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.