All posts filed under “Genetic Engineering

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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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Kill switches prevent escape of genetically modified bacteria

bacterial kill switch

There is considerable public concern about the use of genetic engineering, particularly around the production of food. The most recent example is the FDA-approved, fast growing salmon from the company AquaBounty. This fast growing fish is grown in land-locked tanks as sterile females so that they cannot reproduce if they were to escape into the wild. Interestingly, this fish grows almost twice as fast as salmon in the wild due to the increased production of a fish growth hormone, genetically engineered to be expressed from multiple organs instead of in limiting amounts from the pituitary gland. The basic research that led to this transgenic fish was described over 20 years ago from a Canadian researcher, Dr Garth Fletcher of Memorial University.

What is less appreciated by the public are the numerous examples of genetic modification that result in medicines or other useful and less controversial products. One of the first products resulting from genetic modification was the production of synthetic insulin used to treat diabetes, which is expressed and purified from bacteria.  And the exploding field of synthetic biology has the potential of providing a bounty of new products, medicines, bioremediation strategies using engineered bacteria or biofuel production by microbes. If genetically engineered bacteria are to be employed in the field, at sites of contamination, or in production factories, there is a significant worry about their escape and the possible implications of release into the wild. To address this concern, numerous research groups are designing so-called ‘kill switch’ strategies, which will prevent the growth of these microbes in the environment. A creative new strategy was described earlier this year.

Microbiologists frequently work with ‘auxotrophs’, which are strains that cannot synthesize a specific nutrient, and therefore do not grow without its supply to the microbe. As standard auxotrophs might be capable somehow of scavenging these needed nutrients, ‘synthetic auxotrophs’ were designed that rely on the supply of unusual amino acids that are not commonly found in nature. There are several uncommon amino acids that are coded for in the triplet DNA code, but that are not found very often in proteins. This suggests the possibility to change the genetic code and introduce new codons that would introduce  non-standard amino acids into proteins.

The first stage of the kill switch technology was founded on an earlier landmark paper that used whole genome editing,  where all 321 UAG stop codons were replaced with UAA codons (ref 1). UAG was convenient to alter, since it appears rarely in the genome and only at the end of genes. These strains are referred to as genetically recoded organisms, or GROs.  Note, there were 355 off target mutations created in the process, which did not affect viability but may need to be corrected in future. It should be noted that this GRO strain was now quite resistant to infection from viruses, another advantage to this type of biocontainment strategy. The new genetic code mistranslates viral proteins that hijack the bacterial protein synthesis machinery, which ultimately disables new virus assembly leading to resistance.

Several other sophisticated, genetic manipulations were performed in order to create a growth dependence. It was now possible to reassign UAG as a dedicated sense codon for “plug and play” incorporation of rare amino acids. In order to translate the UAG codon to a  meaningful response by the protein synthesis machinery,  they expressed a specific tRNA synthetase that recognizes the UAG codon and introduces  1-4,4′-biphenylalanine (bipA) into target proteins during synthesis. As there were no longer any functional UAG codons in the genome, they selected a handful of essential proteins and redesigned these genes to encode UAG at various positions. Structure predictions were used to select the candidate essential genes to recode, which were confirmed by protein structure determinations. Mass spectrometry also confirmed the introduction of bipA into target proteins.

 Unfortunately, some synthetic auxotroph strains were still viable at low frequencies when grown in the the absence of bipA, indicating that compensatory mutations occurred which restored viability. In other words, the supposed ‘kill switch’  was not perfect in the earliest attempts. Several additional mutations were needed, including the addition of conventional auxotrophs, to really limit growth and escape of this strain in the absence of the non-standard amino acids. They ultimately identified a highly engineered strain could only survive and produce functional essential proteins when grown in the presence of the non-standard amino acid, 1-4,4′-biphenylalanine (ref 2). This ‘tour de force’ approach created a synthetic auxotroph which now can now be used as the ‘chassis’ for the future development of a particular biotechnology.

In the same issue, a second paper on the same subject was also published. They generally used a similar approach to construct synthetic auxotrophs that were dependent on the exogenous supply of unnatural analogs of phenylalanine (ref 3). The end result was the same, a strain that could not escape its amino acid dependency through mutation or the acquisition of multiple genes through horizontal gene transfer. The NIH standard for an effective kill switch is an approach where survivors do not occur with a frequency of 1 per 108 cells. Other non-auxotroph strategies have been used that meet this minimum requirement.  Environmental microbes capable of degrading environmental pollutants have been designed to express a lethal toxin when in the environment, but only once the pollutants have been degraded. This permits the deliberate use of bacteria for bioremediation of contaminated soils but limits their survival after the job is done. These bacterial suicide strategies were developed to mitigate possible risks and should permit the safe use of microbes in the environment to help resolve environmental or health crises, with the benefit greatly outweighing the risk.

Ref 1. Genomically Recoded Organisms Expand Biological Functions. 10.1126/science.1241459

Ref 2. Biocontainment of genetically modified organisms by synthetic protein design. doi:10.1038/nature14121

Ref 3.  Recoded organisms engineered to depend on synthetic amino acids. doi:10.1038/nature14095

 

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Brewing morphine from glucose in yeast

poppy field free-picture.net

The opium poppy plant Papaver somniferum is currently used as the sole commercial source of the most powerful therapeutic, prescribed opiate pain killers. There has been a long time interest in using synthetic biology to produce opiates as bioproducts, with the goal of controlling the drug, and hopefully producing cheaper, less addictive and more secure medicines. The family of molecules known as benzylisoquinolone alkaloids (BIA) include many plant products derived from tyrosine, such  as pain killers, antimicrobials, anti-cancer drugs  muscle relaxants and cough suppressants.

Within the first six months of 2015, three reports have described a complete biosynthetic pathway using yeast to convert glucose into morphine1,2,3.  Although there have been numerous previous advances in the last several years, it is now possible to engineer the entire morphine biosynthesis pathway in yeast in order to brew morphine instead of beer. While most enzymes in the pathway were derived from the poppy plant, there was also the use of other plant and bacterial enzymes. The ability to combine these enzymes in a single engineered strain still requires significantly more work, but it does bring to the forefront numerous ethical issues. The most intriguing issue revolves around how to prevent these engineered strains from being obtained or possibly reproduced by the drug cartels. Although this area of research has generated gossip of another season of the television series Breaking Bad, featuring a chemistry teacher turned methamphetamine cook and dealer, we will focus here on the synthetic biology behind these discoveries.

Glucose to (S)-Reticuline

During normal metabolism and growth of yeast on glucose, the amino acid tyrosine is naturally produced during the process of amino acid synthesis. The researchers first tackled a long standing problem in yeast, converting tyrosine to L-DOPA1. A plant enzyme was introduced that converts L-DOPA to a fluorescent pigment, whose production can be easily monitored in a biosensor assay looking for pigmented colonies, or using fluorescence to detect yellow yeast cells. It was initially expected that a high throughput screen of cDNA libraries would be necessary, but a bioinformatic analysis revealed two candidate enzymes, one from the button mushroom and another from the sugar beet, that might convert tyrosine to L-DOPA. Codon optimized versions of these enzymes were introduced and yeast strains expressing the sugar beet enzyme were shown to produce L-DOPA, which was converted to the yellow pigment. Further enzyme engineering was carried out using error-prone PCR to generate a library of mutant enzymes, which were successfully screened for variants that had improved yields of L-DOPA1.

The next step was to produce (S)-Reticuline, a central precursor in the biosynthesis of opiates. Using a combination of one bacterial and several poppy enzymes, most of which were previously identified and many shown to function in yeast, (S)-Reticuline was produced. This required the introduction of many non-yeast genes, expressed from strong promoters either from the chromosome or low-copy plasmids, and the use of unique yeast background strains that produced higher than normal amounts of key metabolites required for (S)-reticuline synthesis.

(R)-Reticuline to Morphine

Using knowledge of the morphine biosynthesis pathways in the poppy, a yeast strain was engineered to express 7 poppy plant enzymes, which led to the production of the critical intermediate thebaine, and ultimately the final products, codeine and morphine2.  Cultures were supplemented with (R)-reticuline, because at this time the enzyme that converted (S) to (R)-reticuline had not been described. This kind of pathway engineering can also be tweaked to produce other important final products derived from thebaine, including  opiod antagonists, which are used to treat opiate addiction. Similarly, thebaine is also a starting point for the chemical synthesis of oxycodone, which has a better side-effects profile, compared to morphine.

The most recent paper identified the poppy enzyme responsible for the S-to-R epimerization of reticuline3. With this finding, all enzymes required for morphine biosynthesis were identified. The pathway contains  fifteen enzymatic steps, compared to a previous synthetic biology accomplishment of expressing five enzymes needed for the synthetic production of the antimalarial compound, artemisin.

Final thoughts on brewing morphine.

While the current morphine production from the poppy plant does meet demand, synthetic biologists have made great progress in synthesizing this compound in both bacteria in yeast.  In comparison, bacteria still make higher yields of important intermediates like L-DOPA, when compared to yeasts,  but the overall  yield of opiates are still very low. However, the stage is set to produce a single yeast strain with all necessary enzymes and to optimize the production of high morphine yields from yeast fermentation reactions.

Another major advance in the field of synthetic biology is the ability to synthesize custom stretches of DNA. The relative low cost to order “DNA to go” may lead to abuses from individuals with sinister motives in attempting to create bioweapons, and possibly to attempt to construct their own morphine brewing microbial strains. DNA synthesis companies do regular screening of the sequences they produce, and it has been suggested to now search these orders for enzymes in the morphine synthesis pathway.

References

1.  An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. DeLoache WC, Russ ZN, Narcross L, Gonzales AM, Martin VJ, Dueber JE. Nat Chem Biol. 2015 Jul;11(7):465-71. doi: 10.1038/nchembio.1816. Epub 2015 May 18.

2. Synthesis of Morphinan Alkaloids in Saccharomyces cerevisiae. Fossati E, Narcross L, Ekins A, Falgueyret JP, Martin VJ. PLoS One. 2015 Apr 23;10(4):e0124459. doi: 10.1371/journal.pone.0124459.

3. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy. Farrow SC, Hagel JM, Beaudoin GA, Burns DC, Facchini PJ. Nat Chem Biol. 2015 Jul 1. doi: 10.1038/nchembio.1879

Canadian Synthetic Biology Researchers

Vincent Martin, Professor, Concordia University

Peter Facchini, Professor, University of Calgary