Part:BBa_K4587194

P_B488_05770 - RinA Amplifier Effect Module

Introduction

Citrus greening, or Huanglongbing (HLB), is an agricultural disease in citrus of global impact. The gram-negative bacteria “Candidatus Liberibacter asiaticus” (CLas), along with the affiliated family members like africanus (CLaf) and americanus (CLam), was detected and identified as the main pathogen of HLB [1]. Current methods for citrus greening like chemical drug spreading are time, labor, and finance-consuming. Some synthetic biology attempts to cope with this disease like engineering CTM virus of citrus plants raised much biosafety concerns. In an attempt to find a specific and efficient resolution for citrus greening treatment, we developed an engineered phage that specifically infect the pathogenic bacteria CLas and execute downstream effects, such as produce antimicrobial peptides for pathogen elimination. CLasMV1 is an recently identified phage that exclusively live on CLas and its family members [2]. Interestingly, the CLasMV1 achieved a symbiosis relationship with CLas for unknown reasons. Therefore, the phage CLasMV1 was chosen as our chassis phage for engineering. The engineered phage is supposed to first enter the CLas bacteria and then express effect substances like AMP for bacteria removal or dsRNA for transmission vector removal.

The safety of engineered phage is of the priority concern. In rare cases, the CLasMV1 phage could infect other bacteria and introduce the engineered genome, which might be harmful to local microbiomes. Considering such safety issues, we proposed to add a biosensor to the engineered part of our phage that explicitly recognizes CLas bacteria and exclusively activate the expression of downstream substances. Also, the efficiency of substances expression is another goal we want to achieve in the engineered phage. Upon the above two considerations, we designed this effect module that is inserted in our phage genome to carry out functions we instruct it to do.

Part Design

Transcriptional factors (TF) are excellent biomarkers to be sensed in a intracellular circumstance like this. The MarR family TF LdtR was identified as a unique and important transcriptional regulator in CLas bacteria [3]. Therefore, we proposed to take LdtR as the marker of CLas, which specifically activates the biosensor, B488_05770 promoter, to start the transcription of downstream substances. In order to achieve high efficient expression and magnify the signal, we installed the RinA amplifier system in the genetic circuit. In all, the B488_05770 promoter first senses the CLas marker LdtR, then it gets activated and immediately express RinA activator, which is assembled in the control of B499_05770 promoter. The transcribed RinA activator further acts on the downstream RinA promoter and efficiently transcribe the downstream gene. In this case, the downstream gene is GFP as a reporter (Figure 1). It would be replaced by our optimized and well designed antimicrobial peptide genes or small RNA genes.

<img src="" alt="alt Figure 1" style="width:100%">

Figure 1. Part Design. Schematic view of P_B488_05770 - RinA Amplifier Effect Module.

Part Validity

Both dry ;ab and wet lab data suggest this effect module works both in specificity and efficiency.

Because the biosensor of LdtR transcriptional factor should be promoters that have high specificity and sensitivity with LdtR, we analyzed the published whole transcriptome sequencing dataset, comparing differentially expressed genes (DEGs) with or without LdtR [1] under that threshold of foldchange < 0.9 and p < 0.05>. Under the condition of LdtR inhibition, down-regulation of expression might indicate potential positive regulatory roles of LdtR on the gene's promoter. The gene with unknown function B488_05770 was dramatically down regulated with a fold change of 0.123 when LdtR was inhibited (Figure 2). This suggested strong possibility that B488_05770 promoter might positively and specifically respond to LdtR. This corresponds to the wet lab result, where B488_05770 promoter was installed to control CLasMV1 capsid expression (Figure 3). The expression of phage capsid was almost abolished without LdtR, indicating P_B488_05770 is a sensor that is specific and sensitive to LdtR, so that the whole engineered part of phage can only be activated within the CLas bacteria.

<img src="" alt="alt Figure 2">

Figure 2. DEGs under conditions with and without intact LdtR. DEGs were identified with the threashold of foldchange < 0.9 or > 1.1, adjust p value < 0.05. Volcanal plot was generated by ggplot2. The arrow annotated gene B488_05770.

<img src="" alt="alt Figure 3">

Figure 3. LdtR-dependent activation of P_B488_05770. Western blot results with rabbit anti-FLAG antibody as primary antibody. Bacteria were induced with IPTG for 12 hours under 25°C. Capsid-FLAG has the size of ~55kDa. LdtR: the LdtR expressing E. coli strain background; P_B488_05770: Bacteria with phagemids featuring B488_05770 promoter.

The RinA amplification system is a famous system for signal amplification with many applications [4]. RinA is a transcription activator that act specifically on its corresponding RinA promoter. Here we introduced RinA under the control of sensor, the activation of which would trigger the RinA-RinA promoter driven amplification of downstream genes. The downstream gene, in the case of here, is AcGFP for reporting. Compared to bare system without amplifier, the RinA amplifier system elevated the downstream expression by ~20 folds, suggesting the efficiency of this expression module (Figure 4).

<img src="" alt="alt Figure 4">

Figure 4. RinA amplifier amplifies signal conferred by sensor. Fluorescence intensity at 488nm of sensor-RinA amplifier-GFP system after 24h in LdtR expressing E.coli strain. *: p < 0.001.

Conclusions and Contributions

To sum up, this part can specifically recognize the LdtR marker in CLas bacteria by P_B488_05770, and specifically activate it self, which would be efficiently amplified by RinA amplifier system.

The analysis of the transcriptome data identified a specific sensor for LdtR, which provides a new potential element for citrus greening treatment and prevention. The combination of P_B488_05770 and RinA amplifier system add another piece of evidence for the application of RinA amplifier. The whole module, when put into our engineered phage, is hopeful to offer a new approach for citrus greening treatment and prevention.

References

1: Pagliai FA, Coyle JF, Kapoor S, Gonzalez CF, Lorca GL. LdtR is a master regulator of gene expression in Liberibacter asiaticus. Microb Biotechnol. 2017 Jul;10(4):896-909. doi: 10.1111/1751-7915.12728. Epub 2017 May 15. PMID: 28503858; PMCID: PMC5481520.
2: Zhang L, Li Z, Bao M, Li T, Fang F, Zheng Y, Liu Y, Xu M, Chen J, Deng X, Zheng Z. A Novel Microviridae Phage (CLasMV1) From "Candidatus Liberibacter asiaticus". Front Microbiol. 2021 Oct 13;12:754245. doi: 10.3389/fmicb.2021.754245. PMID: 34721359; PMCID: PMC8548822.
3: Pagliai FA, Gardner CL, Bojilova L, Sarnegrim A, Tamayo C, Potts AH, Teplitski M, Folimonova SY, Gonzalez CF, Lorca GL. The transcriptional activator LdtR from 'Candidatus Liberibacter asiaticus' mediates osmotic stress tolerance. PLoS Pathog. 2014 Apr 24;10(4):e1004101. doi: 10.1371/journal.ppat.1004101. PMID: 24763829; PMCID: PMC3999280.
4: Ferrer MD, Quiles-Puchalt N, Harwich MD, Tormo-Más MÁ, Campoy S, Barbé J, Lasa I, Novick RP, Christie GE, Penadés JR. RinA controls phage-mediated packaging and transfer of virulence genes in Gram-positive bacteria. Nucleic Acids Res. 2011 Aug;39(14):5866-78. doi: 10.1093/nar/gkr158. Epub 2011 Mar 30. PMID: 21450808; PMCID: PMC3152322.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1511
  Illegal XhoI site found at 201
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1032
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 656