Part:BBa_K5250008
PDE-targeting sgRNA
A single guide RNA, part of the CRISPRi system, that specifically targets the Pseudomonas species IsoF PDE PisoF_02645.
Usage and Biology
Cyclic di-GMP is a second messenger which regulates the switches of the cell state between motile and sessile. Phosphodiesterase (PDE) is an enzyme family that regulates the c-di-GMP levels within the cell, more specifically it degrades c-di-GMP.
Phosphodiesterases, which contain either an EAL or HD-GYP domain, are the second enzyme family that controls the c-di-GMP concentration in the cell [2]. They degrade c-di-GMP by either linearizing it into a pGpG or converting it into two molecules of GMP [1]. Our aim is to inhibit the activity of these enzymes to increase the c-di-GMP concentration. Therefore, we aim to target and knock down one of the PDEs encoded on the chromosome of P. sp. IsoF using a CRISPR interference (CRISPRi) system. CRISPRi is a modified version of the CRISPR-Cas9 gene-editing technology and was designed to repress gene expression without cutting the DNA.
Unlike the standard Cas9 protein, which cleaves DNA, the CRISPRi system utilizes a catalytically inactive variant known as dead Cas9 (dCas9). DCas9 can still be guided to a specific DNA sequence by a guide RNA, but it can no longer cut the DNA. Instead, it binds to the target sequence and physically blocks the RNA polymerase and therefore prevents the transcription of the targeted gene. This system thus allows knockdown of a gene encoding for a Phosphodiesterase.
We utilized the comparative analyses of Hailing Nie et al. [1] to identify a suitable PDE for knockdown. Among the P. putida KT2440 mutants they created, the one lacking PDE_0914 showed the highest increase in biofilm formation. After examining the gene bank for Pseudomonas sp. IsoF, we identified the homologous gene in P. sp. IsoF, PisoF_02645. We chose to target this PDE for our knockdown.
The CRISPRi system requires two key components: the modified dCas9 protein and a guide RNA (sgRNA). We were provided with a Pseudomonas sp. IsoF strain, which already contains the gene encoding dCas9 integrated on its chromosome.
Our next step was to design our sgRNA. So the sgRNA was designed by first identifying a PAM motif close to the promoter. This leads to a high efficient knockdown since the DNA polymerase cannot transcribe any nucleotides when it is already inhibited at the promoter. Then we ensured that the 10 adjacent nucleotides were unique across P. sp. IsoF genome, in both orientations forward and reverse, by examining the P. sp. IsoF gene bank. And lastly, we extended the guide sequence by an additional 12 nucleotides.
The final sequence of the sgRNA, excluding the PAM sequence, is:
5’-CAATGAAACCAATACACTATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC-3’.
In this sequence the underlined part represents the guide RNA, while the italicized part represents the tracr-cr RNA.
Characterization
In our genetic construct we want the PDE gene to be knocked down only when xylose is sensed by the bacteria, therefore it is activated by the Pxut promoter (BBa_K5250001). We decided to use a CRISPR interference system (CRISPRi), instead of a gene knock-out to make the genetic alterations reversible. After cloning our sgRNA into the plasmid and transforming it into P. sp. isoF we performed a c-di-GMP assay and a biofilm staining assay.
Cyclic di-GMP assay
Experiment
For the c-di-GMP assay we decided to use the kit from Lucerna Technologies which is based on a fluorescence signal generated by c-di-GMP. The c-di-GMP sensor used in this kit consists of a c-di-GMP riboswitch and a Spinach aptamer. When c-di-GMP binds to the riboswitch, it stabilizes the Spinach aptamer. This allows the fluorophore DFHBI-1T from the kit to bind and emit a fluorescent signal. The fluorescence is then measured using a fluorescence plate reader.
Results
Figure 1: Figure 1.: Fluorescence Intensity from c-di-GMP Assay Across Different Strains, Experiment 1 and 2: The box plot shows the fluorescence intensity measurements for various strains in a c-di-GMP assay, representing the c-di-GMP levels in each strain.
Figure 1. shows the first two assays put together in one graph. The second through seventh strains are our DGC constructs that are intended to increase biofilm formation. We included three different negative controls, P. sp. IsoF with and without plasmid as well as a strain that contains a plasmid overexpressing PDE. Since PDE is inhibiting c-di-GMP production, we expected to see only little c-di-GMP production in this strain. This can’t really be shown from this graph. However, there seems to be a high variance between different values of the PDE, suggesting that more data points would be needed for a conclusive analysis. Further, we included a positive control, P. sp. IsoF YedQ, which is known to increase biofilm production. However, an increase compared to the control can’t really be observed. After analyzing our controls, we decided to handle our results with caution.
Unfortunately, our designed PDE knockdown did not yield an increased c-di-GMP level in either experiment. To address this, we would need to conduct further experiments with a larger sample size while also evaluating the functionality of our PDE knockdown construct, as we suspect it may not have been active.
Biofilm staining
We performed three biofilm staining assays. The first assay only contained the strain with the sgRNA knocking down the PDE. The PDE knock-down strain was also compared to the effect of 5 different diguanylate cyclases (DGCs) that we use in our project. Since our goal is to enhance biofilm formation, we found a method to quantify the biofilm components produced by Pseudomonas species isoF strains. Although biofilms consist of various components, in our assay we focused specifically on staining the polysaccharides in the biofilm as a way to quantify biofilm production. We used Congo red-derived dye, kindly provided by our host lab, and measured the fluorescence of our stained strains under a microscope.
P. sp. isoF wt, P. sp. isoF dCas9 and P. sp. isoF with an empty pBBR1MCS5 plasmid served as negative controls. P. sp. isoF YedQ contained a plasmid with a constitutively expressed DGC YedQ and served as a positive control. P. sp. isoF PDE knockdown refers to P. sp. isoF that contains a plasmid with the sgRNA.
The cultures we wanted to test were pipetted onto agar plates containing Congo-red derived dye that binds to the polysaccharides in the biofilm. After two days of incubation, we examined the plates under a fluorescence microscope, took pictures of the cultures and processed them with ImageJ to quantify the fluorescence levels.
Figure 1: Figure 1a. Represents data from the biofilm assay.
The dCas9 is controlled by a rhamnose promoter which activates our CRISPRi system. We incubated P. sp. isoF dCas9 sgRNA both with and without rhamnose to observe if activating the CRISPRi system would lead to increased polysaccharide production. To determine whether rhamnose itself affects biofilm component production, we also incubated one of our controls, P. sp. isoF dCas9, with and without rhamnose.
The controls showed lowest fluorescence, while P. sp. isoF YedQ displayed the highest fluorescence, as expected. P. sp. isoF dCas9 with the sgRNA showed higher fluorescence than the controls, but lower than P. sp. isoF YedQ. This indicates that the sgRNA inhibits the PDE PisoF_02645, leading to increased c-di-GMP levels and higher polysaccharide production that the control strains.
Despite the fact that the results confirmed our hypothesis, inducing the sgRNA-containing strain with rhamnose did not cause a big difference compared to the non-induced condition. Polysaccharide production also did not increase substantially when dCas9 was induced compared to non-rhamnose conditions. This suggests that the rhamnose promoter in front of dCas9 may be leaky. Discussions with our advisor confirmed that similar results were observed in other gene knockdown experiments.
References
[1] Nie, H., Xiao, Y., He, J., Liu, H., Nie, L., Chen, W., & Huang, Q. (2019). Phenotypic–genotypic analysis of GGDEF/EAL/HD‐GYP domain‐encoding genes in Pseudomonas putida. Environmental Microbiology Reports, 12(1), 38–48. https://doi.org/10.1111/1758-2229.12808
[2]Victor G. Tagua, Maria Antonia Molina_henares, Maria L. Travieso, Rafael Nisa-Marttinez, José Miguel Quesada, Manuel Espinosa-Urgel and Maria Isabel Ramos-Gonzalez. (2022). C-di-GMP and biofilm are regulated in Pseudomonas putida by the CfcA/CfcR two-component system in response to salts. Environmental Microbiology, (24 (1)).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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