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Foldscope – DIY microscope built for less than $1

 

 “Magnify curiosity worldwide”

  Image source, PLoS ONE

  A few weeks ago I excitedly received my Foldscope in the mail. I was one of 8,457 backers in a successful Foldscope kickstarter campaign, that resulted in a first round production of 60,000 origami-inspired paper microscopes. The deluxe kit included the sturdy, water resistant paper microscope frame that is constructed into a moveable stage with a focus “knob”, and includes a spherical micro-lens enabling 140x magnification and ~2 micron resolution.

  The microscope requires no power, is indestructible and can be mounted to a smart phone for digital image recording. The kit also came with numerous sample preparation materials, including slides and coverslips, tweezers, pipettes, filter paper, mesh strainers, scissors and and eppendorf tubes. Slides are prepared, inserted into the frame, and you position your eye to the lens to directly view the sample. Rotating the microscope when placed in front of a light source allows you to view brightfield or darkfield imaging, which has a profound effect on your sample. I have not  yet explored the LED illuminator/ magnifier, which provides an LED light source and supports pre-screening a sample for larger organisms, prior to mounting the slide to the scope.

  The foldscope was recently described in an open-access, PLoS ONE manuscript. Below are several images of what is possible with this system. Microorganisms as small as bacteria can be seen, as well as larger creatures like parasites, amoeba, nematodes, or  lady bug limbs. There are countless examples of student images posted on the foldscope microcosmos website. What I truly love about this system is the unlimited potential for children (and hopefully adults) to explore the unseen world. The foldscope motto is brilliant: “magnifying curiousity worldwide”.

  Fundamental to the mission of the foldscope team is to provide low-cost scientific tools, in order to break down the barrier of access for people to explore their scientific curiosity. While the microscope can be assembled for less $1 in parts, the current price for the deluxe kit is $35US. There are bundle prices for classes, with extremely reasonable prices for 20-100 basic kits. I am hoping to integrate the Foldscope  into a ‘home lab kit’ for Athabasca University online, biology courses. This microscope is a great example of ‘frugal science’,  where through innovation a new product is designed to meet a need, and there is no sacrifice in quality.

  There are so many applications for the foldscope. It has great potential in education and to inspire budding scientists, but can also make a serious contribution to research and solving real world problems. For example, the Foldscope can be used to detect microbial contaminants in rural drinking water supplies, or possibly in diagnosing blood-borne disease like malaria, African sleeping sickness, schistosomiasis and Chagas. My 9 year is very excited about our ongoing search for tardigrades, I hope to present some images soon of the local west coast tardigrades.

  The second round of production is underway, with an ambitious goal of distributing 1,000,000 foldscopes by the end of 2018. I think this product and concept has great potential to be ‘disruptive’. There are exponential possibilities by “providing every child a microscope”. I look forward to witnessing how the Foldscope community uses this tool, and to  learning about future products in their pipeline.

 

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Biosensors to detect macrolide antibiotics in high throughput screening

Wiki Source, DNA binding motif of TetR

  Antibiotics are often natural products discovered in other organisms, including but not limited to bacteria, fungi, plant, frogs and humans. Although they may have different functions in their natural setting, they are exploited by medicine for their ability to kill bacteria.  Their discovery often relies on traditional testing for antimicrobial activity in a crude extract, followed by the purification of the active ingredient. We have progressed the search for new antibiotics and employ elegant new approaches to produce and detect antibiotic activity, combined with high throughput screening methods that permit us to find the’ needle in a haystack’ of tens of thousands of compounds.

Erythromycin,  wiki.  

  Macrolides are a broad-spectrum, bacteriostatic class of antimicrobials that bind to the large 50S subunit of the ribosome and block the translocation step during protein synthesis. Erythromycin was discovered by an Eli Lilly researcher in the late 1940s, who discovered this antibiotic from a Streptomyces isolate recovered from a soil sample.  This polyketide class of antimicrobials also has other important biological activities, including antifungal, antiprotozoal and even anticancer activity. Macrolides have complex structures and are synthesized by type I polyketide syntheses in a modular fashion. Rationally designing these enzymes for novel biosynthetic projects has proved challenging. Rather than engineer these complex modular enzymes to produce new macrolides, the work described here uses a biosensor strategy to detect macrolides with a tailored substrate specificity and increased sensitivity.

  The MphR protein is a TetR repressor protein that represses the expression of a macrolide phosphotransferase resistance protein. In general, the TetR family are transcriptional repressors that bind and repress target promoters, but in the presence of a chemical effector, allow for activation of the target genes. This is a common regulatory mechanism used by bacteria to sense changing environmental conditions and then express appropriate genes to cope with the change.

  In the presence of the macrolide erythromycin, MphR binds the antibiotic, causing MphR to fall off the target promoter and induce expression of the resistance gene. In this system, MphR is the only known bacterial protein that binds to and therefore ‘senses’ the presence of macrolide antibiotics in the cytoplasm. MphR is “de-repressed” in the presence of several natural and semi-synthetic macrolides, including erythromycin and others. Note, resistance to macrolides can also occur through other resistance mechanisms, including efflux pumps or by mutations or modification in the small ribosome subunit.

  Rather than engineer and adjust the macrolide biosynthesis enzymes and pathways to create new novel macrolides, the authors attempted to engineer MphR so that it recognized a different range of macrolides, or better differentiated natural and synthetic compounds, and increased its sensitivity in order to create more effective biosensors.

  The biosensor system was previously described and uses plasmid-based system in E. coli. The plasmid encodes both MphR (biosensor) and the gfp reporter under the control of an MphR- regulated promoter. In the presence of erythromycin, the E. coli biosensor now produces green fluorescence from turning on the gfp reporter. As a proof of concept, error prone PCR and multi-site saturation mutagenesis were performed on MphR, which led to variants of MphR that were up to 10-fold more sensitive to its’ natural ligand, erythromycin. Mutations were recovered in the coding sequence, as well as in the non-coding ribosome binding site (RBS). Utilizing the 3D structure of MphR, mutations were targeted to key amino acids in the repressor that are involved in recognizing macrolides.

 It would be ideal to have an MphR biosensor that recognizes a specific macrolide structure within a complex mixture. Given that MphR has a relatively broad substrate specificity, the goal was to mutagenize MphR and select for variants with a narrowed specificity. The first attempt was to try and find mutants that could differentiate natural from semi-symthetic macrolide structures. After the error prone PCR libraries were constructed and introduced back into E. coli, FACS sorting was  used to screen for constitutive gfp clones, which were excluded, and then erythromycin-responsive gfp expressing clones, followed by determining the kinetics and sensitivity levels. Randomly generated mutants were reconstructed with targeted mutations, which confirmed that MphR variants could now respond to a narrow range of macrolides. The amino acid mutations were mapped to the protein structure, but it wasn’t immediately obvious some of these changes led to a narrow specificity.

The next aim was to engineer MphR variants that could now recognize macrolides that the wild type does not, despite its broad substrate specificity. Several macrolides are produced by Streptomyces venezuelae, such as pikromcyin. The error prone PCR-generated libraries of MphR variants, were screened for those that could now detect pikromcyin. Resulting from this FACS-based screen, a biosensor strain  grown in the presence of pikromycin that expressed a single amino acid variant of MphR, was 123 times more sensitive than the wild type protein.

 Screening for new macrolide structures using conventional approaches and mass spectrometry is very complex, time consuming and costly. The ultimate goal of this project would be to use the relatively simple and high throughput MphR-based biosensor to detect a new macrolide structure.  As a proof of concept, the culture supernatants from a bacterial strain that only produces erythromycin A and not the intermediate B and C products, were recovered and added to the macrolide biosensors. The engineered MphR biosensor was 5 times more sensitive to detect erythromycin A directly from bacterial supernatants that required no purification or concentration.

While the research described here 1 highlights the potential to engineer macrolide biosensors for detecting new substrates with greater sensitivity, others have used a similar approach with the wild type MphR biosensor to detect macrolides from Streptomyces supernatants 2. It is impressive that single or double amino acid mutants can be recovered that are capable of detecting very subtle differences between macrolide structures.

These kind of projects laid the groundwork for future macrolide discovery, relying on the elegant macrolide sensing mechanisms of transcriptional repressor proteins. Biosensors could be used to detect new antibiotics in large collections of semi-synthetic drug libraries or natural products. They are also useful in screening the complex soup of compounds within culture supernatants from other macrolide producing strains, possibly with engineered macrolide biosynthesis pathways, or by screening the culture supernatants from metagenomic libraries. In the latter approach, environmental DNA from any soil sample is cloned and expressed in a lab friendly bacterial host. A library of 1000s of isolates can effectively express all the DNA in a given soil sample, which overcomes our current ability to grow the vast majority of microbes in soil and in most environments. It therefore allows us to access and screen the diverse biosynthetic potential from microbes that are uncultivable. Many new antibiotics will undoubtedly be discovered from this “microbial dark matter“.

References.

1.  Development of Transcription Factor-Based Designer Macrolide Biosensors for Metabolic Engineering and Synthetic Biology. Kasey CM, Zerrad M, Li Y, Cropp TA, Williams GJ ACS Synth Biol. 2017 Oct 12. doi: 10.1021/acssynbio.7b00287. [Epub ahead of print] PMID:28950701

2.  Biosensor-guided screening for macrolides. Möhrle V, Stadler M, Eberz G. Anal Bioanal Chem. 2007 Jul;388(5-6):1117-25. Epub 2007 May 12. PMID: 17497142

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Synthetic biology reporter of bacterial growth rate during an infection

Graphical abstract, Open Access

  The hallmark of chronic infections is the difficulty in treating and curing infection with antibiotics. Biofilms are often the cause of chronic infections,  due to their increased antibiotic tolerance and immune protective, aggregate structure. The antibiotic tolerance of biofilms is attributed mainly to the inability of the antibiotic to penetrate the biofilm, or the slow growth rate of bacteria in a biofilm. A recent study using a synthetic biology approach to examine the bacterial growth rate during an infection has provided some interesting results that challenge the current thinking regarding bacterial growth rates during infections.

  In a brief report published in Cell, Host and Microbe, the team employs an engineered E. coli strain as a reporter for the bacterial growth rate during an infection model that mimics an orthopaedic device implanted into a bone that becomes infected. It is common during various medical procedures for the implant device or catheter to become colonized, and due to the inability to treat this infection, the device needs to be removed and reinserted.

Toggle switch = trigger and reporter elements

  A toggle switch is an example of a synthetic biological circuit engineered in a cell to provide a reproducible and tuneable mechanism to turn on and off specific behaviours or reporters.  In this case, the toggle switch is derepression of the TetR repressor upon exposure to the anhydrotetracycline (ATH) inducer, leading to induction of the Cro repressor. The Cro repressor now represses the cl regulator, both derived from the lambda cl/cro system, which ultimately leads to induction of the lacZ reporter. In the baseline state,  lacZ is repressed, and the colony is white in the classic blue/white screen. During active growth in log phase, after 4 hrs of inducer treatment, all colonies switch to the lacZ expression state, producing blue colonies. The reporter works well in recapitulating the active growth state of log phase cultures, and the frequency of lacZ positive colonies decreases in non-growing or slow growth rates, such as the entry into stationary phase. Even with repression of the cl regulator, active cell division is still needed to dilute the active cl protein at the time of adding the tetracycline inducer.  Therefore,  the lacZ positive phenotype only occurs in actively dividing cells, but slow-growing cells will remain in the lacZ negative state.

  The antibiotic levofloxacin, which kills actively dividing cells best, caused a decrease in cell number, and a decrease in the lacZ positive phenotype. Conversely, it does not kill stationary phase cells very well, and these cells had the lowest  lacZ expression, all of which demonstrates further that the toggle switch is a reliable indicator of growth states.

Are bacteria in a slow growth, antibiotic tolerant state during an infection?

  During chronic infections, it is proposed that treatment fails because it cannot accumulate to high enough concentrations in biofilms, or that bacteria are not actively growing, and therefore tolerant to antibiotic exposure. To test the latter of these two hypotheses, the E. coli reporter was used in this device-associated infection model, rather than Staphylococcus, the more typical cause of orthopaedic implant-related infections. The E. coli infection was maintained for 6 weeks, and after 4 days, about half of the bacterial cells in the infection are actively dividing, and half are not dividing. This was one of the first interesting results, which suggests that cells causing this infection do not mimic a stationary phase culture,  a uniformly non-growing population. Then levofloxacin was used to treat the infection, and although bacterial load decreases compared to untreated  infections (~1 log), it was predicted that the population of actively dividing cells would decrease. However, the opposite was shown, where the infection was enriched for actively dividing cells, which directly contrasts the killing data from in vitro experiments.

  Were bacteria in the infection developing resistance? A few hundred isolates were recovered from the infection and none demonstrated any in vitro levofloxacin resistance. The authors concluded that the presence of non-dividing bacteria at the infection site does not lead to an antibiotic-tolerant infection. This conclusion directly contrasts what we know about the development of resistance under laboratory conditions, and highlights the utility of this kind of growth rate reporter during an actual infection.  When the amount of antibiotic was increased, the infection could be cured, indicating that the non-dividing bacteria either begin to divide at some point, or an increase in concentration is enough to kill. The authors argue that either interpretation supports the unexpected observation that during infection, there are mixed growth rates of actively dividing and non-growing states, and that antibiotic resistance does not necessarily arise from the non-growing bacteria.

  While the authors did not test the other tolerance hypothesis, that bacterial biofilms are the cause of chronic infections, it is assumed that biofilms formed on the inoculated pins. However, no studies were performed to confirm biofilm growth. Even in the presence of biofilms, these infections were cured with higher dose treatments, arguing perhaps that this was not necessarily a biofilm infection. This growth rate reporter would be interesting to examine under biofilm forming conditions, to test some of the underlying assumptions about heterogeneous growth rates in biofilms.

  This straightforward, synthetic biology approach to design a reporter of the bacterial growth rate during an infection will be a useful tool in future antibiotic discovery research. Examining the bacterial response to antibiotics in a host system is ultimately more valuable than studying bacteria growing in a tube of broth.