Detection

Introduction

Based on the previous experiments, we not only stimulated the enrichment and amplification of target nucleic acid in actual application, but also verified the feasibility of RENDR-based detection system in vivo (more details are shown in our Result page). To prove that enrichment and amplification system is practicable and can be really applied to end users, we further conducted in vitro experiments with both laboratory apparatus and our home-built hardware under laboratory conditions (more details are shown in our implementation page). Cell-free gene expression system was constructed to enable the in vitro RENDR-based detection. The in vitro detection was verified by characterization of pirB_g2β and pirB_g1α. Among those, BBa_K4195163 (T7-pirB_g2β_B-T7t) and BBa_K4195158 (T7-pirB_g1α_B-T7t) showed promising results, both of which are based on the AmpC (β-lactamase) reporters to give out colorimetric signals.

Proof of Concept

The overnight culture of colonies containing plasmid BBa_K4195179_pSB1C3 (expressing the conserved region of pirA) or BBa_K4195180_pSB1C3 (expressing the conserved region of pirB) was 10-fold diluted. Then the diluted bacterial culture was lysed by extraction buffer, and the plasmids were enriched by the filter paper placed into the bacteria lysate. After enriching for 1 min, the paper was washed in wash buffer and directly amplified via RPA. Deionized water and undiluted plasmid solution were used as negative and positive controls, respectively. More details about the reagents we used are shown in our Experiment page.

As shown in DNA gel electrophoresis of the RPA products (Fig. 1), target bands (204 bp) can be observed at the position under 500 bp, which proved that the plasmids enriched by filter paper can be amplified by RPA.

Fig. 1 DNA gel electrophoresis of the fiber paper enriched RPA products from bacteria lysate.

We assembled promoter (BBa_K3222000), RBS (BBa_B0034), coding sequence (CDS) of endolysin (BBa_K4195084), terminator (BBa_K731721) and CDS of Gam-his (BBa_K4195085) to obtain the composite part BBa_K4195167 at vector pSB1C3 by standard BioBrick assembly. The constructed plasmids (Fig. 2) are transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Fig. 2 DNA gel electrophoresis of the regular PCR products. Target bands (1483 bp) can be observed at the position between 2000 bp and 1000 bp.

Consequent cultivation of bacteria was processed for preparation of cell lysate. The whole procedure of preparing E. coli cell lysate can be roughly summarized as freeze-thaw cycles. TX-TL (transcription-translation) premix was prepared according to the reference (1), which contains amino acids, ribonucleotides, energy regeneration system and other necessary components. And the ultimate cell-free expression premix is made by mixing the cell lysate and the TX-TL premix together.

We further optimized the concentration of the plasmids added in the cell-free gene expression system and explored the influence of the type of template DNA (linear or circular). Composite part BBa_K4195152 (T7-B0034-GFP-T7t) was used in these experiments. The result showed that the best plasmid concentration was 3 nM, and linear DNA fragment performed better than the circular type (plasmid) of template DNA (Fig. 3).

Fig. 3 Optimization of cell-free gene expression system. a Various concentrations of plasmid were tested. b Comparison between linear DNA fragment and plasmid (circular type). The group of sterile deionized water added was set as negative control (NC).

Before we implement the RENDR-based detection, we firstly verified the ribozyme splicing in the cell-free expression system. Plasmids of pSB1C3 containing part BBa_K4195152 (T7-B0034-GFP-T7t), BBa_K4195155 (T7-B0034-sfG(R)FP-T7t) and equivalent sterile deionized water (ddw) are separately added into the aliquots of cell-free expression premix, then incubated in 30 °C for 6 h. The expression behavior of GFP is observed under blue-light gel imager, which indicates that GFP was successfully expressed in the group of BBa_K4195152 while the expression of ribozyme-containing circuit (sfG(R)FP) seems not obvious (Fig. 4a). We further changed the temperature condition of incubation from 30 °C to 37 °C, and the results afterward were all measured by microplate reader. We found that the ribozyme-containing circuit showed an increased activity of expression after changing the temperature, which means the physiological temperature (37 °C) is more suitable for ribozyme’s function of self-splicing (Fig. 4b).

Fig. 4 The verification of ribozyme splicing in vitro. a Experiment result under 30 °C incubation. b Experiment result under 37 °C. BBa_K4195152 was set as positive control (PC), and the sterile deionized water was set as negative control (NC).

For verifying the feasibility of the RENDR-based detection system, two guides circuits, BBa_K4195163 (T7-pirB_g2β_B-T7t) and BBa_K4195158 (T7-pirB_g1α_B-T7t), were tested in vitro, in which the two circuits all harbor AmpC (β-lactamase) as the reporter. 2 nM BBa_K4195163_pSB1C3 or BBa_K4195158_pSB1C3 and different concentrations of the input (linear fragment) were separately added into the aliquots of cell-free expression premix, then incubated in 37 °C for 2 h. The 2 nM BBa_K4195153_pSB1C3 (T7-AmpC-T7t) was set as positive control (PC). The expression behavior of AmpC was represented by the relative absorbance unit (RAU). In microplate reader, data were collected once per minute for 20 min, and the peak signal was recorded. For these two guides, we found that when the input concentration increased, the intensity of output signals also showed an increasing trend (Fig. 5). This result suggested that the RENDR-based system could function for input sequences sensing in vitro, which was consistent with the characterization in vivo. Consequently, the validity of RENDR-based system for detecting input sequences has been verified and the promising result motivated us to implement the system in our home-built hardware for the further more practical use.

Fig. 5 Verification of cell-free RENDR-based detection system. a The expression behavior of T7-pirB_g1α_B-T7t under different input concentrations. b The expression behavior of T7-pirB_g2β_B-T7t under different input concentrations.

Based on the previous results, we carried out the test in our home-built hardware. Primarily, we obtained the standard working curves for the AmpC and NanoLuc luciferase reporters with using the purified enzymes. Then we also performed the in vivo characterization for NanoLuc and the in vitro examination for AmpC reporter to sense the added input sequences. Learn more information about our hardware and the results in Hardware page.

Conclusion

Our experimental results provided preliminary proof that the RENDR-based detection system can detect and report the presence of toxin gene in water. Further characterization should be focused on the RENDR-based detection system with the RPA products as input template. Since we have obtained promising results in cell-free expression system, it is convincing that we showed the potential of the detection system to be further optimized into a standardized bacterial disease detection platform, with the fundamental principle of post-transcriptional regulation.

Treatment

Introduction

We have successfully proved that the endolysin Lysqdvp001 (encoded by edl060) could kill the Vibrio parahaemolyticus-alternative strain Vibrio alginolyticus by directly incubating the bacteria with the purified endolysin (Learn more from our Result page). Based on this, we further implemented the OMVs-assisted delivery of the plasmid harboring edl060 for killing the pathogen like a horizontal gene transfer (HGT) mode, as what we have described in our Design page. For careful verification, we firstly tested whether the plasmid could be enveloped into the OMVs or not through polymerase chain reaction (PCR), and then demonstrated the killing effect of the delivered endolysin-encoded plasmid to Vibrio alginolyticus via spot assay.

Proof of Concept

The bacteria harboring BBa_I0500_pSB1C3 (araC/pBAD) was cultivated to extract OMVs by common extraction protocols. After extraction, spot assay on LB agar plate (without any antibiotics supplemented) was performed to verify that the OMVs-containing sample is sterile. The result of spot assay showed there is no colony growing on the plate, which means the OMVs-containing sample has no bacterial contamination (Fig. 6a). Subsequently, routine polymerase chain reaction (PCR) was used to confirm that the plasmids (BBa_I0500_pSB1C3) were enveloped into the OMVs, in which the extracted samples were set as the PCR template. The target band (1524 bp) can be observed at the position around 1500 bp (Fig. 6b).

Fig. 6 Verification of OMV’s ability to package plasmid. a The image of LB agar plate of spot assay for bacterial contamination test. b DNA gel electrophoresis of the PCR products. 

For delivering the endolysin-encoded plasmid to kill the pathogen-alternative Vibrio alginolyticus, we took the first step to verify that the plasmid could be transferred into the bacteria through a OMVs-mediated way. The OMVs, extracted from the culture harboring BBa_K4195115_pUC57-Simple (encoding endolysin Lysqdvp001), were spotted onto the LB agar plate to check if it was sterile (top half of the plate in Fig. 7a), prior to be incubated with Vibrio alginolyticus. The incubation mix was then diluted and spread on the LB agar plate containing ampicillin for overnight growth. Colonies were picked from the plate, followed by colony PCR for verification of the transformation-like plasmid delivery. As shown in the gel image, target bands (2250 bp) can be observed at the position between 2000 bp and 3000 bp (Fig. 7b), suggesting that OMVs can be regarded as a vector for plasmid delivery to other strains.

Fig. 7 OMVs can be regarded as a vector for delivering plasmid. a The plate image of spot assay for bacterial contamination test, in which no colonies can be observed in the dashed circles on the top half of the plate. b DNA gel electrophoresis of the colony PCR products of BBa_K4195115_pUC57-Simple.

For testing the killing effect of the OMVs-delivered plasmid harboring edl060 (BBa_K4195115_pUC57-Simple), the colonies of Vibrio alginolyticus on the plate after the delivering experiment were picked and cultivated overnight. Then the culture was diluted then spotted on the LB agar plates containing ampicillin with or without inducer L-arabinose (for inducing the expression of endolysin). Four colonies were tested, which were consistent with the group numbers. Beside the spot assay, we also implemented colony PCR for verifying whether the colony harboring the target plasmid or not. We found that the groups, which had positive bands in colony PCR, showed an L-arabinose-dependent manner of mortality (Group 2, 3 and 4 in Fig. 8). In contrast, the bacteria survived after induction in Group 1 (Fig. 8), which coincides with the negative result in colony PCR. Thus, the killing effect of the transferred plasmid was verified and it is convincing that the OMVs can deliver plasmid to kill Vibrio alginolyticus as expected.

Fig. 8 The result of spot assay to test the killing effect of the OMVs-delivered plasmid harboring edl060. ns: no significance, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Conclusion

We have confirmed that the plasmid encoding endolysin Lysqdvp001 could be delivered into the target strain via OMVs and such delivery could lead to the cell death after inducing the expression of endolysin. This was a very significant step for validating the implementation of our Treatment part in OMEGA. For increasing the efficiency of plasmid packaging, we have tried to construct a hypervesiculation strain with different approaches (Learn more from our Result page). Such incorporation of plasmid DNA into OMVs has been reported (2, 3), especially as a way of HGT to transfer antibiotics-resistant gene among some pathogens (3). While few researches for killing pathogens in the plasmid-deliver way via the vector OMVs have been published. A concern about potential HGT to transfer the ampicillin-resistant gene was raised, however, we have designed an extra CRISPR array for self-targeting in our project (Learn more from our Design page). We hope the validity of this idea would be further verified, since we have no more time to carry out the related characterization. In summary, the critical aspect of the Treatment part for killing the pathogen Vibrio parahaemolyticus-alternative strain Vibrio alginolyticus has been covered, and we hope our results will inspire other iGEM teams and researchers in the field of pathogens specific killing and the plasmid-transformation of non-classic chassis, thus contributing to the successful applications of synthetic biology to solve local problems.

Prevention

Introduction

Based on the previous experiments, we successfully proved that the displayed receptor rFET can bind the binary toxin subunit PirB and rLvAPN1 can bind both PirA and PirB on the surface of bacteria (more details are shown in our Result page). Due to the stronger interaction between rLvAPN1 and PirB than PirA (4), we considered it promising to neutralize the toxins when displayed on OMVs. Hence, the characterization at the level of OMVs were performed around the ClyA-rLvAPN1 (BBa_K4195130) through a simple but feasible experiment, dot blot (Fig. 9), which has been applied to confirm the presence or absence of displayed antigen on the exterior of OMVs (5, 6).

Fig. 9 The illustration of dot blot to probe the binding event on the surface of OMVs. The secondary antibody is HRP-conjugated so that the positive results can be read out via chemiluminescence imaging.

Proof of Concept

The OMVs were firstly extracted from the culture of engineered bacteria harboring BBa_K4195130 after induction. Subsequently, the OMVs-containing samples were directly spotted onto the nitrocellulose (NC) membrane. Then the NC membrane was incubated with purified his-PirB and anti-His-tag antibody in turn and finally probed by the HRP-conjugated secondary antibody. By comparing the chemiluminescence imaging results of OMVs-containing samples of different origins, we could characterize whether the displayed rLvAPN1 on OMVs is functional or not.

Fig. 10 The imaging results of chemiluminescence (dot blot analysis) to probe the binding event on the surface of OMVs.

Conclusion

For achieving the goal of preventing AHPND with the toxin-binding receptor-displayed OMVs, we have taken efforts to demonstrate it through various aspects from receptor’s display to function of receptor on bacterial surface and OMVs’ surface. Our favorite ClyA-rLvAPN1, showed a good performance in binding toxins, which offered a promising solution for those secreted-toxin related disease. However, it should be noted that the quantitative characterization was rarely used in our experiments and to what extend the OMVs decorated with displayed receptors could function in the real conditions should be further explored. Consequently, we have confirmed that the ClyA-mediated displayed receptor rLvAPN1 can bind to toxins, which composes a necessary component of the Prevention part in OMEGA.

Reference

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