A Solution to Research in Low Resource Areas

Shaharyar Lakhani published on July 3, 2019:

While science produces many advancements across various fields, it is undeniably costly and intricate. Therefore, only people with the right equipment and facilities are able to carry out high level experiments. Dr. Sanchita Bhadra and her team in the Ellington Lab at the University of Texas at Austin are trying to resolve this problem by making products that can aid in carrying out scientific research in environments where these facilities aren’t present. This would allow researchers to conduct experiments all over the world without needing high-tech and costly equipment.

A lot of molecular biology research is dependent on enzymes, such as Taq DNA polymerase and Mu-MLV reverse transcriptase among others, which are typically purified using technically involved procedures and can only remain function at certain temperatures and breakdown otherwise. Usually, these enzymes require constant cold and controlled environments for extended storage. To cut down on production time and cost, Bhadra and her team have found an alternative solution; “We’ve developed a way to replace purified enzymes with enzymes expressed inside dried out bacteria called ‘cellular reagents’, which are cheaper and easier to make without complex instruments or processes. Cellular reagents are also easier to transport, and store due to their ability to stay at room temperature for several months whilst retaining functionality, comparable to that of purified enzymes,” says Vylan Nguyen, a researcher part of Dr. Bhadra’s team. Additionally, due to the significantly simplified production protocols, the reagents could be produced locally, thus saving time and money on distribution. Dr. Bhadra’s team used the enzyme Taq DNA Polymerase to test the success of these cellular reagents. The team expressed Taq in E.coli, which they then dehydrated. When PCR assays were performed using the original commercial pure Taq enzyme and the Taq cellular reagents, the results were comparable. Vylan states that “users, particularly students learning new protocols, would not have to be concerned with keeping enzymes at freezing temperatures throughout their experiments.” The team has subsequently developed cellular reagents for many other common molecular biology enzymes including Bst DNA polymerase, KlenTaq polymerase, Phusion polymerase, and Taq DNA ligase, and demonstrated their efficient use in common molecular biology procedures, such as reverse transcription PCR, diagnostic techniques, such as real-time PCR, and even synthetic biology applications, such as plasmid construction using Gibson assembly.

Dr. Bhadra, Nguyen, and the rest of the team are currently working on ways to make the experiments as user friendly as possible for both lower budget and low resource environments, as well as for teaching purposes. To do so, they are creating an educational kit with a lab assignment where users can set up a PCR reactions using cellular reagents. Included in the kit would be a template and corresponding primers to work as a “control” of sorts that users can practice with, along with extra cellular reagents if the users have their own templates they want to test to perhaps make their own diagnostics. Additionally, the team is trying to make the procedure require as few steps as possible, to enhance ease of use and minimize errors.

The kit is intended for use by students and instructors in low-resource areas due to its cost effectiveness and ease of use. This would especially prove beneficial in giving research opportunities to aspiring researchers in need of lab resources. For larger labs, these cell reagents can also be made in bulk, and each PCR tube could perform up to ten separate reactions. With the help of Dr. Bhadra’s team and their efforts, more people can be involved in scientific research around the world.

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The Biosecurity Battle

Shaharyar Lakhani published on June 28, 2019:

There are always two applications of scientific research; the intended application, and well... the unintended. Assuming the former is being used to advance and benefit the world, the latter, when in the wrong hands, can result in the misuse of information or technology with malicious intent. We are approaching a time where so much can be done with information as it is published and disseminated. However, if the right precautions are taken beforehand, scientific progress can continue with less risk. 

In order to build community awareness of risk and threat, the Engineering Biology Research Consortium (EBRC) held a “Malice Analysis Workshop” on Wednesday, June 19th. The workshop was sponsored by the Center for Systems and Synthetic Biology at the University of Texas at Austin. The objective of the workshop was to help researchers “identify potentially malicious applications of various projects, mitigation options, and what to do if you identify something and don’t know how to proceed.” 

Clem Fortman and Douglas Friedman led a group of about 35 graduate students and postdoctoral fellows, and taught strategies for how to mitigate information misuse. After discussing about the importance of this issue for the first hour of the workshop, the attendees were split up into small groups and using abstracts of their own research, given a rubric to analyze the potential ways for their projects to be used nefariously. While the harmful implications of scientific research are often overlooked, each team had to come up with various misuses of their information and find ways to prevent this misuse. Following a quick lunch break, a team leader from each team presented what they had thought up in front of the entire room and stood to answer questions that others had regarding biosecurity relating to their scenario. 

Jonny Riggs, an attendee of the workshop, analyzed a project involving small molecules commonly found in air pollutants that can stimulate harmful side effects through inhalation. “The risk of these pollutants is that terrorist groups could potentially weaponize them and release them into populated areas,” remarked Jonny. However, Jonny and his team had an altruistic goal, wanting to prevent any harm from reaching people due to these molecules. The team discussed the likelihood of the information being obtained, the expertise and equipment needed to synthesize and purify the molecules, and possible remediation techniques should something go wrong. Although projects like these do have the potential to be dangerous, Kasia Dinkeloo, another attendee, was “confident that the expertise, resources, and environment needed to conduct research for most of these projects would preclude them from misuse.”

Overall, the workshop was beneficial to many scientists like Kasia and Jonny. “As a plant scientist, I rarely think about how the research I do would be anything other than positive,” explains Dinkeloo. “The workshop provided a lot of useful insight on how we can be better stewards of the technology we create, by understanding that there are always two sides to research we conduct,” she further states. With an interactive approach, all the attendees were able to play out real world scenarios and benefited a great deal.

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Speeding Up Evolution Using Artificial Intelligence

Shaharyar Lakhani published on June 24, 2019:

Artificial intelligence is a powerful tool that is impacting almost every facet of our existence, including and especially science and technology. An artificial neural network, a computer system modeled on the human neural system, allows a computer to figure out what constitutes a particular subject on its own. Take a cat for instance; telling a computer that a cat is defined as an animal with whiskers, a tail, and paws is one way for the computer to identify a cat, but this way can lead to many errors since there are so many variations of cats. However, if a computer were fed multiple images of different cats, it would itself be able to come up with what constitutes a cat and continuously update its perception with every image.

The team of Raghav Shroff, Danny Diaz, and Austin Cole in the Ellington lab are applying this same concept not to cats, but to amino acids in the context of the proteins they comprise. Their project aims to use convolutional neural networks to recognize the ‘amino acid-ness’ of individual amino acids in a protein structure. This is a very different way of looking at protein structures, and draws inspiration from Wen Torng and Russ Altman (Stanford). Instead of calculating the physical or energetic properties of how each amino acid fits into a protein, they used neural nets to essentially ask protein structures on a position by position basis, “What amino acid would fit best here?” Just like the cats example, the team feeds in images and first lets the neural network learn what amino acids are already present at different spots in a protein. Most of the time, the answer matches the amino acid that’s already there, giving them confidence that their network is working correctly. However, in some cases the answer is different … and this gives the team the insight that maybe there is beneficial mutation waiting to happen! They call the project “JMBLYA” because like the dish, they mix various components together to make a final product, in this case the mutated amino acids to make a protein. Various tests of JMBLYA have now proven it to be a time machine of sorts, where it can predict the future evolution of a protein to be more stable, and allows us to intervene in the present to make the beneficial mutation.

The biotechnology implications of this insight are enormous, and the team has already used this method to alter the structure and function of three different proteins: the antibiotic resistance protein beta-lactamase, a blue fluorescent protein (shown below), and the enzyme phosphomannose isomerase. They are working towards ever more difficult protein targets of industrial and biomedical relevance, and have formed a company, AI Protein Solutions, to work with others on how to speed up evolution. Their entrepreneurial spirit has already led to negotiations on the use of their (now very blue) blue fluorescent protein, which they call “Blue Bonnet,” a nod to their Texas origins. Into the future, as computational power grows, we are confronted with the odd prospect of machines knowing more about our evolutionary futures than we do, and one of the reach goals of the team is to begin to predict the evolution of the human proteome (including mutations that abet or resist cancer) for millions of years to come.

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A New Biological and Financial Ecosystem for Plastic

Shaharyar Lakhani published on June 24, 2019:

Plastics have been in popular use for only around 50 years, yet they have managed to saturate the planet. The toxic pollutants in plastics, combined with the long time they take to degrade, have adversely affected land, water, and air pollution. Nearly 300 million tons of plastic are produced every year, with the majority going to waste and ending up in landfills or the ocean. If the current trend continues, by 2050, the total weight of plastic in the ocean will be more than that of fish!

But what if there was a way to turn this plastic into something positive? To have its pervasive presence in the ecosystem actually be a functioning part of that ecosystem? A group of researchers in the Center for Systems and Synthetic Biology at the University of Texas at Austin are working on exactly that: developing microbes that could potentially degrade plastics and convert them to other, useful products. At the heart of these efforts are enzymes released by microbes that would begin the plastics degradation process, converting inedible solids into foodstuffs for these and other bacteria. Hannah Cole, a graduate researcher in the Ellington and Alper labs is improving the stabilities of plastic-degrading enzymes, and is working closely with chemical engineers (Nate Lynd and his group) to study the effects of these enzymes on different plastics (yes, most plastics are as different from one another as are the people who use them!).

Precisely because of the diversity of plastic waste, both in terms of the type of plastic and in how it appears in the environment, parallel efforts for identifying new plastic-degrading bacteria and fungi are being carried out by Dr. Moriah Sandy and a team of undergraduates in the Freshman Research Initiative program. In particular, the team is exploring ways to use these bacteria and fungi to break down nurdles – small pellets of plastic runoff from manufacturers – gathered from the shores of Port Aransas. To cross the “nurdle hurdle” and literally eat away at the plastics problem, the Bioprospecting Stream is carrying out metagenomic analyses of the microbes present in contaminated environments and then carrying out competitions to see which bacteria is the most hungry for a given type of plastic.

Into the future, these researchers envision the development of custom bacteria that are capable of eating one or more plastics, and that can act in packs (or consortia) to eat virtually any plastic product in a landfill. Safety ‘switches’ built into the bacteria will prevent their leaving the landfill. The bacteria themselves can potentially serve as harmless feedstocks for other organisms, or can become part of a new economy in which plastics are converted to other, more biologically friendly materials – a ‘green’ version of the giant refineries of the petroleum industry.

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Changing the Genetic Code to Improve Biomedicine

Shaharyar Lakhani published on June 13, 2019:

Proteins are the workhorses of life, and are comprised of strings of amino acids, which in turn are specified by the triplet codons of the genetic code. Currently, only 20 amino acids make up the genetic code in most organisms. In order to expand the functionalities of proteins, genetic engineers have been attempting to expand the genetic code with new amino acids that offer novel chemistries.

There are already some known departures from the genetic code, including the amino acid selenocysteine, in which the sulfur atom of cysteine is replaced by the element immediately below it in the Periodic Table, selenium. Selenocysteine augments the chemistry of proteins in a number of ways, including changing their ability to catalyze reactions, shuttle electrons, and form very stable bonds. For example, while the disulfide bridges (shown below) in antibodies can be readily broken during storage or in the body, similar bonds based on selenium (diselenide bonds) are much more stable.

A researcher at the University of Texas at Austin, Dr. Ross Thyer, is working to enhance the stability of therapeutic proteins, giving them longer shelf lives and better pharmacological properties. Dr. Thyer and his team started with an engineered bacteria from the labs of George Church (Harvard) and Farren Isaacs (Yale) that lacks a single codon (normally used to ‘stop’ protein biosynthesis), and have introduced machinery that incorporates selenocysteine at this now ‘blank’ codon. By replacing the cysteine codons in a gene encoding a therapeutic antibody, an antibody can be produced which contains diselenide rather than disulfide bonds. Dr. Thyer and other researchers have started a company, GRO Biosciences, to further develop this innovation.

As an analogy, Dr. Thyer suggested that having a new amino acid is akin to having a new Lego piece; when you’re building a structure and you have 20 lego pieces to work with, you can only do so much, but adding that 21st piece opens up many new possibilities. Selenocysteine can now be added in different places to make many protein structures beyond stronger, more stable antibodies. Whether bacteria with access to a larger Lego set can build their own new structures is a question for the future.

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Anna J. Simon’s Publications

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1. Simon A.J.,* Zhou Y.,* Ramasubramani V., Glaser J., Pothukuchy A., Gollihar J., Gerberich J., Leggere J., Morrow B.R., Jung C., Glotzer S.C., Taylor D.W., Ellington A.D. (2019). Supercharging enables organized assembly of synthetic biomolecules. Nat. Chem. 11, 204-212.

2. Simon A.J., Morrow B.R., Ellington, A.D. (2018). Retroelement-based genome editing and evolution. ACS Synth. Biol., 77, 2600-2611.

3. Simon A.J., Walls-Smith L.T., Plaxco K.W. (2018). Exploiting the conformational-selection mechanism to control the response kinetics of a synthetic hydrogel. Analyst 143, 2531-2583.

4. Simon A.J., Walls-Smith L.T., Freddi M.J., Fong F.Y., Gubala V., Plaxco K.W. (2017). Simultaneous measurement of the dissolution kinetics of responsive DNA hydrogels at multiple length scales. ACS Nano 11, 461-468.

5. Ricci F., Vallée-Bélisle A., Simon, A.J., Porchetta A., Plaxco K.W. (2016). Using nature’s “tricks” to rationally tune the binding properties of biomolecular receptors. Acc. Chem. Res. 9, 1884-1892.

6. Simon A.J., Ellington A.D. (2016). Recent advances in synthetic biosafety. F1000 Reviews, 5-2118.

7. Mao X., Simon A.J., Pei H., Shi J., Li J., Huang Q., Plaxco K.W., Fan C. (2016). Activity modulation and allosteric control of a scaffolded DNAzyme using a dynamic DNA nanostructure. Chem. Sci. 7, 1200-1204.

8. Watkins H.M., Simon A.J., Sosnick T.R., Lipman E.A., Hjelm R., Plaxco K.W. (2015). A random coil negative control reproduces the discrepancy between scattering and FRET-based experiments of denatured protein dimensions. Proc. Natl. Acad. Sci. USA 112, 6631-6636

9. Simon A.J., Vallée-Bélisle A., Ricci F., Watkins H.M., Plaxco K.W. (2014). Intrinsic disorder as a generalizable strategy for the rational design of highly responsive, allosterically cooperative receptors. Proc. Natl. Acad. Sci. USA 111, 15048–15053.

10. Simon A.J., Vallée-Bélisle A., Ricci F., Watkins H.M., Plaxco K.W. (2014). Using the population-shift mechanism to rationally introduce “Hill-type” cooperativity into a normally non-cooperative receptor. Angew. Chemie 53, 9471-9475.

11. Watkins H.M., Simon A.J., Ricci F., Plaxco K.W. (2014). Packing density effects on the folding thermodynamics of a surface-tethered biopolymer: an experimental study of folding in an ultra-crowded regime. J. Am. Chem. Soc., 136, 8923-7.

12. Swasey S.M., Karimova N., Aikens C.M., Schultz D.E., Simon A.J., Gwinn E.G. (2014). Chiral electronic transitions in fluorescent silver clusters stabilized by DNA. ACS Nano 8, 6883-6892.

13. Mao X., Wei M., Zhu C., Lu J., Gao J., Simon A.J., Shi J., Huang Q., Fan C. (2013). Real time in vitro regulation of DNA methylation using a 5-fluorouical conjugated DNA-based stimuli-responsive platform. ACS Appl. Mater. Inter., 5, 2604–2609.

14. Barbero R., Carnelli L., Simon A., Kao A., d’Arminio Monforte A., Ricco M., Bianchi D., Belcher A.M. (2013). Engineered yeast for enhanced CO2 mineralization. Energy and Environmental Science, 6, 660-674.

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