Team:Wageningen UR/Results/DSF Sensing Circuit

Xylencer

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DSF sensing circuit

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The goal of this subproject is to design a synthetic circuit for the Phage Delivery Bacterium (PDB) in order to detect Xylella fastidiosa. Upon sensing of X. fastidiosa, the synthetic circuit will be switched on, resulting in AcrIIA4 production. Production of this anti-CRISPR will alleviate CRISPR-dCas silencing, allowing for phage proliferation. This project is focused on the signal transduction by the presence of X. fastidiosa, which results in GFP production. Once the functionality of the synthetic circuit is proven, the GFP will be replaced by the AcrIIA4. Unfortunately, the overall system was not able to be switched on upon sensing of X. fastidiosa. We showed that certain parts of the synthetic circuit are operating and functional. We demonstrated that the X. fastidiosa sensing protein RpfCch is sucessfully transported to the membrane. Additionally, GFP is efficiently transcribed, but GFP translation is inhibited by the riboswitch.

Introduction

X. fastidiosa uses quorum sensing molecules to communicate among species in a cell density-dependent manner. The quorum sensing molecule X. fastidiosa uses is part of the family of diffusible signaling factors (DSF) [1]. DSF molecules are typically 2-cis unsaturated fatty acids, produced by a variety of unrelated species like Burkholderia cenocepacia and Pseudomonas aeruginosa, but also by X. fastidiosa [2]. The PDB will be a related species to X. fastidiosa, namely Xanthomonas arboricola, which also uses DSF to communicate among species [3].

Figure 1: Schematic overview of the genetic circuit
  • Background arrow_downward

    DSF sensing is based on a two-component system, consisting of RpfC and RpfG, both present in X. fastidiosa and X. campestris [4-8]. Perception of DSF happens via a membrane protein called RpfC [9]. RpfC is a histidine kinase that, upon detection of DSF, alters its conformation and autophosphorylates in the cytosol. This autophosphorylation will phosphorylate and, in turn, activate RpfG. RpfG is a phosphodiesterase which cleaves the second messenger molecule, cyclic-di-GMP (c-di-GMP) into c-GMP. In X. fastidiosa and X. campestris, c-di-GMP affects the cAMP Receptor-Like Protein (CLP) which is a transcription factor regulating the production of mannanase (ManA) [10,11]. This ManA is negatively regulated when c-di-GMP is bound to CLP. Therefore, cleavage of c-di-GMP upon sensing of DSF should, therefore, result in transcription of the gene controlled by the manA promoter. The Dundee iGEM 2014 team tested this system in E. coli, but they observed leaky expression of GFP, which they tested via western blot. They hyphotesized that a high homology of CLP in E. coli, could explain the amount of leakiness. Such high levels of leaky expression are not desired and therefore other c-di-GMP effectors were reviewed. In order to solve this problem, the c-di-GMP riboswitch was chosen to tightly regulate gene expression. C-di-GMP riboswitches are conserved in multiple different eubacterial species [12]. The c-di-GMP riboswitch of choice for this study is the Vc2 c-di-GMP riboswitch, naturally present in Vibrio cholerae, due to its clearly described properties in terms of structural characterization [13,14].

    Figure 2: Schematic representation c-di-GMP riboswitch.

Approach

The protein responsible for DSF sensing in both X. fastidiosa and X. campestris is called RpfC. There is a high level of structural similarity between both species’ RpfC proteins. Both organism are able to sense each other’s DSF molecule, although in lower efficiencies compared to their natively produced DSF [15]. In a previous study, a chimeric protein called RpfCch was constructed. This protein was made from the X. fastidiosa N-terminal transmembrane domain (TMD) and the C-terminal histidine kinase part from X. campestris. This strategy allowed for sensing the native X. fastidiosa DSF, without the need of using X. fastidiosa RpfF [16]. A protein model for this RpfCch was constructed (Fig. 3) where the anchoring in the membrane is clearly visible. At our modeling page we describe how these models are constructed.

Figure 3: Modeled version of the DSF sensing RpfCch protein. The red and blue planes represent the outer and inner cell membranes respectively.

In Cloning Approach the different constructed parts are described. In the proposed system, the BBa_K3286204 and BBa_K3286205 composite parts are representing the 2 plasmid systems. The 1 plasmid system is a combination of both parts in on one plasmid backbone.

  • Cloning Approach arrow_downward

    Golden Gate was used as cloning method to create the desired constructs. The designed circuit was tested in E. coli as the chassis organism. The constructs were cloned in two different plasmid systems for analysis (Table 1 and Table 2).

    The BBa_K3286204 and BBa_K3286205 composite parts were cloned into either pSB1C3 or different SEVA plasmids [17,18] and studied in E. coli DH5alpha. Different scenarios were anaylysed, in which the main differences are the plasmid copy numbers and therefore varieties in gene expression, but also antibiotic resistance markers and origin of replication incompatibility (Table 1).

    Table 1: Schematic overview of the different constructed plasmids.
    Plasmid Plasmid System Ori Marker
    pSB1C3 Both 2P systems pMB1 (Derivative) Chloramphenicol
    pSEVA23 1 pBBR1 Kanamycin
    pSEVA44 1 ColE1 Spectinomycin
    pSEVA62 2 (Vc2 +GFP) RK2 Gentamycin
    Table 2: An overview of the parts used for different systems.
    1P system > 2P RpfCch+RpfG > 2P Vc2+GFP
    Promoter J23100 Promoter J23100 Promoter J23108
    RBS B0034 RBS B0034 Vc2 riboswitch
    RpfCch RpfCch sfGFP
    RBS B0034 RBS B0034
    RpfG RpfG
    Promoter J23108
    Vc2 riboswitch
    sfGFP
    Table 1 and Table 2 are describing the constructs which were built in order to get the desired system. The only combination tested for the two plasmid system was pSB1C3 with RpfCch + RpfG and pSEVA62 with the Vc2 riboswitch + GFP. Although, a negative result was observed after induction with DSF.

Results

Our proposed system was not able to generate the expected fluorescent signal, at least optically, in none of the tested conditions, independently on the distribution of the several elements, the types of DSF, and its respective concentrations (1, 5 and 10 µM). On the other hand, the high copy number plasmid pSB1C3 showed GFP expression in both induced and uninduced states (Fig. 4). Alternatively, different approaches were performed to determine the functionality of the riboswitch part by part.

Figure 4: Four strains that contained the Vc2 riboswitch on the plasmids pSEVA23 (top left), pSEVA44 (top right), pSEVA62 (bottom left) and pSB1C3 (bottom right) with different systems. E. coli was grown on LB agar and not induced with DSF. Fluorescence is visualized by a blue light transilluminator.

First, the TMD and the complete RpfCch protein were both fused to GFP. This to observe whether the RpfCch proteins were being located in the membrane. Results were visualized with fluorescence microscopy and demonstrated the successful transcription, translation and transport of RpfCch to the membrane (Figure 5).

Figure 5: Fluorescent microscopy pictures of the RpfCch (TMD) - GFP fusion. (A-C) Negative control of a strain not expressing GFP. (D-F) Images from a strain constitutively expressing GFP. (G) Image of the RpfCch-sfGFP fusion (BBa_K3286206). (H-I) Image of the RpfCch(TMD)-sfGFP fusions(BBa_K3286207).

The second strategy aimed to test whether GFP translation was inhibited by the Vc2 riboswitch. RNA isolation and cDNA synthesis were performed. cDNA was used as a template in PCR for GFP amplification. Successfully, GFP was transcribed when the Vc2 riboswitch was present in both pSEVA23 and pSB1C3 (Fig. 6). The optical fluorescence can be visualized in Fig. 7.

Figure 6: Amplification of the cDNA with GFP specific primers. The ladder that was used was a 1 Kb+ ladder from NEB. The expected band should be 207 basepairs (bp). (1-3) pSEVA23 1 Plasmid system. (4-6) pSB1C3 samples with BBa_K3286205 (7-9) Positive control contained a constitutively expressed GFP. (10-12) Negative control, which is an E. coli Dh5Alpha strain. (13,14) Two positive colony PCR controls with engineered with a GFP sequence. (15) Negative colony PCR control. (16) Positive control on an extracted plasmid containing a GFP sequence.
Figure 7: The four strains tested for GFP transcription were pSEVA23 (top left), pSB1C3 (top right), positive control (bottom left) and a negative control (bottom right). E. coli was grown on LB agar and not induced with DSF. Fluorescence is visualized by a blue light transilluminator.
  • Third Approach arrow_downward

    In the third strategy, we wanted to test overexpression of the phosphodiesterase PdeH [19]. Constitutive expression of this PdeH would lead to lower levels of c-di-GMP [20]. Possibly resulting in GFP translation due to riboswitch alleviation. Unfortunately, the sequencing results of this construct were incorrect due to an insertion of around 30-40 nucleotides between the RBS and the start codon, this will have a strong negative impact on enzyme production. This part was dropped due to time constraints.

Conclusion

With the current efforts, the proposed system is not able to be switched on when induced with DSF. On one hand, the leakiness of the high copy number pSB1C3 suggests that the amount of riboswitch transcribed is excessive that the native c-di-GMP levels are not enough to repress it under non-induced conditions. On the other hand, the low copy number plasmids used in this study might transcribe a minimal amount of riboswitch. However, it is not enough to overcome the repression of c-di-GMP after induction of DSF. To overcome this repression, a phosphodiesterase could be overexpressed constitutively. Unfortunately, the inability to build the correct plasmids prevented our study to execute this strategy. Alternatively, the pSB1C3 2 plasmid system with BBa_K3286205 could be regulated by a weaker Anderson promoter.

From this study, it can be concluded that the sensor RpfCch is getting translated, transcribed and transported to the membrane. Additionally, cDNA synthesis revealed that GFP is transcribed, but not translated for at least the pSEVA23 1 Plasmid system. The pSB1C3 2 plasmid system with BBa_K3286205 showed leaky GFP production on plate, which was as well confirmed with cDNA synthesis.

As a conclusion, we are close to have a functional DSF sensing system. In this study, we have already proven that the riboswitch is functional and that the RpfCch is located in the membrane. These are key elements in developing a functional system. Therefore, the following step by linking the AcrIIA4 for phage repression to this system is within reach.

  • References arrow_downward
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