All posts filed under “Biosensor

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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.

 

 

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Crowdfunding to support synthetic biology research projects

Image Source

   One of the most talked about and frustrating science topics on social media and during coffee break,  is the low success rates by science granting agencies. The current finding climate for basic research  in Canada, the United States, and in many places has reached an unprecedented low. The golden era of 25-30% success rates is a thing of the past, and if you are unlucky to have missed those peak years to develop your career, you are likely a mid or early career scientist facing this new reality.

   It is extremely demotivating to approach writing a grant when you have an approximate 10% chance to secure funding. Researchers are not bothering to miss out on the narrow window of summer heat by  writing grants with fall deadlines. To combat this universal challenge, many scientists are now exploring crowdfunding as an alternative approach to raise money to support basic research projects. While biotech start-ups are an obvious candidate to use crowdfunding, I am referring to early stage, idea-based projects, that may or may not have commercialization potential.

  The first crowdfunding science project in 2013 was to raise money to construct a bioluminescent “Glowing Plant”. The pitch was to use light producing plants as a natural light source and to replace electric or gas light sources. The novelty of the crowdsourcing idea to fund a science project was enough to raise almost half a million dollars, well above their intended goal of $65,000. The potential of synthetic biology  captured the imagination of funders and didn’t suffer the fear of producing a genetically modified organism.  As recently discussed in the Atlantic, the project has ended, run out of money and was not successful in creating a final product. It was not surprising, doing science is hard and they were unable to achieve their scientific goals.

   While kickstarter hosts the infrastructure for many crowdfunding projects, there are now specialized crowdfunding providers that feature research projects, including experiment.com or petridish.org.  They present projects in diverse research fields, with a wide range of budgets, aimed to support principal investigator faculty members, startups and iGEM teams. A successfully crowdfunded project was based on studying climate change in the Falkland islands, and its impact on penguins (and other animals). The project raised over $10,000, which was roughly half of the actual total budget needed to complete the project. There seems to be a trend to ask for a portion of the actual budget needed. It may make the funding more tempting, knowing that there are phases and the upfront costs are smaller. I see parallels in standard grant competitions, where we undercut the actual budget, either angling to be high value, or lacking in confidence due to low success outcomes.  Upon final analysis, this scientist indicated that crowdfunding is not easy, it requires a huge social media campaign, and in this case, a large twitter following was probably a key piece to success. In addition, there is constructing a website, making a captivating movie and being available to respond to timely questions from potential backers.

   The stage has been set and I was happy to discover that there are many really interesting  synthetic biology projects hoping to crowdfund their way to some preliminary data,  additional funding and ultimately a successful startup. I support these innovators and am excited for the day when biology-based startups grow into a mature stage of success.

My top 3 crowdfunding synthetic biology projects

Engineered skin microbes to sense glucose and produce insulin to cure diabetes

  Dr Suzuki Yo is a faculty member at the J Craig Venter Institute and raised $50,000 to for his crowdfunding project titled “Needles be Gone for Type One Diabetes Patients“. This was a particularly interesting collaborative approach involving a researcher at the infamous and private JCVI with a unique crowdfunding venue, the Diabetes Research Connection.  Normal skin microbes were recently reported to live 3-6 mm below the skin surface, and often in proximity to blood vessels. Prior to this metagenomic study, most skin microbes were thought to be closer to the surface. Using these bacterial members of the skin microbiome as the chassis for the sensor, the approach is to engineer these host friendly microbes with a glucose sensing machinery, and to couple high glucose measurements with the controlled production of insulin. In this way, skin microbes could in principle replace the function of pancreatic beta cells and effectively “cure” type I diabetes, by restoring normal regulation of blood sugar levels.  This is an exciting and unexpected approach so I am not surprised this project raised significant funds, and the upside of the pitch is eliminating the use of needles to deliver insulin. The short-term goal is to demonstrate the proof of concept by painting the glucose sensing, insulin producing microbes onto the skin surface of mice, and to monitor glucose levels in a mouse model.

Aptapaper – aptamer detection of bacterial proteins in paper assays

A University of Michigan iGEM team has been successfully funded in two crowdfunding projects with the aim of building paper-based detection assays of bacterial pathogens. The most recent project is focussed on the detection of Mycobacterium tuberculosis, which causes tuberculosis in up to 10 million people each year. The project relies on the use of aptamers, which are short stretches of DNA that bind to specific proteins. In this case, the target protein is likely a conserved M. tuberculosis protein. The binding of two separate DNA molecules to a target protein facilitates  ligation, subsequent conversion to double stranded DNA using in vitro transcription and cell free expression to produce a reporter protein. The reporter could be as simple as an enzyme that cleaves a substrate to produce a coloured product, and therefore a coloured spot on paper.  Paper-based assays are inexpensive and have long term stability at room temperature.

DNA typewriter to revolutionize data storage

Since there are more bits of data than grains of sand on the earth, we obviously need new tools to store the data! As our data increases with time, we are faced with a lack of suitable storage and resources to keep up. In principle, this is a proposed encryption system, where text can be encoded within the DNA molecule. Many are pursuing this new medium, given the small scale, stability and abundance of DNA. The program will need to convert english words into a stretch of DNA sequence, where each word fragment is named a BabbleBrick. Once the word fragments have been isolated, they will need to be assembled by a ligation method. The modularity of the system allows for combining BabbleBricks in any order, to construct any sentence of text. All the world’s data can be stored in 1 gram of DNA, in place of millions of USB sticks.

While all of these projects are exciting, the crowdfunding model comes with a few challenges. The typical incentive to seduce a financial backer is to offer some kind of reward. For more straightforward projects, the tangible project being made is often gifted to backers. There is a far greater risk of success for these projects, so it can be difficult to provide a reward. When you include a few dozen or few thousand investors in the crowdfunding, do they have any role in ownership or patents? By simply disclosing the idea, the innovator may be prevented from obtaining patent protection. As all scientists are well aware, the ability to carry out a proposed research is rich in failure, and early stage ideas like these may not reach the finish line. Others may question whether raising a few thousand dollars is worth the risk.  I find the opportunity to pursue crowdfunding an excellent funding path for the right project.