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”

Summer Friday Blog: This Week We Travel to Hawaii and Peshtigo, WI to Learn about Firestorms

Fire-whirlThis week’s video takes us to a forest fire on Mauna Kea, a dormant volcano on the island of Hawaii. One of the firefighters captured this amazing video of a fire whirl that erupted as the air temperature near the ground grew very hot. Fire whirls like this one are caused by extreme heat rising from the ground rather than a confluence of atmospheric events, but they can be be every bit as destructive as atmospheric tornadoes and cause a forest fire to continue to burn out of control.

There are written records of fire tornadoes including several that developed after lightning struck an oil storage facility near San Luis Obispo, CA, USA in 1926. In 2003, scientists confirmed true fire tornadoes in Australia associated with the Canberra fires. In this case the fire tornadoes produced damage consistent with the intensity of an F2 tornado. In The Great Pestigo Fire in 1871, the town of Peshtigo, WI, may well have been consumed by fire tornadoes. Dry weather conditions and slash and burn farming practices contributed to this devastating fire (as they did the more famous Chicago fire that occurred on the same day). Strong winds carried a forest fire into the mill town of Peshtigo, WI, and researchers theorize that cut timber and wooden structures of the town fueled such intense heat that a massive fire whirls formed, consuming the town. You can read some compelling stories about the Peshtigo fire here and here.

Understanding the conditions under which firestorms and fire tornadoes form hopefully will lead to a better understanding of how forest and brush fires spread and allow scientists and fire control experts to develop more effective methods of control.

If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions

14231183 WB MS Protein Interactions Hero Image 600x214

Robert Hooke first coined the term “cell” after observing  plant cell walls through a light microscope—little empty chambers, fixed in time and space. However,  cells are anything but fixed.

Cells are dynamic: continually responding to a shifting context of time, environment, and signals from within and without. Interactions between the macromolecules within cells, including proteins, are ever changing—with complexes forming, breaking up, and reforming in new ways. These interactions provide a temporal and special framework for the work of the cell, controlling gene expression, protein production, growth, cell division and cell death.

Visualizing and measuring protein:protein interactions at the level of the cell without perturbing them is the goal of every cell biologist.

A recent article by Thomas Machleidt et al. published in ACS Chemical Biology, describes a new technology that brings us closer to being able to realize that goal.

Continue reading “If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions”

Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) .

Continue reading “Targeting MYC: The Need to Study Protein:Protein Interactions in Cells”

Thawing Out to Sing: The Story of the Wood Frog

Wood Frog_Northern WisconsinOne of the hallmarks of the arrival of Spring in Wisconsin is the cacophony of evening croaks and calls from the Spring Peepers and Chorus frogs. Indeed frogs and toads are ubiquitous around the globe, and many of us who have become life scientists (even those of us who have relegated ourselves to the world of macromolecules, cell signaling networks, and nucleic acids) probably spent some time in our childhood chasing and catching frogs.

But what happens to those frogs and toads over the harsh winter months in places like Wisconsin? Well, their strategies are species-dependent, but at least some of them overwinter by freezing, and the story of one species, the Wood Frog, is quite amazing. Think about it. It freezes from the inside out. No heart beat, no circulation, completely dormant. Then in response to some unknown signal (day length? temperature? angle of the sun?), bodily functions slowly resume. What kind of cell signaling cascade controls that response?

Here is a video from NOVA about the Wood Frog and its amazing deicing event. The next time you are out on a Spring or Summer evening and you hear a chorus of frogs calling, you can think about the incredible molecular story behind the event and be even more impressed!

A NOVA Video about the Wood Frog:

 

Get Ready to Celebrate Pi!

26562342_lWhat will you be doing on 3/14/15 at precisely 9:26:53? Twice the clocks will align with the first few digits of my favorite irrational and transcendental number (3.141592653…) or π. Over 1 trillion digits to the right of the decimal point have been calculated, and they still go on, never making a pattern, never repeating.

Take any circle, that blueberry pie that your grandmother baked would be a good choice. Measure the circumference and divide by the diameter and you will have Pi, the mathematical constant that represents the ratio of circumference to diameter in a circle. You probably encountered Pi in your early mathematics education when learning the formula for the area of a circle (2πr2).

In celebration of Pi, here are a few Pi diversions to brighten your day. Continue reading “Get Ready to Celebrate Pi!”

Choosing the Best Luciferase Vector for Your Experiment—Now Made Easier with the Vector Selector

4621CAGenetic reporters are used as indicators to study gene expression and cellular events coupled to gene expression. They are widely used in pharmaceutical and biomedical research and also in molecular biology and biochemistry. Typically, a reporter gene is cloned with a DNA sequence of interest into an expression vector that is then transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. A good reporter gene can be identified easily and measured quantitatively when it is expressed (in the organism or cells of interest).

Bioluminescent reporters are ideal for these types of studies because they have a number of important features including:
• Measurements that are almost instantaneous
• Exceptional sensitivity
• A wide dynamic range
• Typically no endogenous activity in host cells to interfere with quantitation

However, one factor that is critical for the success of a bioluminescent reporter assay is the vector.

At Promega we offer several different luciferases as reporters, and the genes for those luciferases are available in a variety of vectors. The vectors may vary in the promoters used or the presence or absence of sequences for rapid degradation. Often seemingly small changes in the vector can make a big difference in the suitability of the vector for a given experimental system. Do you need a reporter with a short half-life to detect rapid changes in gene expression? Are you studying a specifically localized protein? Do you wish to perform a transient or stable transfection?

To make finding the best reporter vector for your experimental system easy, we have developed the Luciferase Reporter Vector Selector. Using this online tool, you can narrow the choices of available vectors by promoter type, application (in vivo imaging, cancer pathway analysis, etc), availability of selectable marker, and type of luciferase.

So, as you design your luciferase reporter experiment, keep in mind this handy tool to help you choose the best luciferase vector for your needs.

What Things Are You Thankful for in Science?

What are you thankful for in science?
What are you thankful for in science?
As the social media lead for Promega, I keep my eye on trends in new media. I have personal accounts that I keep mostly to see what other people are doing. I try hangouts, social networking and other things so that I have an idea of developing practices outside of the biotechnology industry. One activity that has been popular over the last couple of years during the month of November in the United States is the Facebook post of “30 days of thanksgiving”.

I wondered what “thanksgiving” looks like to the research scientist. So I asked:

What are the things you are thankful for in science?

The answers have been as varied as the people I talked to ranging from little things like water bath floats to really big things, like the renewal of your research funding or achieving tenure.

Here are some of the answers from my informal inquiries:

“Tube floaties for water baths.”

—E.V., genomics product manager

“I was always thankful for Geiger counters.”

—K. G., science writer

“Thermal cyclers and Taq Polymerase. As an undergrad I watched someone sit with a timer and move their tubes between water baths at 3 different temperatures, opening tubes and adding polymerase at the end of each cycle. Modern PCR is SOOO much easier.”

—M.M., research scientist

“I am thankful for competent cells. I remember preparing the CaCl2 and doing slow centrifugation. Also thankful for serum-compatible transfection, rapid ligations and online journal access (no longer have to traipse over to the university library to get papers photocopied- uuurrrgggghhh).”

—R.D., technical services scientist

“How about T-vectors for cloning? I was no molecular biologist, but could make a T-vector work.”

—K.K., science writer

“I am thankful for open-access journals and the ability to read the full article without an institutional subscription.”

—S.K., science writer

“I am ever so thankful for ONLINE ORDERING! So awesome. Throw in online technical manuals, on-line support tools, on-line calculators – all are awesome!!”

—A.P., director, scientific courses

“I am thankful for automated sequencing- manual sequencing was laborious and hazardous!!!”

—R.G., technical services scientist

Do any of these resonate with you? What are you thankful for as a scientist? Let us know in the comments.

Fold It Up and Discover a Whole New World

FIGURE 1: Foldscope design, components and usage. (A) CAD layout of Foldscope paper components on an A4 sheet. (B) Schematic of an assembled Foldscope illustrating panning, and (C) cross-sectional view illustrating flexure-based focusing. (D) Foldscope components and tools used in the assembly, including Foldscope paper components, ball lens, button-cell battery, surface-mounted LED, switch, copper tape and polymeric filters. (E) Different modalities assembled from colored paper stock. (F) Novice users demonstrating the technique for using the Foldscope. (G) Demonstration of the field-rugged design, such as stomping under foot.
FIGURE 1: Foldscope design, components and usage.
(A) CAD layout of Foldscope paper components on an A4 sheet. (B) Schematic of an assembled Foldscope illustrating panning, and (C) cross-sectional view illustrating flexure-based focusing. (D) Foldscope components and tools used in the assembly, including Foldscope paper components, ball lens, button-cell battery, surface-mounted LED, switch, copper tape and polymeric filters. (E) Different modalities assembled from colored paper stock. (F) Novice users demonstrating the technique for using the Foldscope. (G) Demonstration of the field-rugged design, such as stomping under foot.

Scientific inquiry —looking at the world and asking questions about what we observe—is a natural human behavior. Why is the sky blue? What would happen if I did this Mom? Ask any grade school teacher. Kids do science naturally. They are not afraid of questions. They are not afraid of nature. They are not afraid of experiments and data collection.

One other things kids do really well is: fold paper. I never cease to be amazed at the elaborate fortune tellers, hoppers, boats, hats and other creations that my daughter and her friends make at a moment’s notice out of virtually any scrap of paper they can find.

Recently members of the Prakash Lab at Standford University announced the Foldscope: an optical microscope that is printed and folded from a single flat sheet of paper. These microscopes, which can provide magnification of up to 2000X, can be produced for less than $1.00/each. Furthermore these scopes weigh less than 10g (a couple of coins), require no external power source, can be dropped from 3-stories without damage, and can even be stepped on.

These characteristics make the Foldscope ideal for field work, particularly in remote locations where access to power and other resources is difficult. Prakash and colleagues have published their work in a PLOS One paper and have demonstrated many uses for these Foldscopes including high-resolution brightfield microscopy, fluorescence microscopy, and darkfield microscopy. Continue reading “Fold It Up and Discover a Whole New World”

Freedom to Focus: Maxwell® Rapid Sample Concentrator

Wish I had one of these when I was at the lab bench...
Wish I had one of these when I was at the lab bench…

Back in the dark ages, when I was a graduate student, my idea of “automated” plasmid DNA extraction involved performing home-brew, “toothpick preps” in “strip tubes” or , if I was really feeling ambitious, a 96-well plate.

I would get just enough DNA to check for the presence of an insert, but the quality of the DNA was too low and the quantity too small to even consider using it for any other downstream experiments like amplification.

And increased throughput for other nucleic acid extraction needs? Nope. If I wanted genomic DNA, RNA or high-quality plasmid DNA, I spent time with columns and tubes, giving each sample my undivided individual attention.

Remember cesium chloride preps for RNA isolation? Even with the advent of column purification, which greatly simplified and standardized my protocols, nucleic acid purification was still a manual task that required a lot of time and effort to get the high-quality product I needed.

Doing the experiments that would answer the questions that I really wanted to ask (those “downstream experiments”), meant spending time at the bench performing careful (if tedious) work to isolate and clean up the highest quality nucleic acid possible. Even then inconsistency in sample prep could wreak havoc on downstream work.

Fortunately, for the modern scientist, personal, bench top automation, has progressed far beyond the toothpick and the strip tube to quality-tested, reliable nucleic acid extraction platforms like the Maxwell® Rapid Sample Concentrator (RSC).

The Maxwell® RSC improves sample preparation consistency, eliminating variability associated with manual handling, and your downstream results will reflect this consistency.  With the RSC you can extract DNA or RNA from up to 16 samples in approximately 1 hour and viral total nucleic acids in less than an hour.

The instrument is easy to use: simply load the sample, push a button and walk away. Cross contamination is minimized and the instrument is supported by the Promega technical support and service you have come to trust over the past 35 years. 


Want to know more about how the Maxwell® RSC can give you the freedom to focus on the work that interests you the most? To learn more, click here.