Manipulating Microbiota: A Synthetic Biology Exploration of the Gut

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Microbial cells outnumber the cells of our own bodies approximately 10:1, these microbes that live on our skin and along the epithelial linings of our internal tubes make up our microbiota*, and they can have major effects on our health. Most of our microbiota are commensal organisms, living in harmony with our body, but if you suppress our immune system or greatly reduce their populations with large doses of antibiotics, and you will soon see the effects of disrupting our microbiota.

There is much interest in the microbiota that inhabit our bodies. For instance several studies have indicated that intestinal microbes can play a big part in obesity, with changes in the makeup of the microbiota being a major risk factor (1). But many of these organisms are hard to learn about—the ones that inhabit the deep folds of our gut thrive in moist, warm, anaerobic conditions with lots of specialized nutrients, conditions that are very hard to replicate in the laboratory. For that reason, we don’t know much about many of the microbes that are the most abundant within us.

The Human Microbiome Project begun in 2008 by the National Institutes of Health (2) seeks to understand human microbiota and their relationship to human health. To do this, the researchers leading the project took a metagenomic approach—using advanced DNA sequencing technologies to sequence the genomes of human microbiota and get a look at the human microbiome—without culturing the microbes.

But to truly understand their biology, and to perhaps exploit what we learn to enhance human health we need to be able to manipulate these organisms. In particular, biologists who are interested in synthetic biology would like to use these micro-organisms to monitor what is going on in our bodies, particularly our guts. What better monitor for these hard-to-access places than an organism that is already well adapted to live there? 

Continue reading “Manipulating Microbiota: A Synthetic Biology Exploration of the Gut”

All You Need is Pla (for Pneumonic Plague)

Yersinia pestis. By Mrs Robinson at bg.wikipedia (Transferred from bg.wikipedia) [Public domain], from Wikimedia Commons.
Writing about Yersinia pestis or the Black Death, has earned me a reputation among Promega Connections bloggers. I am interested in what researchers have been able to piece together about the causative agent of ancient plagues, what modern research shows about how Y. pestis spreads in the body and the continuing reservoirs in modern times, resulting in publication of eight blog posts on the subject. Understanding Y. pestis bacterium is of continuing interest to researchers. How did Yersinia pestis evolve from the humble Yersinia pseudotuberculosis, a pathogen that causes gastrointestinal distress, into a virulent pneumonic plague that is a global killer? One strategy for answering this question is to look at the genomic tree of Y. pestis and trace which strains had what characteristics. In a recent Nature Communications article, Zimbler et al. explored the role of the plasmid pPCP1 in Y. pestis evolution and the signature protease Pla it expresses. Continue reading “All You Need is Pla (for Pneumonic Plague)”

Getting the Most DNA from Your Plasmid Prep

dificult_cloningPlasmid DNA preparation is one of the fundamental techniques of molecular biology research. It goes without saying that you want to maximize your plasmid DNA yield as much as possible. Below are a few tips to help you get the most out of every Plasmid prep.

Increase Your Culture Volume

Most plasmid prep systems can process a range of volumes. For example, the PureYield™ Miniprep System can process 600μl to 3 ml of culture. Note: Exceeding the recommended culture volume can result in decreased yields because the increased biomass can lead to insufficient lysis and lysate clearing problems.

Use Optimized Culture Conditions

Media: We recommend growing cells in 1X Luria-Bertani (LB) medium. The use of rich medium, such as 2X YT, CircleGROW®, Terrific broth, or LB-Miller medium, which contains more NaCl, can significantly increase yields, provided that the biomass is within the acceptable range.

Culture incubation times: Low-density bacterial cultures yield relatively low amounts of DNA. Overgrown cultures produce suboptimal yields and excessive chromosomal DNA contamination. Do not use cultures grown longer than 18–20 hours. Continue reading “Getting the Most DNA from Your Plasmid Prep”

Designer Bacteria Detect Cancer

Every day scientists apply creative ideas to solve real-world problems. Every so often a paper comes up that highlights the creativity and elegance of this process in a powerful way. The paper “Programmable probiotics for detection of cancer in urine”, published May 27 in Science Translational Medicine, provides one great example of the application of scientific creativity to develop potential new ways for early detection of cancer.

The paper describes use of an engineered strain of E.coli to detect liver tumors in mice. The authors (Danino et al) developed a potential diagnostic assay that uses a simple oral delivery method and provides a readout from urine, all of which is made possible by some seriously complex and elegant science. Continue reading “Designer Bacteria Detect Cancer”

Your Health has a Season

Photo of pasque flowers
Pasque flowers in a northern hemisphere garden in spring.

As the seasons change so does the general state of health for many of us. The further from the equator we live, the more pronounced these effects are. For instance, did you know that blood pressure elevation for many people increases with the distance they live from the equator, an effect most pronounced during the low sunlight season (winter in the northern hemisphere)?

A report published online in Nature Communications May 12, shows evidence of changes in cellular physiology with the seasons. Todd et al. published a study entitled: “Widespread seasonal gene expression reveals annual differences in human immunity and physiology”, where they note,

“Here we find more than 4,000 protein-coding mRNAs in white blood cells and adipose tissue to have seasonal expression profiles, with inverted patterns observed between Europe and Oceania.”

Let’s Take a Look at the Research

Todd et al. looked at ethnically and geographically distinct populations, including subjects from Australia, The Gambia (Africa), Germany, the UK and Iceland. Individuals from the various studies were infants, adults with type 1 diabetes and asthmatics in the range of 18-83 years of age. The authors analyzed RNA from peripheral blood mononuclear cells and subcutaneous adipose tissue biopsies, as well as examining peripheral blood cell counts and circulating levels of proinflammatory cytokines. Continue reading “Your Health has a Season”

Animal or Plant? FISH Labeling Reveals Horizontal Transfer of Algae Gene into Sea Slug Chromosome

There are times when I ask myself why I chose a career in science. This happens on what I call “grass is greener” days. On these days I dream of other careers—like National Geographic reporter or Caribbean tour guide–which all sound way more exciting than scientist. Admittedly these alternative careers are not ones that many people have the privilege of attaining, but sometimes reality gets to take a vacation. Fortunately, science is a fast-moving, always-changing field. As much as I might occasionally dream of exotic jobs in far away locations, science always pulls me back in with something new and unexpected. Because as much as we’d like to think we know, the truth is there is so much more that we don’t.

Image from: Pelletreau KN, Weber APM, Weber KL, Rumpho ME (2014) Lipid Accumulation during the Establishment of Kleptoplasty in Elysia chlorotica. PLoS ONE 9(5): e97477. doi:10.1371/journal.pone.0097477
The sea slug Elysia chlorotica. Image from: Pelletreau K.N., et al. (2014)  PLoS ONE 9: e97477.

A case in point—sea slugs. These unfortunately named, exotic looking creatures have some surprising secrets.

Continue reading “Animal or Plant? FISH Labeling Reveals Horizontal Transfer of Algae Gene into Sea Slug Chromosome”

Could This be the Next Generation Ebola Virus Vaccine?

Ebola virus has received a lot of press in the last year due to the extended epidemic outbreak in Africa. Ebola is part of the family of Filioviruses (filamentous virus) and causes hemorrhagic fever that leads to internal bleeding and loss of bodily fluids. As the epidemic in Africa has illustrated so starkly, once the virus infects a large enough population, the human suffering it causes is devastating to individuals and communities. Because no treatment other than palliative fluid support is available to those infected by Ebola virus, virologists have focused attention on potential therapeutics and vaccines. The vaccine strategies now in clinical trials are based on a single Ebola virus glycoprotein, GP, and involve a DNA-based vaccine or innoculation with an Ebola protein expressed from a viral vector. How effective and safe this approach may be for protection from Ebola virus infection is currently under investigation.

Based on the history of effective vaccines, Marzi et al. was interested in testing a whole-virus vaccine for Ebola (EBOV). A whole-virus-based vaccine like smallpox or measles uses an attenuated or inactivated virus. The advantage of this method is that all the proteins as well as the nucleic acid are available for immunological reaction, offering broader-based protection than a single protein. In the recently published Science report from Marzi et al., a replication-incompetent Ebola virus was used as the basis for a whole-virus vaccine that was tested for its efficacy in nonhuman primates.

Continue reading “Could This be the Next Generation Ebola Virus Vaccine?”

Culturing the Unculturable Bacteria

Culturing bacteria is't always this easy.
Culturing bacteria is’t always this easy.

It is estimated that all the bacterial species so far described represent only a tiny fraction of the total. The rest remain unknown to science because they are “unculturable” in standard (or known) laboratory media. Given that many antibiotics were first isolated from environmental bacteria, it seems likely that these as yet unknown organisms could also be a rich source of potential new drug candidates. The desperate need for new strategies to combat multi-drug resistant infections gives impetus to studies investigating how we can culture some of these “unculturable” bacteria and uncover their potential as a source of much-needed new treatments. 

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Tracking the Beginning of a Pathogenic Bacterial Infection

Yersinia pestis by U.S. Center for Disease Control [Public domain], via Wikimedia Commons.
Understanding the course of a pathogenic infection involves not only understanding what ultimately kills the host or how the bacterium or virus enters the body but also how it establishes itself in the host organism. What is the receptor that allows a virus to enter the cell? Which cells does a bacterium first target or how does it evade an immune response? While other studies of bacteria like Yersina pestis have looked at imaging the bacterial burden in model mice, questions remain about how this bacterium gets from the skin after an infected flea bites to the draining lymph nodes, where the bacteria replicate and enter the bloodstream and infection becomes fatal. A recent PLOS Pathogens article examined how the nonmotile Y. pestis disseminated itself starting from a tiny innoculation mimicking a flea bite on a mouse ear and following pathogen interaction with the host from skin to lymph node. Continue reading “Tracking the Beginning of a Pathogenic Bacterial Infection”

Death in the Stars: A Virus Decimates Sea Star Populations Along the Pacific Coast

Starfish BlueA killer is lurking in the waters off the pacific coast. Silent and lethal, it leaves its decimated victims in tidal pools. They first began to appear in the early summer of 2013. Limp and curled, missing some or all of their limbs, the bodies were little more than globs of slimy tissue.  They were hardly recognizable as what they once were—Sea Stars. Continue reading “Death in the Stars: A Virus Decimates Sea Star Populations Along the Pacific Coast”