Biologists Swimming in a Mathematical Ocean: Modeling Gene Expression

Math is everywhere in today's society, and advanced mathematics have fueled the engineering efforts which underly almost all technology. In the past, biology may have been a refuge for scientists that were math-adverse. This is not longer true; instead, biologists should openly embrace higher mathematics and employ these techniques in a variety of ways. Perhaps the most relevant application to the synthetic biologists is for developing mathematical models of gene expression and cellular behavior.

Biologists make models all the time: any hypothesis, for example, is a reflection of some model held by the scientist which describes how a certain system works. Many times, this model may be implicit in a description for a biological process. More powerful models are explicitly formulated using the language of mathematics. Leveraging mathematics allows quantitative predictions of a cell's behavior or gene expression pattern to be made with great precision. Engineering the metabolism of a cell by modulating expression of many different genes requires sophisticated and (as much as possible) accurate models.

How then, is the a biologist with limited mathematical training to understand models of gene expression, let alone craft ones themselves? Diving into advanced literature on the subject can lead one to practically drown in a sea of differential equations, boolean logic, and matrices. I must even admit to having trouble staying afloat at first: my formal mathematics education stopped at 'Calculus II for Bio Majors" at Cook College, Rutgers (a course which covered matrices but not differential equations). Fortunately, my brother is quiet adept at this sort of math and has provided me with some excellent tutorials.

Even if you don't have a skilled mathematician in the family, there are resources to help you learn gene expression modeling (or any modeling of a biological system). Below I feature links to several of these resources. The resource which I have found to have the best blend of exhaustive yet accessible explanation is a thesis from 2010 written by Hosam Abdel Aleem. I'm sure there are other articles and publications out there that are as good, if not even better, than Dr. Aleem's work, but his is a delightful read and quiet approachable (it's also freely available).

An Algebraic Approach to Modelling the Regulation of Gene Expression

https://www.escholar.manchester.ac.uk/api/datastream?publicationPid=uk-ac-man-scw:108029&datastreamId=FULL-TEXT.PDF

This thesis throughly explains the philosophy behind modeling, as well as how to construct mathematical models of gene expression in detail. The author covers not only modeling by differential equations (continuous), but also boolean models (binary) and his own methods (discrete but with multiple values). I highly recommend this read to anybody that is interested in modeling gene expression but doesn't know where to start.

In future posts, I will provide more detail of my own experience learning some of this material, including some step by step examples of how to create a model, solve or analyze the associated equations, and calculate the results. Until that time, I'd like to wish all of my readers a very Happy Thanksgiving!

For more links to resources for mathematical modeling of gene expression, select 'Read More'. Did I miss an important resource, or do you have a favorite method for modeling transcription and translation? Please share your thoughts below as a comment!

Bacteria learn how to take a pulse: programming microbes to convert digital light signals to analog gene expression.

What do telecommunications, power delivery, and your audio system* all have in common? For starters, their underlying electrical systems use digital pulses, alternating ON and OFF states over time. These pulse patterns and the way they change, known as pulse width modulation or PWM, can encode and transmit information. Now, research from a team of British and American scientists have made a surprising addition to the list of systems that can decode information in pulse widths: Escherichica coli (E. coli), a bacteria normally found in our gut. 

In an article recently published in the Journal of Molecular Biology, the research team describes genetic modifications to E. coli that enable it to read the pulse width modulation of alternating green and red lights. The gene expression of a reporter protein represented an analog output in response to this digital pattern of light color. In essence, scientists have been able to replicate in bacteria a process important in electrical engineering.

The creation of a system in E. coli capable of decoding PWM information is a significant step forward in the field of synthetic biology. This field, which sits at the intersection between biology and engineering, attempts to design artificial sensing and gene regulatory networks in bacteria. Perhaps most exciting, however, is the potential to use microbes like this one described in this study as an interface between digital signals from machines and the biological activity of cells.

For more detail and commentary about this study, please select 'Read More'. Which ways do you think PWM sensing in E. coli should be used? How would you continue this study? Comments are welcome below!

*not all audio systems utilize PWM, if I am not mistaken

Impressions from iGEM WCJ 2013

The International Genetically Engineered Machine competition, or iGEM, is an annual event in which teams of undergraduates compete to develop the best synthetic biology project. Their results are presented, and prizes awarded, at conference events dubbed iGEM jamborees. 

The iGEM 2013 event featured hundreds of teams. After qualifying at regional jamborees in North America, Asia, Latin America, and Europe, many teams converged at the Stata Center in MIT between November 1st and November 4th for the World Championship Jamboree.

I attended (as a volunteer) the Championship Jamboree this year. It was a great experience, and is something I recommend to anybody that is interested in the field of synthetic biology but cannot themselves join an iGEM team. In the rest of this post, I will share my impressions of the Jamboree and highlight some of my favorite teams and projects from this year.

If you are interested in learning more about all of this year's projects, and see their presentations from the World Championship Jamboree, you can visit the iGEM 2013 livestream channel for archived videos. HD videos and other files (including photos) should be or will be available on the main iGEM website. (For example, the finalists and medalists presentations are available, both video and poster files).

What do you think about iGEM, and which team or project was your favorite? Please share your thoughts in a comment below!

Note: I do not own, or claim any rights to, the official iGEM logo shown above; it was taken from igem.org

Paradigms in Synthetic Biology Part II: Semiotics and Economics

In the first post in this series (See PartI: Analog to Digital), I point out how synthetic biology will require researchers and engineers to remain flexible with regards to the conceptual framework they use. Indeed, entirely new concepts may be necessary for this field, which is the intersection of engineering and molecular biology (which is not as fully understood as other sciences which have been grappled by engineers). Treating a genetic network or transcription circuit as components that follow digital logic has it's advantages, but also it's limitations (mostly due to the real physical nature of transcription and molecular biology occurring in the cell). Other paradigms, such as analog computing, can also be applied and sometimes inform more powerful designs in synthetic biology.

Beyond different types of computing, it may be helpful to borrow concepts and framework from a wide variety of fields. Below, I discuss several other fields and concepts which may find use in synthetic biology. One prism through which molecular biology can be viewed is that of semiotics; the study of information and signs. Even the field of economics may have concepts which biologists can find useful. After all, economists need to model complex studies, which many different actors / agents, with a great deal of uncertainty.

How do you see Synthetic Biology? Is there a certain paradigm or field that you think Synthetic Biologists can borrow useful concepts from? Does all of this dense, abstract blather simply amount to hot air? Feel free to share by leaving a comment below.

I recently attended the iGEM 2013 world championship jamboree. Stay tuned for a future post reflecting on my experience!

Synthetic Biology Paradigms, Part I

Is any one conceptual framework is sufficient for advancing the field of Synthetic Biology? Will a new paradigm be needed eventually? We can apply a diverse set of paradigms to think about how biology works: Analog devices (clocks, gears, etc) or digital circuits (processor), biochemical pathways where the flow of substrates can resemble the flow of fluids through a complex network of pipes, or even complex mathematical constructs (like those developed by Wolfram) can all be used to represent or draw parallels with different aspects of molecular biology.

Synthetic Biology is sometimes described as molecular biology with an engineering perspective. Indeed, many leading researchers in the field have backgrounds in electrical engineering or some related discipline. Much of the earlier work done by pioneering synthetic biologists has been to formulate biological phenomenon in more familiar engineering terms. These efforts has enabled engineers to leverage their expertise during the design of complex artificial genetic networks and cell behaviors. Are the promises of synthetic biology within our grasp, limited only by a need for the import of more engineering knowledge?

Despite the success of an engineering paradigm in synthetic biology, I believe that harnessing the full potential of this field will require new concepts and perspective. Synthetic Biology can and should grow from conceptual frameworks borrowed from engineering disciplines, but also must not be constrained by them. 

Incorporating ideas and concepts from fields other than the digital logic of electrical engineering can only serve to strengthen the efforts of a synthetic biologist. In this series of articles, I will detail how perspectives from Molecular Biologists, as well as Economists and other Engineers (particularly those that deal with Analog systems) may prove to be an invaluable part of the synthetic biologist's conceptual toolkit. The remainder of this article (which you can view by selecting 'Read More') details a recent advance in synthetic biology: the design of an analog genetic circuit by the Sarpeshkar Laboratory Group. I also discuss how Synthetic Biology can mimic and learn from nature, and why keeping an open mind and a flexible vocabulary may be important.

Paper of the Week at JBC

A stalled ribosome (Dark Blue) is rescued through translation of the tmRNA ORF (Magenta). Proper positioning of this region of tmRNA in the A-site is achieved by the C-terminal tail of SmpB (Yellow) which is connected to the body of the SmpB protein (Orange) by a flexible glycine residue (Yellow gymnast). Generated with PDB files 4ABR, 4ABS, and 3J18.

Although I usually don't use this blog to herald my own accomplishments (i.e. I didn't mention successfully earning my doctorate earlier in the year), I cannot hide my excitement that my first author article, "Active and Accurate trans-Translation Requires Distinct Determinants in the C-terminal Tail of SmpB Protein and the mRNA-like Domain of Transfer Messenger RNA (tmRNA)", has been selected for paper of the week.

As a paper of the week (an honor bestowed on less than 5% of all JBC articles, I am told), there is a neat summary of the article on the JBC site. The summary / synopsis piece is entitled "How Two Molecules Keep Ribosomes Moving". Along with this preview, there is also short profile about me.

Unfortunately, reading the actual, finished article requires a subscription to the Journal of Biological Chemistry. A word of caution to interested readers: it is written for an expert scientific audience (as almost all research articles for scientific journals are). However, it is possible to view the earlier, 'online' version of the article; in addition, the JBC capsule provides a bite-sized summary of the work.

The below is copied from the article page at the JBC website. Although I wrote most of it, I claim no copyright. You can find the original text at http://www.jbc.org/content/early/2013/08/28/jbc.M113.503896

Capsule

Background: tmRNA and small protein B (SmpB) rescue stalled ribosomes through a template switching mechanism.

Results: Changes to the SmpB hinge, SmpB C-terminus, or tmRNA ORF affect ribosome rescue activity and accuracy.

Conclusion: Proper positioning of SmpB and tmRNA make distinct and supplementary contributions to ribosome rescue.

Significance: Template switching requires concerted action of distinct SmpB, tmRNA, and ribosomal determinants.

A Blogroll for Synthetic Biology

The emerging discipline of synthetic biology holds tremendous potential for both basic research and for delivering powerful, novel solutions to real world problems (both in medicine and in other industries). This field has grown from a handful of researchers at Princeton, MIT, Harvard, and Universities in California (with pioneers such as Dr. Drew Endy, Dr. George Church and Dr. Ron Weiss leading the way) to include laboratories all over the world. It is my belief that this field will represent the future of biology and will be a vast and critical part of the economy in the years to come.

There are several great resources on the web to learn about synthetic biology and keep abreast of the latest developments. In addition to sites such as syntheticbiology.org, I present below a blogroll of great sites to visit for anybody interested in this amazing field. (Image above is modified from synthetic biology.org; I claim no rights to this image). Of course, I haven't included my own blog, even thought I already have several posts about synthetic biology (for example, my article about DNA synthesis)

My Top Three:

Oscillator

http://blogs.scientificamerican.com/oscillator/

Part of the scientific american family of blogs, Oscillator mostly focuses on Synthetic Biology and is a great resource for interesting articles and news about the field. I especially like some of the perspectives given by the main author Chistina Agapakis (whom you can follow on Twitter).

Peccoud Lab Journal Club Page
http://peccoud.vbi.vt.edu

http://peccoud.vbi.vt.edu/category/journal-club/

For the aficionado or seasoned expert, this is a great resource where recent papers about, or related to, synthetic biology are discussed. This is a great feature to have on a laboratory group website which deserves emulation by other scientists.

Dreamer Biologist's Blog

http://dreamerbiologist.wordpress.com/blog-2/

This blog, maintained by an undergraduate with a passion for biology (and synthetic biology in particular), features articles on a range of topics. He maintains a separate section with material solely devoted to synthetic biology (http://dreamerbiologist.wordpress.com/synbio/), and the title of the biology I think captures the essence of what this field is: a call for biologists to dream up novel solutions to real world problems, and imagine new technologies based upon the power of life.

Please continue reading the article to get my full list of synthetic biology blogs and sites (Click 'Read More'). They are really worth a read! 

Do you know of a blog or site that I have missed? Please share the link and a description below by leaving a comment.

E. coli Biosensors: Going for the Gold

All that glitters is not gold, and the shine of most modern gold deposits are hidden underneath layers of dirt, soil and sand. Finding these deposits usually requires expensive and time-consuming chemical analysis of soil samples. Recently, an international team of reseachers met this challenge of gold exploration and prospecting by turning a common gut microbe, Escherichia coli (E. coli), into a miniature gold detection device.

In a recent paper published in PLoS One, researchers from the University of Nebraska and their collaborators in Australia detail how they have genetically modified E. coli to act as a gold biosensor by borrowing the golTSB genes from a closely related microbe, Salmonella typhimurium. By pairing these gold recognition genes to a known enzymatic activity, researchers can detect and quantify small amounts of gold by simply measuring a change in the color of the bacteria-containing solution. The gold detection limit for this biosensor is on par with that of the chemical analysis currently used in the industry, which is slower and involves much more expensive instruments.

These proof-of-concept studies, which were partly funded by both Newmont Exploration Proprietary Limited and Barrick Gold of Australia Limited, are the latest step towards the development of a quick, accurate and specific biosensor that will make examining potential gold mining sites easier and faster. The authors of the study demonstrate that their biosensor can be used to determine the concentration of gold in a soil sample or a sample containing multiple metals. This is an improvement over earlier research of prototype biosensors, which only demonstrated detection in relatively pure samples.

For more detail and commentary about this study, please select 'Read More'. Do you think cell based biosensors will revolutionize gold exploration? Comments are welcome below!

Nanopore Sequencing: Towards Reading and Writing?

Nanopore sequencing is an emerging technology that promises fast, easy and affordable way to 'read' the bases in DNA. While researchers are seeking the $1000 genome, nanopore sequencing (once refined) may be able to deliver under budget and on a time scale of minutes, not hours or days.

Other bloggers and science writers (at BiteSize Bio, among others) have done a great job covering this technology (several of which I complied at the end of this article in a short 'webibliography', or bibliography of websites). Here, I would like to speculate on the use of a nanopore for the synthesis (or writing) of a DNA sequence. 

FInding a cheaper and faster way to synthesize a DNA sequence is a big challenge, and one that with a growing urgency. I've previously highlighted the importance of meeting this challenge and some current attempts at solutions. The ideal solution may currently be residing in the realm of science fiction. As I mentioned in the previous article, solutions that employ a controllable polymerase have great potential. A recent article from the Akeson laboratory (Olasagasti, 2012; PMC3711841) shows that this may be possible. Indeed, Akeson and colleages are able to electronically control both the threading of DNA through a nanopore, as well as the synthesis of the threaded DNA. 

Select 'Read More' to see the rest of the article. What are your thoughts on nanopore sequencing? Do you think that it is feasible that this technology can be adapted in some way for a next-generation DNA sequencing solution?

A Webibliography of Microbiology Blogs

There are many places on the web to read about microbiology and molecular biology. From science news breaks to commentary on research and everything in between, below are my favorite blogs and sites devoted to the field (other than my own, of course). This list is often referred to as a blogroll, although here I am calling it a webibliography (a condensed version of the phrase used by a friend and fellow blogger, Michael Goeller, at the Kenilworthian).

My Top 5 Greatest Hits (with a bullet!)

Microbe World: A terrific hub for everything microbiology. This site contains blogs and news feeds, as well as excellent podcasts such as TWiM (This Week in Microbiology). Sister podcasts include the more specific TWiV and TWiP (This Week in Virology and Parasites, respectively).

MicroBEnet: A great blog, which I have included in this greatest hits collection for the reason given in the note below. This blog focuses on various aspects of microbiology, with a particular focus on those microbes that share our dwellings (the microbiology of the built environment, as the author himself describes it.) This focus is connected with a program setup by the Sloan Foundation.

Of particular interest in the MicroBEnet blog is the Microbiology Blogroll post, which is similar to the very collection of websites you are currently reading. The blogroll post is much more comprehensive than my own list (although it lacks the CSHL blog), although it is unannotated. Instead, the most recent post for each blog is shown.

Small Things Considered: This blog, which seems to have a title that plays off of an NPR game show, is an excellent read. It is supported by the American Society for Microbiology (ASM). Here you can find microbiology news ranging from the TWiM press family, as well as reviews and commentary on primary literature (look for the research blogging icon) and everything in between. 

Research Blogging, Biology: A sort of biology blog aggregation site which lists posts from a variety of authors and topics with a common thread: they all are either review or commentary on primary research. You may have noticed the research blogging icon on other sites; all of these posts are also listed at this website. 

CSHL Lab Dish:  A news blog for Cold Spring Harbor Laboratory, a preeminent research organization (can be considered the birthplace of molecular biology). Although infrequently updated, the quality posts are worth the wait. My favorite are the SCIENCE SHORTS: brief and straightforward 5 minute talks on a particular topic, prepared for a general audience.

Select 'Read More' to see the rest of my annotated list of microbiology websites. Do you have a favorite site that I have missed? Please feel free to share it by leaving a comment below!

NusA to save the day during heat shock: commentary and review of Li, et al 2013

A review of Li, et al Escherichia coli transcription termination factor NusA: heat-induced oligomerization and chaperone activity (2013) Scientific Reports, vol 3 (2347) PMCID: PMC3731644

In previous blog posts, I have taken a look at two slightly dated articles concerning drug development against mycobacterium tuberculosis. Here I will continue my series of reviews but with a new direction. My last review provided a new perspective on a study that had already received press from others. For this review, I have chosen a much more recent paper, which has not been analyzed (to my knowledge) by another other blogger, science journalist, or microbiology enthusiast.

Why have I chosen a paper on NusA molecular biology in E. coli? Other than the fact that the article is fresh off the press (from the Nature sub-journal Scientific Reports), the claims (which are fairly well supported) the authors make are another example of how bacterial proteins often function as swiss army knifes: they have multiple functions, sometimes not revealed until environmental conditions are changed or cellular stresses are introduced. 

I will begin this review with a short summary, known as a Capsule. This style of synopsis / abstract is being pioneered by the Journal for Biological Chemistry (JBC), and I think it is a great idea for making summaries primary research literature more accessible to a general audience. One way to think of it is a shorter abstract, written not for experts but for the public (and policy makers, I suppose!). Authors of manuscripts submitted to JBC must provide a capsule statement; here, the capsule below is my own, not written by the authors of the NusA study in Scientific Reports (and not conforming to JBC's strict 60 word limit).

Capsule

Background:  NusA is a protein factor known to be involved in transcription termination and anti-termination (transcription is part of the process of turning genetic information in DNA into proteins and enzymes).

Results: Upon heat shock, NusA forms oligomers (multiple copies of the same protein factor bound together) which help prevent other proteins from aggregating.  

Conclusion: NusA contributes to the heat-shock resistance in E. coli by acting as a buffer to protein aggregation.

Significance: Describes a new role for NusA and expands the knowledge of how bacteria cope with stress; these abilities (in general) are important for many bacteria, including pathogenic bacteria that must resist stress from our immune system and medicines.

I invite readers to form their own capsule of this article, especially if you disagree with my choice of areas to emphasis.

Manuscript Highlights

1. NusA is the latest example of a multi-functional protein with latent chaperone 'buffer' activity during heat-shock. GreA, another transcription related factor, is also recent example.

2. NusA oligomerization (distinct from aggregation), mediated by the C-terminal repeat domains, is thought to be responsible for the chaperone buffer activity.

3. NusA's role in heat-shock conditions is not demonstrated under physiological conditions; experiments are done in vitro or with over-expressed and tagged NusA. This may reflect technical limitations.

Select 'Read More' to see the rest of the review

Protein Folding can be fun in more ways than one

Although this blog often features serious discussions of scientific research, I am myself a big fan of board and card games. In fact, I am an avid chess player, dedicated enough to play in (and sometimes place) in tournaments in the New York area. I even run another blog devoted to Chess and the ways in which it intersects with Science.

One feature of gameplay that attracts me is how it can be adapted for educational uses. In indirect and abstract ways Chess has taught me a multitude of lessons. Other games, such as Scrabble and Boggle (and some iPhone adaptions or derivatives) may help to improve one's vocabulary, even if just slightly. More to the point, however, I recently began to think of ways to devise some games that will make certain concepts in Microbiology and Molecular Biology easier to grasp and fun to learn.

Here I am showcasing the first of these efforts: a game about protein folding. Protein folding describes the manner in which a protein, which is a polymer (or string) of amino acids in a particular sequence, folds into a 3-dimensional shape. For a refresher on the relationship between our genetic material and a protein's shape, please refer to my crash course on the central dogma of molecular biology. The 3D configuration of a protein is extremely important in microbiology and molecular biology. A majority of the behaviors a microbe carries out are executed by proteins, and protein structure usually dictates function.

Before giving you a description of my invention, which is meant solely for edutainment, I need to point out another game that revolves around protein folding. FoldIt, designed by David Baker's laboratory group, is at once an entertaining computer game and a brilliant idea. Since it is difficult for computers to accurately determine the structure of a protein from the amino acid sequence alone, Baker uses FoldIt to enlist the help of people all across the world in this task. By taking protein structures and turning them into puzzles, FoldIt has players tweak the shapes of proteins and rewards them with points for more stable structures. Apart from actually being quite fun (especially if you are a fan of puzzle games), playing this game actually advances scientific research and protein structure prediction.

My game, which I tentatively call "Protein Power" (for lack of a better name), is different. It is a board game designed simply for fun and to teach some concepts behind protein folding. It resembles in some ways both Scrabble and primitive protein folding models (ones in which polar and hydrophobic beads were placed upon a grid). The screen shot above gives you a glimpse into the game: you place amino acid tiles upon a board and score points for connections or interactions between them: hydrophobic interactions are rewarded, salt bridges score points as well. Hopefully, through the game players will become familiar with the idea that protein folding involves hiding hydrophobic residues from water and forming energetically stable structures. At the very least, I hope they have fun playing and that the gameplay is not too derivative (I think it's actually fairly original). Unlike Foldit, it is a multiplayer, strategic board game.

Select 'Read More' to see the rules for the game and example gameplay.

Waging Warfarin against Tuberculosis: A Review of Dutton et al, 2010

A review of Dutton et al. Inhibition of bacterial disulfide bond formation by the anticoagulant warfarin. PNAS (2010) vol. 107 (1) pp. 297-301 PMCID: PMC2806739

This PNAS study from a few years ago looks at the potential antibacterial use of a popular anticoagulant, warfarin. Although my attention was drawn to this article at first through a journal club, I think that this is an interesting paper to review for several reasons. First, it is a paper published in a relatively high profile journal, and it also note worthy because it is research that has a more direct connection to clinical applications and drug development. 

This work has also been reviewed by other blogs and science writers (in a more timely fashion than I have done). For example, see a guest post in Small Things Considered blog written by two graduate students entitled "All is fair in love and warfarin". These students do a good job of summarizing the article, especially for a wider audience. A more in-depth review (and slightly more critical) entitled "Warfarin: Not Just Rat Poison" is also worth a read.

Here, I would like to add my commentary to this work, going further than the above two reviews / summaries in pointing out some flaws. The very rationale of this work is questionable, and might provide a glimpse into the misalignment of public interest and the motivation of the scientists. This review also happens to continue an unwitting trend of reviews on anti-tuberculosis drug papers, following my review of Shi et al, 2011, entitled "Grainy Westerns and Fuzzy Logic".

Manuscript Highlights

1. High doses of warfarin (and lower doses of the anticoagulant phenindione) are capable of inhibiting the growth of Mycobacterium smegmatis and tuberculosis (only warfain data for M. tb)

2. Mycobacterium VKOR, the homolog of the human target of Warfarin, is active when expressed in E. coli. The activity of this enzyme in these experiments is sensitive to Warfarin.

3. Surprisingly, inhibition of Mycobacterium VKOR by warfarin does not appear to be the reason why this drug inhibits M. tuberculosis growth. Should either VKOR or Warfarin really be the focus of drug development?

Synopsis:

In this study, Dutton and colleagues explore the use of the anticoagulant warfarin to inhibit the Mycobacterium tuberculosis VKOR enzyme (MtbVKOR). This work could lead to the development of novel, VKOR-targeting anti-tuberculosis therapeutics. This bacterial enzyme, MtbVKOR, is the ortholog of the human target of warfarin, Vitamin K epOxide Reductase (VKOR). While human VKOR is involved in the clotting pathway and bacterial VKOR is thought to be responsible for intracellular disulfide bond formation, the underlying enzymatic activity for these orthologs are very similar. In a previous study, the authors demonstrated that MtbVKOR is active in disulfide bond formation when expressed in E. coli, thereby providing a convenient system for studying the properties of this enzyme in vivo.

Utilizing this heterologous E. coli / MtbVKOR expression system, the authors demonstrate that high doses of warfarin can inhibit the activity of the Mycobacterium enzyme in this setting. This demonstrates that the bacterial and human VKOR enzymes share more than just some sequence similarity. Selection and analysis of warfarin-resistant variants of MtbVKOR supports this conclusion. Finally, the authors demonstrate that warfarin has antibacterial properties against Mycobacterium species, although they cannot identify VKOR as the target in this case.

Select 'Read More' to see the rest of the review.

Building a Better Future, One Base at a Time

Synthetic Biology holds promise to revolutionize biotechnology and many other industries. Broadly defined, it is an engineering approach that seeks to design artificial genes, gene regulatory circuits, genomes, and useful and novel cellular behaviors. This includes efforts in recent years to program bacteria to specifically invade tumor cells as well as the production of microbial derived artemisinin. In addition to these impressive feats of bio-engineering, synthetic biology also gives researchers a way to better understand the design principles by which normal cells function. After all, it is much easier to understand how a system works once you have succeed in constructing one of similar complexity.

The scale of gene construction involved in synthetic biology can be quite large. In a multi-million dollar effort by the J. Craig Venter Institute, researchers successfully synthesized an entire bacterial genome and transplanted it into a cell. While the synthetic genome contained mostly natural sequence (the genome was physically synthetic, but the information contained within was a copy of what nature and evolution has already produced), future efforts may involved rewriting of vast sections of the genome. These grand examples of synthetic biology highlight a major obstacle facing this emerging discipline: the costs involved in the actual de novo synthesis and assembly of DNA.

Over the past decade and a half, tremendous advances in DNA sequencing technology have been made. The original human genome project took years of efforts by many researchers, with a final price tag estimated at approximately 4.4 billion dollars (current dollars, adjusted for inflation) or slightly more than $1 per base pair. Due to the improved scale of sequencing coupled with several technological revolutions, scientists are rapidly approaching the coveted $1000 genome, which would be approximately a million fold improvement in terms of cost (or $0.000001 per base)

DNA synthesis technology and capabilities currently sit where DNA sequencing was a decade ago, when the completion of the final draft of the human genome was announced. As a matter of fact, synthesis technology is arguably behind even this mark. The Venter Institute's creation of Mycoplasma laboratorium (also known as Mycoplasma genitalia JCVI-1.0) is estimated to have taken a small team of researchers nearly a decade and $40 million dollars to complete. Since this genome is only half a million dollars, this represents a cost of $80 a base pair. Most smaller projects demand less intense and less iterative assembly efforts, but synthesis of even short oligos is still between $0.25 and $1.0 per base, depending on the size, scope, and quality of the synthesis efforts.

Clearly, there is room for improvement in DNA synthesis technology. This improvement will be critical to the advance of the field of synthetic biology, which hold tremendous promise for a variety of industries. Where will the improvement come from? For DNA sequencing, improved scale and efficiency of existing technology was important, but not enough for the incredible leap in capability and cost-effectiveness. Both evolution and revolution (innovative second and third generation technologies) was necessary to achieve the current level of capability. 

So far, the horizon for new, ground-breaking technology for DNA synthesis is not clear. The chemistry used in the synthesis of oligomers has remained largely unchanged for years. There are a few new approaches which may hint at how DNA synthesis can be made more reliable and affordable. Here, I'll survey a few of the newest developments. This includes the MOSIC method, the research by Dr. George Church's group at Harvard and its implementation at Gen9, as well as Cambrian Genomics and some other creative ideas. 

Select 'Read More' to learn more about emerging DNA synthesis technologies.

Grainy Westerns and Fuzzy Logic: A Review of Shi, et al 2011

A review of Shi et al. Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science (2011) vol. 333 (6049) pp. 1630-2

PMCID: PMC3502614

This study was recently published in the prestigious journal Science attempted to explain the mode of action for an important antibiotic, Pyrazinamide, used in treating tuberculosis. Due both to the high profile of this work, and the conclusion they draw regarding trans-translation, I will provide below a review of this article.

The interested reader should be aware of one important fact when approaching this article: there are different versions of it, depending on how you access it. The open access version at Pubmed is outdated; a more recent version (with updated Figure 3) is available from the journal Science itself. 

Manuscript Highlights:

1. Demonstrates binding of ribosomal protein S1 to POA (active form of Pyrazinamide drug)
2. Suggests mechanism of action for POA: binding to S1 inhibits trans-translation.
3. Quality of trans-translation related data (western blots) make the conclusion drawn by the authors questionable.
4. Alternative explanations to trans-translation rescue of stalled ribosomes in their in vitro system are not ruled out, undermining the conclusions drawn.

Synopsis:

In this study, Shi and colleagues sought to identify the target and antibiotic mechanism of the anti-tuberculosis drug Pyrazinamide. This drug is important in treatment of tuberculosis, particularly in clearing persister cells through combination with other compounds. In order to accomplish this task, the authors used affinity chromatography to capture M. tuberculosis proteins that are capable of interacting with the drug. Through this approach, ribosomal protein S1 is identified as the primary target.

Using ribosomal protein S1 (RpsA) as a lead, the authors attempt to explain the mode of action for PZA. Binding studies using isothermal titration demonstrate more conclusively that PZA is capable of binding RpsA Furthermore, a PZA resistant strain with mutant RpsA genes that cannot bind the drug is identified. Finally, the authors use an in vitro translation system to assess the effect of PZA on translation and trans-translation. The conclusion drawn from these studies is that PZA specifically inhibits trans-translation, but only in the context of M. tuberculosis ribosomes.

Perspectives on Scientific Publishing

I'd like to kick off this blog by highlighting a sentiment made by several others: the model of academic publishing is broken. It is a business model in which publishing companies receive content, copyright, editing services, and peer review services from (mostly) government paid researchers on a volunteer basis. These same companies take the content and place it behind a paywall, selling the work back to universities through subscriptions, and denying the taxpayer which funds most of these transactions (namely, the research, the researchers, and the university) any access to the results. At least, this is the often repeated compliant about the publishing industry.

There have been efforts to change from some. Digital publishers have arisen with different business models, such as open access publishing. In this model, the copyright is not always forfeited by the authors of the manuscript, and the publisher derives profit not from subscriptions but rather through fees assessed by the authors themselves. This model is logical, since there is usually great incentive for the authors to publish, enough for them to devote precious grant dollars or other funds towards these fees. After all, publishing articles in peer reviewed journals is important for maintaining funding from government agencies.

Most of these new models claim represent some improvement over traditional publishing, although each is unique. These include PeerJ, Frontiers, and PLoS One. You can read more about PeerJ in a insightful post at TechDirt.com. Likewise, the Chronicle of Higher Education ProfHacker blog has an interesting interview with the founder of Frontiers.

You can read more about the issue and make up your own mind. I think that publishing companies should adapt to new technologies, but that their business model or presence is not necessarily a problem in research. The editing and peer review services offered by these companies is important for scientific research; as technology improves, these services should be augmented in ways that more fully engage the readership of academic journals as well as the authors. In particular, I would like to see an effort to promote scientific communication from graduate students, who usually are responsible for executing the experiments and gathering the data that supports a manuscript.

By more actively engaging graduate students, and making them aware of the role of communication in the scientific community as well as their own careers, publishing companies will not only be empowering an important segment of this community but fostering good relationships with future lead researchers.

Introducing Another Bacteria Blog

Welcome to "Just me and Eubacteria", a blog about Microbiology, Molecular Biology, and related fields. 

Within the confines of these pages, I hope to share interesting facts and stories about bacteria and the people who study them with a general audience. Also featured here will be commentary on molecular biology research in bacteria, as well as some of my own observations. Although I will endeavor to make each post accessible to the widest possible audience, the jargon heavy and highly specialized nature of contemporary research complicates this task.

I can write about some of these topics with at least a modicum of authority, as I have spent the last six years studying E. coli as a graduate student in the laboratory of Dr. Wali Karzai. During this time, I have worked on various aspects of the trans-translation ribosome rescue system. Unfortunately, the secretive and competitive nature of research prohibits me from discussing the details of my unpublished work. Eventually I hope to share the details of the more interesting projects, after they have been accepted in a peer reviewed journal. Thankfully, at least some of my work, opinions, observations, and perspectives are free from such restrictions.

This blog stems from my long standing interest in synthetic biology and bacterial gene expression. I believe that increasing our understanding of how bacteria do what they do allows us to better realize the potential benefits that come from engineering them to accomplish a specific task. Alongside recent developments in the fields of synthetic biology, I will also share my interests in the study of infectious bacteria. While synthetic biology holds great promise for the future, the study and treatment of bacterial infections (especially antibiotic resistant ones) is a very serious and immediate concern.

Of course, for a topic so broad and important, there are many other interesting blogs and websites available. Some of the notable examples include the excellent website Microbe World, the ASM blog Small Things Considered, and fellow blogspot users Of Bacteria and Men and Twisted Bacteria. Rather than simply parroting these great sources, I hope to provide my own insights on the field. In addition, you can look forward to plenty of reflections on graduate education and the way science is conducted. During my fledging career as a scientist, I have witnessed many interesting projects and results, as well as a great deal of stupid ideas, cheap papers, and bad science! Most of this seems to be hidden from the public; many other sources seem intent on only celebrating science. While scientific research is a wonderful endeavor, I will attempt to present it in a more honest light.

Finally, I should note that this blog is technically an offshoot of an earlier project of mine, Science on the Squares. This blog, currently on hiatus (undergoing renovations soon returning) had a dual focus on Chess and Science. I am an avid chess player, and I found it appropriate to blog about Science alongside my favorite hobby, sometimes evening drawing analogies between these two intellectual pursuits. Because of the dual audience, I have written some great introductory articles to molecular biology for Science on the Squares. These include a introduction to the central dogma, an explanation on how data in this field is collected and (should be) analyzed, and comments about Occam's Razor. If you feel you need a refresher on any of these topics, I strongly encourage you check out those pages.

If you'd like to learn more about me, you can also check out my personal website.