Difference between revisions of "Part:BBa K1031803"

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Pu-B0031-sfGFP-Terminator (XylR)

For detailed information concerning XylR and Pu promoter, please visit 2013 Peking iGEM Biosensor XylR

Instruction

Gene cluster and chemical pathway

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.

Fig 1The 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 Ps2 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.

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

Protein structure

The XylR protein consists of FOUR domains (Fig. 3): Domain A is the sensor domain, which will conduct a conformational change upon effector binding. Previous studies showed that Domain A inhibits the DNA binding affinity of Domain C before the 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 effector-binding 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.

Fig 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.

Promoter structure

Pu promoter which is activated by XylR, is σ-54 dependent. It is composed of three elements. The UBS (Upstream Binding Site) site which is responsible for interacting with XylR transcriptional factor. The IHF binding site which allows IHF to participate, enhancing transcription efficiency. -24 and -12 region interact with σ-54 factor of RNA polymerase, enabling the formation of open complex. (Fig 4)

Fig 4 Structure of Pu promoter. The UAS of this promoter shown as blue sequence in the blue frame interacts with DmpR. IHF binding site is shown in green in the green frame. The orange sequence indicates σ54 binding site as -24 region and -12 region. The G in red represents +1 site.

Mechanism

XylR is capable of forming tetramer when bound with ATP [7]. Without ATP binding, the XylR dimers bind to two sequence-specific DNA sites. 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].(Fig 5)

Fig 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.

Previous engineering effort

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 usually needs fine-tuning. Promoter engineering is considered to be an answer. A XylR-conrolled 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].

Sequence and Features

Sequence and Features

Construction

Pc promoter J23114 is selected to initiate the transcription of XylR. Based on this circuit, we constructed a library of RBS (Ribosome Binding Site) including B0031[1], B0032[2], B0033[3] and B0034[4] for tunning for expression level of reporter gene sfGFP. K1031803 consists of Pu promoter, RBS B0031 and reporter gene sfGFP (Fig 2).

Fig 2 Construction of reporter circuit Pu-B0031-sfGFP. The orange arrow represents Pu promoter for XylR. The green oval stands for RBS B0031. sfGFP coding sequence is shown with dark blue, while terminator B0015[5] is in dark red.