis on XylR Domain B has been performed in previous studies[9]. One XylR mutant, referred to as XylR28 in ref. [9], carries 4 point mutations in Domain A and Domain B. These point mutations endow XylR with a remarkably improved response to 2.4-DNT and TNT and a reduced response to its natural inducer, m-xylene, indicating the directed evolution may provide possibility to engineer XylR to respond to compounds that it doesn't naturally sense[6].

As discussed above, the XylR/Pu pair needs fine-tuning. Promoter engineering is considered to be a method. A XylR-controlled Pu promoter shows a high basal level. But the cognate promoter of XylR homolog, DmpR, has a fairly low basal level. We found that XylR could activate the Po promoter of DmpR. A hybrid promoter has been accordingly designed using the binding site of XylR from the Pu promoter. This design has shown that the basal level of the hybrid promoter is low and the XylR binding affinity is high[12].

We obtained wild type XylR from Biobrick BBa_I723021 designed by iGEM07_Glasgow. To find the optimal performance of biosensor XylR/Pu, we combined Pr-XylR coding sequence with 8 reporter circuits adopting different inducible promoters with Ribosome Binding Sites at different intensity. They are Pu promoter naturally activated by XylR with B0031, B0032 and B0034; Po promoter which is originally regulated by DmpR with B0031, B0032 and B0034; Po' promoter specially designed for XylR with B0031 and B0032. Results showed that the XylR biosensor adopting Pu promoter with RBS B0034 obtained the optimal performance. Then this XylR biosensor was subject to ON-OFF test to determine its detection profile (Fig. 5).

As shown in Fig. 5, the performance of XylR is quite different from the previous studies. The conventional inducers for XylR, for instance, TOL, m-Xyl and 3-CITOL, could not elicit any significant responses. It was probably because that these hydrophobic aromatic compounds can vaporize and permeate the sealing film used in our experiment. Regarding this problem, we performed the ON/OFF Test using centrifuge tubes, rather than the conventional 96-well microplate. Results indicated that the XylR biosensor indeed give response to conventional hydrophobic compounds such as TOL and m-Xyl if the vaporization could be prevented (Fig. 6).

In summary, we have successfully constructed the XylR biosensing circuit. The aromatics-sensing profile of XylR biosensor is considerably narrow (Fig. 5b), making it a convenient biosensor for the presence of 4-chloro-benzyl-aldehyde and 3-methyl-aniline. Notably, we are the first iGEM team that demonstrates the ability of XylR to really work as a biosensor; this is probably due to our proper fine-tuning and the method to avoid the vaporization of hydrophobic aromatic compounds.

Figure 1. The regulatory network of TOL pathway, including the xyl gene cluster, XylS and XylR. XylR is the master regulator that regulates Pu promoter (controls "upper pathway", xylMABC) and Ps1 promoter (controls the expression of XylS, thus to indirectly activate the expression of "downstream pathway“, xylXYZLTEGFJGKIH). The xylene or its derivatives are supposed to be the typical inducers of XylR.

Figure 2. The TOL degradation pathway. The supposed inducers of XylR, toluene and its derivatives, are highlighted in blue.

Figure 3. Schematic organization of XylR protein domains. From N-terminal to C-terminal: the sensor domain A, the linker domain B, the dimerization domain C and the DNA binding domain D.

Figure 4. The mechanism of σ54-dependent transcription activation by XylR. Step1, RNAP recruitment by σ54; XylR has formed dimers when binding to DNA. Step2, formation of XylR tetramer, coupled with ATP hydrolysis. Step3, RNAP ready to initiate transcription. Step4, transcription start with σ54 released. See the main text for more detailed explanation of transcription activation at the Pu promoter.

Figure 5. The induction ratios of all 78 aromatic compounds obtained from the ON/OFF Test using Test Protocol 1. (a) XylR biosensor could respond to several aromatics whose induction ratios are not sufficiently high. This result was inconsistent with previous studies [5], which is probably due to the vaporization of hydrophobic aromatic compounds, as we discussed in the main text. (b) The aromatics-sensing profile of XylR biosensor. The aromatic species that can elicit strong responses of XylR biosensor are highlighted in orange in the aromatics spectrum (For the convenience and clearance of data demonstration, 4-chloro-benzoate, 4-bromo-benzoate and salicylic acid are not included here for they can already be well sensed by other biosensors). The structure formula of the typical inducer(s) is also presented around the spectrum. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself. Click Here for the full names of aromatic compounds.

Figure 6. The induction ratios of hydrophobic aromatics compounds obtained from the ON/OFF Test using the new protocol (the same with Test Protocol 1 except that the experiments were performed in centrifuge tubes to avoid the vaporization).

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