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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

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   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.

 

 

 

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CRISPR Antimicrobials: A bacterial defense strategy turned on itself

Phage introduction of CRISPR-Cas9, DNA break and eventual cell killing.

Phage introduction of CRISPR-Cas9, DNA break and eventual cell killing.

Bacteria are innovators. Life as a bacterium means lethal and frequent chemical assaults from antibiotic exposure and physical assault from the most abundant “biological entities” living on earth, bacteriophages. The myriad of resistance mechanisms used in response to these threats illustrates the creativity of bacterial survival. The CRISPR-Cas system is one shining example of how microbes resist killing by viruses. Bacteria recognize foreign DNA injected from viruses and degrade it, along with keep a record of all viral DNA sequences from previous infections, as a kind of ‘memory’ for the bacterial immune system.

 Components of a CRISPR system.

Encoded on the bacterial chromosome of numerous species, the CRISPR immune system has 3 main components:

  • An enzyme that cuts double stranded DNA (Cas gene)
  • A short RNA guide (tracRNA)
  • CRISPR array of “clustered regularly interspaced short palindromic repeat” sequences separated with “spacer” regions that contain the target DNA

The original function of CRISPR/Cas system is to degrade incoming plasmid or viral DNA, but it has been developed as a powerful genome engineering tool. It is easy and cheap to employ this pioneering system towards cutting any desired target DNA, which explains its widespread and rapid usage in many academic research labs. The CRISPR technology permits DNA cutting, editing, introducing mutations, deletions or insertions, with unprecedented precision. There can be “off-target”mutations that arise and this is an area where the technology needs refining to limit the creation of potentially harmful DNA changes.

Game changing applications of CRISPR and the patent race to own it.

The list of potential applications is growing and underscores its transformative potential. CRISPR may give the field of gene therapy a boost, with the ability to correct numerous human genetic diseases including blindness, Duchenne muscular dystrophy, Cystic Fibrosis, sickle cell anemia and others. CRISPR gene drives are being developed to engineer sterile mosquitoes to help eliminate vector-borne diseases like malaria, dengue fever, West Nile and Zika. Plants could be edited for resistance to drought and pathogens, which can proceed without the addition of any foreign genes, but rather editing existing genes. This approach to not introduce foreign genes offers a solution to the problems associated with GM organisms. The other huge opportunity is to engineer T cells to combat HIV infection or to treat cancer. Since HIV infects T cells, it is proposed to engineer T cells to either resist HIV infection, or to redirect HIV immune responses. As part of the oversimplified “cancer moonshot”, CRISPR is also being considered for use in treating cancer.

Despite the many hurdles faced to obtain these diverse goals, it is no surprise that there is a mad rush to patent the CRISPR technology and researchers at the Broad Institute (MIT) (Feng Zhang) and the University of California Berkeley (Dr Jennifer Doudna – along with Emmanuelle Charpentier) are engaged in a combative patent dispute. The value of the technology is difficult to estimate but the impact will be comparable to the invention of PCR by Kary Mullis, and will also likely lead to a Nobel prize. Professor, open science advocate, and co-founder of the Public Library of Science (PLOS) journals, Michael Eiesen, has been refreshingly honest and critical of the tactics used in the CRISPR patent debate. He even went so far as to propose that neither group should be able to patent a technology that was government funded research, and therefore ultimately owned by the tax-paying citizens.

Novel CRISPR antimicrobial treatment that does not involve antibiotics.

Widespread antibiotic resistance is an increasing, global problem due to the excessive use of antibiotics in hospitals and more importantly, in agriculture. The arms race between microbes and antibiotics is a losing battle for antibiotic discoverers. As soon as a new antibiotic is discovered and employed, there is often the immediate discovery of clinical resistant isolates in response to treatment. It has also been shown than antibiotic resistance even precedes the clinical usage of antibiotics, as bacteria need to defend against antimicrobial attack our in the real world. Resistance is out there.

As an alternative to continuing our search for new antibiotics (which is ongoing in new and exciting ways), scientists are developing new approaches to kill microbes and treat bacterial infections. The CRISPR-based antimicrobial approach described here is one such alternative approach to killing microbes without antibiotics. A bacterial defense strategy against viruses is being turned on itself.

Early work showed that the CRISPR-cas system can be directed to cleave DNA sequences in the bacterial genome, which led to irreparable damage and bacterial death (1). Recent studies have moved this concept further by developing a system to deliver cytotoxic, CRISPR-Cas9 systems to microbes. Phagemids are plasmids derived from phage genomes, viruses that specifically target bacteria. The phagemids have been engineered to express all the components of a CRISPR system, in addition to the information required to package the phagemid DNA into a phage particle. Bacteriophages are experts at landing on the outer bacterial surface, injecting its DNA into the bacterium, taking over the bacterial machinery and producing hundreds of viruses. The factory ultimately bursts, leading to death and release of an army of viruses to maintain the attack on neighbouring cells.

CRISPR Antimicrobials.

In back-to-back papers in Nature Biotechnology, two groups have published remarkably similar reports on the use of the Streptococcus pyogenes type II Cas 9 protein as a killing enzyme (2,3). The basic concept was to develop a CRISPR-mediated approach that can cause specific bacterial death. The element of specificity is particularly interesting in light of our increasing awareness of the dangers of broad-spectrum killing of healthy and helpful commensal, microbiome members. Drugs that cause little collateral damage is a new principal of antimicrobial design, which is a large shift from conventional thinking that broad-spectrum is best. Also, these papers explored the use of CRISPR to limit the transfer of antibiotic resistance genes.

Both approaches used phage-derived vectors to encode the CRISPR components of the the Cas9 enzyme, the short RNA guide (tracRNA) and a CRISPR array containing the desired cut sites within the spacer regions. When directing cleavage of kanamycin or ampicillin resistance genes, both groups could successfully show a loss of antibiotic resistance in the target organism, after delivery of the phagemid-encoded CRISPR systems. The same principal was applied to monitoring the efficiency of transformation, by measuring the number of viable cells in strains containing the target genes compared to strains that lacked the target. There was a 1000-fold decrease viability (and in the efficiency of conjugation) in hosts that contained the targeted cleavage site, indicating that successful DNA disruption and killing. Interestingly, there was never a complete killing of the target population in these experimental conditions.

To further examine the feasibility of this approach, various infection models were used to determine the capacity of CRISPR killing when applied to pathogens during an infection. In both a skin infection and the Galleria larval infection models, the pathogen numbers could be reduced by CRISPR-Cas antimicrobials, which permitted skin healing and survival of the larvae. The final experiments were to test the system when exposed to simple, mixed communities, in order to show specific pathogen targeting and non-targeting of commensals.

Although phages are being revisited for use as an alternative to antibiotics, engineered phages with CRISPR capacity have perhaps the added value of narrow spectrum killing. The biggest obstacle to improving these transmissable, programmed antimicrobials is to improve the method of delivery. It is not unreasonable to image a scenario where due to the low costs of DNA synthesis, custom, specific CRISPR-Cas-phage particles could be synthesized within a few days, and used as needed to treat any possible infectious disease.

References

1 . CRISPR design for next-generation antimicrobials. Beisel CL, Gomaa AA, Barrangou R. Genome Biol. 2014 Nov 8;15(11):516. doi: 10.1186/s13059-014-0516-x.

2.  Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW, Duportet X, Fischetti VA, Marraffini LA.                  Nat Biotechnol. 2014 Nov;32(11):1146-50. doi: 10.1038/nbt.3043..

3. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases.Citorik RJ, Mimee M, Lu TK. Nat Biotechnol. 2014 Nov;32(11):1141-5. doi: 10.1038/nbt.3011.