Tag Archives: bacteria

CRISPR: Accelerating the pace of molecular biology

CRISPR stands for Clusters of Regularly Interspaced Short Palindromic Repeats. Dr. Jennifer Doudna was one of the first researchers to see these short palindromic repeats in bacteria and archaea where she speculated that they were being used as a form of molecular immune system to protect these organisms from viruses.

Even bacteria get sick, so having a protection against invading viruses is a matter of life and death to a cell. Recall that viruses are essentially genetic material that will reproduce itself again and again after it hijacks a cell. Viruses may have protein coats or membranes to protect them outside of the cell, but inside, they are little more than DNA. If this DNA can be damaged or destroyed, then the virus is rendered harmless.

Screen Shot 2015-07-27 at 9.54.10 PMTo the right is a clip from Dr. Doudna’s video illustrating the repeated elements (in black) flanking a variety of ‘other DNA’. This ‘other DNA’ is what the cell will use to identify  foreign DNA – presumably from retaining the genomic material from an earlier exposure either in the lifetime of the single cell or its parents.

So, how does it actually work?
Two videos do an excellent job of explaining how CRISPR works. A short, simple video from MIT gives a non-technical explanation (a good place to start).

MIT’s McGovern Institute

Jennifer Doudna explains the system in greater detail…

Basically, the natural system uses two RNA molecules to target specific DNA sequences in the genome and recruit a protein that acts as an endonuclease to cleave this target:

crRNA – a ‘targeting’ molecule
tracrRNA – an adaptor RNA that recruits CAS9 to the bound crRNA
CAS9 – an endonuclease enzyme that will bind and cleave DNA once recruited by the RNAs

Doudna’s lab improved the system by combining the two RNA molecules into a single RNA that still effectively recruits CAS9 but is easier for researchers to manipulate in the lab. This last element is essential because manipulating this RNA sequence gives researchers the power to target any DNA sequence in the cell.

As stated above, the system was originally identified in prokaryotic organisms where it appears to allow targeting of the viruses that attack them. CRISPR uses ‘stored’ DNA as the targeting RNA and then brings in CAS9. CAS9 binds to the targeted DNA and cleaves it resulting in one of two possibly outcomes. 1) the virus is destroyed and is no longer a problem, 2) the virus is cut, but then repairs itself – hopefully in a way that introduced fatal mutations.

How might this translate into clinical medicine?
The possibilities are endless, however a few low-hanging fruit present themselves immediately. Among these are therapies for sickle cell anemia (and a host of other blood disorders). Because sickle cell anemia is caused by a single base pair mutation, it is conceivable that hematopoietic (i.e. blood) stem cells can be isolated, the faulty gene repaired, and then re-introduce the corrected stem cell back into the body (possibly after the faulty stem cells have been ablated).

The newly altered and re-introduced stem cells now do the rest of the work for you by finding their place in the body where they reside while continually producing cells with the desired genetic changes.

The key is that these RNA molecules are quite simple to make exactly and in pure form (i.e. they can be manufactured chemically rather than needing cells to do the job for us and then we have to clean up all the extraneous contaminants). Most labs will design the molecules in-house and then order the constructed molecules from a ‘core lab’ that specializes in doing just that.

Jacob Corn, of UC Berkeley has compiled a simple protocol that anyone with a modicum of molecular biology training could follow. Find that protocol here.

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Posted by on July 27, 2015 in Uncategorized


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More on the Lac Operon

A while ago I wrote two posts about the Lac Operon here. The first pointed to an animation by McGraw Hill Publishers that did a pretty good job illustrating how the operon works. In the second post, I highlighted the notion of polycistronic messages (more than one gene per mRNA molecule) and how this allows for control of a number of related genes at once – a trait not shared by eukaryotic cells. In that second post, I also finished with a graph of how cells grow in the presence of glucose and lactose.


Cell Growth in the presence of glucose + lactose – As glucose is depleted, cells adjust to lactose digestion

One feature of that graph (reproduced here) that is notable is a little bump in the growth rate as glucose runs out and the cell converts to lactose digestion. A second important feature is that the rate of growth slows when the cell is burning lactose as its primary fuel.


Together, these features suggest that the cell is regulating lactose digestion very closely. In fact, there are two primary mechanisms of this regulation to appreciate. The first is that the lactose-digesting enzymes are controlled together on an operon that is regulated by lactose itself (or at least we can assume so for simplicity’s sake). In the absence of lactose, no lactase enzymes are made and no lactose is used as fuel. The reason for this is obvious when you look at the slope of cell growth under glucose metabolism (left) and lactose metabolism (right). Clearly, growth is SLOWER when lactose is used as fuel.

Therefore, so long as there is glucose, it is pointless to digest lactose at the same time. So it is best to only turn on the lac operon in the ABSENCE of glucose – regardless of whether lactose is present of not.

If glucose is absent and lactose is absent, turning on lactase enzymes is still useless. However, slow growth is better than no growth. So we should have a mechanism to turn on the operon when there is lactose in the environment.

Here’s a matrix of ideal regulation:

Screen Shot 2014-03-31 at 2.18.05 PM

How can a little, mindless bacteria achieve this exquisite control?

Simple: By using two regulators. One for glucose and one for lactose. Only when both conditions (glucose-, lactose+) are met do we make lactase.

Structure of the Lac Operon


First, lactose itself serves as an inducer. In the absence of lactose, a regulator protein binds to a DNA site between the polymerase binding site (the promoter) and the structural genes (the enzymes). When the regulator binds, its presence physically prevents the progress of RNA Polymerase.

When lactose is present, it binds the repressor protein in a way that causes its shape to change in a way that can no longer bind the DNA. The repressor then drifts away from its binding site allowing RNA Polymerase a clear shot to the structural genes.


However, RNA Polymerase is not always parked on the promoter waiting for the repressor to be removed. Its binding requires another protein to help stabilize its interaction with the DNA. This second protein is the CAP protein. The Catabolite Activated Protein. However, CAP alone will not bind either. It requires a signaling molecule called cyclic AMP (cAMP). cAMP is readily broken down when glucose is in the cell, so it only accumulates when glucose is absent. In that case, cAMP accumulates and binds to the CAP protein, which then binds to the CAP site. This site is located adjacent to the promoter, but on the side away from the structural genes. When CAP binds, it assists in recruiting the RNA polymerase to the promoter.


Therefore, if only one condition is met, it is insufficient to promote gene transcription. Only when the CAP+ cAMP protein is bound will the Polymerase be recruited. And only when lactose is present, will the repressor protein let the Polymerase pass.


In terms of the matrix we set forth above, we can see that these molecular interactions result in exactly the regulation that is optimal:



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Posted by on March 31, 2014 in Uncategorized


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A quick followup on that lac operon post

Last week I posted a quick link about operons for my micro class to check out before taking their quiz on bacterial gene regulation This post is intended to complement that one. To go back to that post, click here. If there’s one thing to remember about operons it is that bacteria, lacking a nuclear membrane, regulate their genes differently than Eukaryotes. Having a nuclear membrane separates transcription and translation into two distinct compartments allowing for more subtle tweaking of Eukaryotic mRNAs before they are exported for translation.


Click on this figure to go to a good description of how polycistronic genes work

One thing this does is it makes it very beneficial to package genes with related function closely on the genome and use a single regulatory region to control them all together. They wind up getting packed so closely together that they are actually expressed as a single messenger RNA – known as a polycistronic (meaning ‘many gene’) message.

Upstream of this polycistronic cassette are regulatory elements. One element common to all regulatory elements is the promoter. The promoter consists of several elements which ‘promote’ the binding of an RNA polymerase to the DNA. Additional regulatory elements exist to ensure that this polymerase only transcribes the genes if they are needed. In doing so, the cell conserves energy and components (e.g. Amino Acids) for only necessary processes.

In the case of the paradigm lac operon, lactose is a fuel source, but not as good as glucose. Therefore, enzymes to digest lactose are only needed when lactose is present, but glucose is not. In order to interrogate both conditions, two additional regulatory elements are present.

First, the operator sequence. This sequence binds a repressor protein that physically blocks the polymerase’s path in the absence of lactose. However, if lactose is present, the sugar binds to the repressor, causing a conformational (shape) change that causes the protein to release its grip on the operator sequence.

Second, a catabolite activator protein (CAP) will only bind to the DNA behind the RNA polymerase if cAMP is present. Let’s not get too distracted, other than to say that cAMP levels are high in the ABSENCE of glucose, and low when that sugar is present. When cAMP binds to the CAP protein it can now bind the DNA and do it’s other job: making a nice binding site for the RNA polymerase. Without CAP, the polymerase binds very inefficiently.

Together, the production of lactase enzymes (those that digest lactose) is exquisitely controlled in a way that conserves the most energy.

ImagePs – take a look at this graph and tell me why (not mechanistically, but rationally) the cell does not make lactase enzymes when both glucose and lactose are present.

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Posted by on March 15, 2014 in Uncategorized


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Lac Operon

I’m typically not a fan of McGraw-Hill’s animations because they are rather dry and un-engaging, however, this one does a good job of describing conditions under which the lac operon will be either ‘on’ or ‘off’ and illustrating how positive and negative regulatory elements work together so that the operon is on when needed, but conserves energy by remaining off when it is not needed.

more on this later….

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Posted by on March 10, 2014 in Uncategorized


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A damn fine cup of coffee

I was looking through my next lecture for Microbiology (which I realize I still need to post online) and was reminded of an older post that I made pointing to a virtual lab on streak plating of bacteria.

I recommend my students check that out if they have not plated bacterial cultures in the past. You can find that post here.

Also, as always this time of year, I like to reflect on some of the great pleasures of life. And, because these things often become conversations in my classes, I’d like to share a short video that couples two of my favorite things: Coffee and Twin Peaks

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Posted by on January 15, 2014 in Uncategorized


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A thoughtful article on the use and abuse of antibiotics

ImageEarlier today, I found this well written article on the era of antibiotics by Maryn McKenna (Published November 20, 2013). While I’m not sure I agree with everything in it – and have been spending time tracking down some publications to support or refute some data cited here (particularly in regards to the use of antibiotics in agricultural animals), the  summary of how antibiotics were first discovered and used and how researchers including Flemming feared an end of antibiotic usefulness, paints a vivid portrait of the problem at hand.

While we might typically think of antibiotics as being prescribed in a clinic following a positive test for strep throat or some other bacterial infection, that is just one example of their use. One element of this paper that I found particularly insightful was how easily overlooked are the myriad uses of antibiotics in situations such as surgical procedures or following chemo- / radiation therapy.

 British health economists … recently calculated the costs of antibiotic resistance. To examine how it would affect surgery, they picked hip replacements, a common procedure in once-athletic Baby Boomers. They estimated that without antibiotics, one out of every six recipients of new hip joints would die.

Let me know what you think.

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Posted by on November 24, 2013 in Uncategorized


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Paul Offit’s Vaccine class

ImageIn my microbiology class, students read Paul Offit’s ‘Vaccinated’, an excellent account of the life and work of Maurice Hilleman, creator of many vaccines in common use today. I don’t know how many of my students are aware of it, but is a free online university offering courses in a number of subjects taught by senior faculty from many distinguished universities. Today marked the first day of Paul Offit’s Vaccine course, which covers topics related to the history, development, use and misinformation surrounding vaccines. From the course website:

1) History of Vaccines – Viruses

2) History of Vaccines – Bacteria

3) Current and ‘Alternate’ Schedule

4) Common Questions About Vaccines

5) Vaccines in the media

6) Creation of a Vaccine – Case Study: The Rotavirus Vaccine

7) Vaccine Exemptions

I can’t promise that this course is still open, but I expect it is – and best of all it’s FREE.

Furthermore, if you sign up and only decide to watch one or two class lectures, there’s no risk: just drop out or simply stop signing in. So, if you have any interest in the subject, or would simply like to see what it’s like, go onto and type ‘vaccines’ in the search bar.

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Posted by on September 3, 2013 in Uncategorized


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A great old government film touting the discovery and promise of Penicillin:

Penicillins are a group of drugs naturally produced by penicillium molds. The antibiotic activity of penicillium was first observed by Alexander Fleming in 1928. He recognized the value of what he was seeing, but was unable to isolate the molecule that mediated the activity and therefore could not perform appropriate trials. It was not until Howard Florey and a team of researchers including Ernst Boris Chain and others from the Sir William Dunn School of Pathology, University of Oxford managed to isolate and purify the substance that its great promise became evident. (later Fleming, Florey and Chain shared the Nobel Prize in Medicine for their work)

Alexander_FlemingA number of naturally occurring penicillins exist, each characterized by a beta-lactam ring joined to a variable R-group. These drugs may be effective in the treatment of certain, susceptible (mostly) gram positive bacteria. The mechanism of action is the inhibition of peptidoglycan crosslinks in the bacterial cell wall, such that organisms cannot produce new cell wall and wind up shedding the wall during division. Without the cell wall, the bacteria is highly susceptible to immunological mechanisms and is readily killed.

ImageUnfortunately, many otherwise susceptible bacteria produce an enzyme (penicillinase / beta-lactamase) that cleaves the beta lactam ring structure leaving it ineffective. This single enzyme can be easily passed from one bacteria to another via a sex pilus or transformation rendering them non-susceptible to the antibiotic.


This enzyme breaks the β-lactam ring and deactivates it’s antibacterial properties. Because beta-lactam is central to the activity of penicillins, cephamycins, and carbapenems, all of these antibiotics can be rendered ineffective by organisms possessing this enzyme.

To counter this beta-lactamase activity, Clavulanic Acid can be used as an inhibitor of this enzyme. Clavulanic acid acts as a competitive ‘suicide inhibitor’, covalently bonding to the active site of the β-lactamase and irreversibly inactivating it. Compounds of the drug (Penicillin) and the enzyme inhibitor (Clavulanic Acid) are available as Amoxicillin under a number of brand names.

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Posted by on May 21, 2013 in Uncategorized


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Miller, Urey and Venter

The Spark of Life

Life is a funny thing. Despite our primary connection and concern with it, life defies a simple definition. This seems absurd because it is intuitively obvious whether the dog lying at your feet is alive or dead. But do you use the same criteria in judging if the tree in the front lawn is alive? What about a frog frozen in the Siberian winter? Somehow the line still seems fairly bright – even if difficult to define.

You might be a little more hesitant if you’ve spent time in a biology classroom contemplating the fringes of life. Biologically, there are two general ideas that define life. The first is the ‘cell theory’ that defines the cell as life’s smallest unit. Second is a list of characteristics that life may possess. These include such things as organization, homeostasis, growth, response to stimulus and reproduction (among others.) Some simple thought experiments illustrate how some of these characteristics may exist in dead things, while others may be absent in living things.  But when taken together, they provide support for, or argue against a thing’s life. Consider: a fossil has evidence of both cellular structure and organization, but fails to respond to stimulus or grow; a single celled amoeba responds to stimulus but does not grow (at least not beyond its single cell ‘body’); a castrated bull that has almost all of the listed characteristics save reproductive ability. Which of these is alive?

In contrast, consider a virus. It fails almost all of the tests and is nearly always considered to be unliving, yet is very well organized, can reproduce when infecting a host cell and does respond to some stimuli as its DNA may harmlessly reside in a cell until conditions induce it to ‘awaken,’ start reproducing itself and finally kill the host cell as it bursts from the sheer number of virii produced.

Life, then, seems dangerously close to Justice Stewart’s 1964 definition of pornography – or rather, his failure to define it – “I know it when I see it.”

It’s amusing that the definition of life even eludes biologists. ‘Biology’ itself translates as ‘the study of life,’ yet those of us who study it can’t say exactly what it is in every case.

So it is doubly complicated when scientists puzzle the origin of life. We don’t know exactly what life is AND current estimates place the origin of life back 3.5 billion years. This leaves us with fossils, assumptions about the early Earth’s atmosphere and laboratory experiments meant to mimic early conditions. We are also in something of a quandary because of Louis Pasteur’s demonstration in 1862 that showed quite definitively that spontaneous generation does not occur  under present conditions– only life can beget life. Yet this must have happened once for us to be here pondering our origins.

In 1922 the soviet scientist, Alexander Oparin, was pondering the origin of life on Earth. At the time, evidence was beginning to suggest the environment of the early Earth as being a reducing environment consisting of ammonia, methane, hydrogen and water vapor. Oparin determined that this was ideal for the chemical evolution required to generate the amino acids and other complex molecules that could later make life possible. One reason this atmosphere was ideal was the lack of oxygen that can be very destructive to molecules that become oxidized in its presence.

Stanley Miller

It took thirty years for Oparin’s theory to be actually tested in a laboratory and it was not Oparin himself who did the experiment. Instead, it was the Americans, Stanley Miller and Harold Urey who, in 1952, constructed an apparatus that sterily re-created the Earth’s early atmosphere within a self-contained glass tubing. Water was heated to form vapor and an electrical spark was provided to simulate lightening. Under these conditions Miller and Urey witnessed the generation of five amino acids as well as a number of sugars and fats – all building blocks necessary for life.

This does not prove that the chemical precursors of life formed this way, but it does demonstrate that, under these conditions, molecules necessary for life could self-assemble. Regardless, this was not the generation of life in the laboratory, but only a step showing that macromolecules required for life can spontaneously form under the proper conditions.

Beyond this, we know some of the things that are required of life as we have defined it. There must be the generation of some device to carry information from one generation to another.  Why do we require this? Because there needs to be a way for the new organism to ‘know’ how to do the things it needs to do to stay alive. That way is through inherited information

It is interesting that from the beginning we assume that life must proceed in generations rather than as a single organism going on indefinitely – but considering how fragile a single life may be, generations may be the only way. This information is almost uniformly encoded in DNA. Further, the code used to store this information is also (for practical purposes) universal. Together, these facts argue strongly for the origin of life as a single event.

There must also be a compartmentalization of this information and the machinery required to replicate it within a membrane – thus creating a cell. It’s another interesting thought experiment to contemplate whether life is possible without a membrane. However, it is difficult to conceive how a lifeform could occur without the ability to concentrate itself and hide its resources away from potential predators.

So, life is difficult to define and we have only hints about how it originated. An interesting question is, ‘can we create life ourselves?’ The answer is: of course, it’s so easy that it doesn’t require a lab at all.  All our youthful energies are directed at making new life (or perhaps avoiding doing it so we can earn enough money to pay fertility doctors to do the same thing once we’ve gotten to old to do it ourselves.) But that’s not what I mean. More specifically, “Can we create life in the lab synthetically… from scratch?”

More than just an idle curiosity, this is more a question of testing what we know about life and its requirements. Biology has been a reductionist science since the structure of DNA was modeled by Watson and Crick in 1953 and developed as a tool of molecular biologists by the 1980s. Creating a living organism in the lab is a test of our ability to rejoin these reductionist ideas into a unified, functional model.

A step in this direction was taken in 2002, when Eckard Wimmer’s group made a synthetic poliovirus and demonstrated that DNA made in the lab was just as infectious as naturally derived poliovirus DNA. This advance was met with skepticism based on the fact that the only novel element of this work was the chemical source of the DNA. All other elements of the experiment had been previously demonstrated and were not at all unexpected (these are the two elements that make for good science– novelty and unexpectedness.) Furthermore, in terms of creating life, a virus is generally not accepted as alive for reasons we discussed above.


Craig Venter

Enter Craig Venter. In 2000, his Celera Genomics company was the first to sequence the entire human genome. Since then he founded the J. Craig Venter Institute and Synthetic Genomics as vehicles for deriving synthetic life. In 2010, his group was successful in generating an entire microbial genome chemically and then using it to direct life functions within a new cell that previously had its own DNA removed.

This might not sound like much of an advance over Wimmer’s work with a synthetic virus, but it certainly brought its own challenges, one of which originated from the fact that the ‘host’ cell used was not the same as the ‘donor’ DNA. This led to some trouble in overcoming certain molecular defenses set up by the cell in order to repel parasites such as viral infections. After overcoming these obstacles, the cells were observed replicating the introduced DNA, manufacturing the proteins it encoded and dividing into daughter cells.

This work also began to define just what genes were required to accomplish this goal and possibly address the question, “what are the minimal requirements for cellular life?”

A possible utility for the project would be tailoring this bacterium for the production of recombinant proteins, fats, etc. These bugs can then be used to make medicines, remediate ecological damage and perhaps even serve as medical therapies themselves. Since we have created it from scratch, we know that there is no danger of having it ‘escape’ and mutate back into a virulent form.

So, despite the difficulties associated with even knowing what life is and how it originated, we have not let these questions prevent us from using what we do know to accomplish amazing tasks that were unthinkable just a few short decades ago: creating life in the laboratory.

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Posted by on October 30, 2012 in Uncategorized


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