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A protease-based biosensor for the detection of schistosome cercariae.

Webb AJ, Kelwick R, Doenhoff MJ, Kylilis N, MacDonald JT, Wen KY, McKeown C, Baldwin G, Ellis T, Jensen K, Freemont PS - Sci Rep (2016)

Bottom Line: Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings.Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide.Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

View Article: PubMed Central - PubMed

Affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, London, UK.

ABSTRACT
Parasitic diseases affect millions of people worldwide, causing debilitating illnesses and death. Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings. A common signature of parasitic diseases is the release of specific proteases by the parasites at multiple stages during their life cycles. To this end, we engineered several modular Escherichia coli and Bacillus subtilis whole-cell-based biosensors which incorporate an interchangeable protease recognition motif into their designs. Herein, we describe how several of our engineered biosensors have been applied to detect the presence and activity of elastase, an enzyme released by the cercarial larvae stage of Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide. Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

No MeSH data available.


Related in: MedlinePlus

B. subtilis whole-cell biosensors are maintained post-lyophilisation.(a) Strains containing the plasmids encoding the biosensors, as well as the empty vector control, were lyophilised, reconstituted, and expression of the biosensors quantified using flow cytometry. The left hand panel, the middle panel and the right panel represent the LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON biosensors respectively. Empty vector (EV), Labelled TEV, Labelled ELA and Labelled CON are the untreated but labelled samples for LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON respectively. Flow data analysis was carried out using FlowJo (vX 10.0.7r2) software. (b) Summary of flow cytometry data. Mean Geometric means were: LytCCWD-TEV 272.7 ± 31.8, LytCCWD-ELA 355 ± 10.6 and LytCCWD-CON 311.3 ± 51.7.
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f6: B. subtilis whole-cell biosensors are maintained post-lyophilisation.(a) Strains containing the plasmids encoding the biosensors, as well as the empty vector control, were lyophilised, reconstituted, and expression of the biosensors quantified using flow cytometry. The left hand panel, the middle panel and the right panel represent the LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON biosensors respectively. Empty vector (EV), Labelled TEV, Labelled ELA and Labelled CON are the untreated but labelled samples for LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON respectively. Flow data analysis was carried out using FlowJo (vX 10.0.7r2) software. (b) Summary of flow cytometry data. Mean Geometric means were: LytCCWD-TEV 272.7 ± 31.8, LytCCWD-ELA 355 ± 10.6 and LytCCWD-CON 311.3 ± 51.7.

Mentions: To enable easy and cost effective transportation of the biosensor strains, especially in settings lacking reliable cold chain access, we wanted to show that these strains would be viable and maintain the plasmids encoding the biosensors after the process of lyophilisation. According to previous reports B. subtilis and related Bacillus strains are viable after the lyophilisation process and do maintain plasmids that they carry2930. To test whether our host B. subtilis strain, WB800N, would survive the process we decided to lyophilise the strains carrying the plasmids encoding the three biosensors, as well as the empty vector control, and analyse whether the strains survive and maintain their plasmids. Cells were grown overnight, and aliquots (1 ml OD600 nm 1.0) were resuspended in 50 μl fresh sterile LB medium before being lyophilised. Lyophilised cells were resuspended in sterile dH2O (thereby reconstituted in LB medium) and allowed to recover at room temperature for 2 hours. The cells were then added to fresh LB broth and induced to express the sensors as described in the materials and methods section. All strains survived the process and the plasmids were maintained as evidenced by the labelling of the cells with His-PE as measured by flow cytometry (Fig. 6).


A protease-based biosensor for the detection of schistosome cercariae.

Webb AJ, Kelwick R, Doenhoff MJ, Kylilis N, MacDonald JT, Wen KY, McKeown C, Baldwin G, Ellis T, Jensen K, Freemont PS - Sci Rep (2016)

B. subtilis whole-cell biosensors are maintained post-lyophilisation.(a) Strains containing the plasmids encoding the biosensors, as well as the empty vector control, were lyophilised, reconstituted, and expression of the biosensors quantified using flow cytometry. The left hand panel, the middle panel and the right panel represent the LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON biosensors respectively. Empty vector (EV), Labelled TEV, Labelled ELA and Labelled CON are the untreated but labelled samples for LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON respectively. Flow data analysis was carried out using FlowJo (vX 10.0.7r2) software. (b) Summary of flow cytometry data. Mean Geometric means were: LytCCWD-TEV 272.7 ± 31.8, LytCCWD-ELA 355 ± 10.6 and LytCCWD-CON 311.3 ± 51.7.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835807&req=5

f6: B. subtilis whole-cell biosensors are maintained post-lyophilisation.(a) Strains containing the plasmids encoding the biosensors, as well as the empty vector control, were lyophilised, reconstituted, and expression of the biosensors quantified using flow cytometry. The left hand panel, the middle panel and the right panel represent the LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON biosensors respectively. Empty vector (EV), Labelled TEV, Labelled ELA and Labelled CON are the untreated but labelled samples for LytCCWD-TEV, LytCCWD-ELA and LytCCWD-CON respectively. Flow data analysis was carried out using FlowJo (vX 10.0.7r2) software. (b) Summary of flow cytometry data. Mean Geometric means were: LytCCWD-TEV 272.7 ± 31.8, LytCCWD-ELA 355 ± 10.6 and LytCCWD-CON 311.3 ± 51.7.
Mentions: To enable easy and cost effective transportation of the biosensor strains, especially in settings lacking reliable cold chain access, we wanted to show that these strains would be viable and maintain the plasmids encoding the biosensors after the process of lyophilisation. According to previous reports B. subtilis and related Bacillus strains are viable after the lyophilisation process and do maintain plasmids that they carry2930. To test whether our host B. subtilis strain, WB800N, would survive the process we decided to lyophilise the strains carrying the plasmids encoding the three biosensors, as well as the empty vector control, and analyse whether the strains survive and maintain their plasmids. Cells were grown overnight, and aliquots (1 ml OD600 nm 1.0) were resuspended in 50 μl fresh sterile LB medium before being lyophilised. Lyophilised cells were resuspended in sterile dH2O (thereby reconstituted in LB medium) and allowed to recover at room temperature for 2 hours. The cells were then added to fresh LB broth and induced to express the sensors as described in the materials and methods section. All strains survived the process and the plasmids were maintained as evidenced by the labelling of the cells with His-PE as measured by flow cytometry (Fig. 6).

Bottom Line: Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings.Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide.Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

View Article: PubMed Central - PubMed

Affiliation: Centre for Synthetic Biology and Innovation, Imperial College London, London, UK.

ABSTRACT
Parasitic diseases affect millions of people worldwide, causing debilitating illnesses and death. Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings. A common signature of parasitic diseases is the release of specific proteases by the parasites at multiple stages during their life cycles. To this end, we engineered several modular Escherichia coli and Bacillus subtilis whole-cell-based biosensors which incorporate an interchangeable protease recognition motif into their designs. Herein, we describe how several of our engineered biosensors have been applied to detect the presence and activity of elastase, an enzyme released by the cercarial larvae stage of Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide. Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

No MeSH data available.


Related in: MedlinePlus