Transforming Bacteria


As mentioned before, my primary work at the lab involves trying to gain insight into genes, their expression and their interactions. One challenge is telling when and where a gene is being expressed. A great indicator of gene expression that we commonly look for is the very first product of expression, mRNA. It is impossible to tell if a certain gene's mRNA is being produced just by looking under a light microscope, so we must utilize methods that make mRNA easier to visualize. A great way to do this is to make probes for the mRNA. Probes are complimentary RNA strands designed to hybridize to the mRNA of interest. Probes contain special nucleotides that are either fluorescently labeled, radioactively labeled, or, in our case, contain antigens for antibody labeling. This allows us to see where genes are being expressed easily under a microscope.

~Raising a Plasmid~

Last week I received a letter in the mail containing a simple piece of filter paper wrapped in plastic. No, not another birthday card, this was my plasmid, the complimentary DNA I needed to make RNA probes. How we get that DNA from a piece of filter paper, create thousands of copies, and eventually make probe is an interesting technique that employs the services of microbiology’s favorite bacteria: Escherichia coli.

Don’t let the name scare you. Some strains of E. coli live peacefully in your intestine, while others, especially the dreaded 0157:H7 strain found on spinach leaves the other week, will make you horribly ill. In the lab, E. coli is the cattle of the microbiology world. We grow and raise them and they, in turn, produce product for us. Of course, we only use harmless, competent E. coli stains. Never, not even in all the years of practical jokes against unsuspecting undergrads, have we even touched the 0157:H7 strain.

The E. coli we use are said to be competent because they have the amazing ability to take-up DNA from their environments. The process of taking up DNA from the environment is called transformation, something competent cells are, well, competent at.

Once the plasmid has been eluted from the filter paper, it is introduced to the E. coli cells in a heat bath for half a minute. The heat makes the bacterial membranes more permeable, and it is hoped that many of the cells will take up the plasmid DNA in a marvelous transformation at this point. We then allow them to grow and reproduce for a while, undisturbed.

The plasmid is ingeniously designed. It contains the complimentary base pairing to our mRNA of interest, but it also contains an ampicillin resistance gene. This is essential, because it is the only way we are able to select for bacteria containing the plasmid. You can see a picture of a typical plasmid below, the "insert" being where our probe DNA would go and the ampicillin section represents the ampicillin resistance gene:


After letting them grow for a day, we plate the bacteria on medium containing the antibiotic ampicillian. Those cells which contain the plasmid will also have the resistance gene and survive the plating without a hitch. Those that do not will die, helping us filter out the unwanted cells. It’s a little experiment in microevolution every time! We also plate positive and negative controls, to make sure there were no errors made while making the plates. I imagine my plated bacteria to be like little pigs, all hogging out before we extract the bacon—the probe—a few days later.

If the cultures grow well, we are then able to transfer a colony to a liquid medium and grow up the selected stock. Creating liquid medium is an interesting process in itself, as all liquid and jars must be sterilized before use in bacterial transformations in a HUGE machine called an autoclave. The autoclave is like a gigantic high heat dishwasher and I’m terribly frightened of it. I managed to embarrass myself the last time I went in to collect my growing medium. My "procedure" for collecting medium after sterilization involves standing as far away from the freakish metal steam trap as I can while cranking the door open slightly, one foot outside of the autoclave room, then running into the hall as steam rushes out of the autoclave. Last time I spotted one of my coworkers giggling at the sight of me from across the hall. Ah well, better safe than sorry, with the autoclave. One must then wait ten minutes for all the steam to clear before collecting the sterilized equipment to avoid burns.

When the bacteria are done growing it's time to collect the "bacon." First we spin the liquid cultures in the mega centrifuge—a machine that looks rather like a washing machine (although I don't think my clothes would survive the 6,000 Gs). The pellet (solid at the bottom of the bottle) from the centrifugation is saved and the liquid growing medium is disposed. Next we lyse the bacterial cell walls with enzymes, and then expose the material to several washes and filters until all the bacterial DNA and cellular components are cleared and only the pure plasmid remains.

I still have yet to make probe from the plasmid, which is a story for yet another week.

And now, for those of you who are truly geeky, here’s a little section on how the ampicillin bacterial selection works.

~Antibiotic Mechanisms~

Like penicillin, ampilcillin functions by irreversibly inhibiting the enzyme transpeptidase, which is essential to the production of the peptidoglycan cell wall of E. coli bacteria.

Transpeptidase contains a serine amino acid at its active site, and it is this component that makes it susceptible to the ampicillin molecule.

In the picture below I’ve circled a four membered ring containing three carbons, a nitrogen, and an oxygen double bonded to a carbon.


This ring is especially susceptible to nucleophilic attack as it is somewhat unstable. The alcohol of the serine molecule at the transpeptidase active site acts as a nucleophile, opening the ring. The hydrogen from the serine alcohol is transferred to the ring nitrogen and the carbonyl carbon binds to the alcohol oxygen of the serine molecule. The result is a disabled transpeptidase, covalently bound to the antibody:


This kills the bacteria, as they are unable to make and repair their cell walls without transpeptidase. That is, it kills all bacteria but those that have been transformed. The plasmid codes for the enzyme beta-lactamase, which acts on ampicillin in much the same way transpeptidase does. The serine alcohol of beta-lactamase attacks the ring, then interacts with water to produce an inactive antibiotic (the picture is for penicillin, but the mechanism against ampicillin is the same):




Sources:
Craig Stevens (lab mentor)
David Cole (biochemistry professor)
Ampicillin: wikipedia
Enzyme mechanism: http://www.antiinfectieux.org/antiinfectieux/PLS/Beta-lactames/PLS-Betalactames-mecanisme-action.html
Plasmid: http://teachline.ls.huji.ac.il/soreq/targil2/Q2-3.html
E. coli 1: http://www.rowett.ac.uk/edu_web/Gut_reactions3.html
E. coli 2: http://www.corante.com/loom/archives/evolution/

One of the genes I’m researching in the Stenkamp lab is the sonic hedgehog gene. We're interested in deciphering how it regulates retinal development. Many people laugh when they hear that I’m studying sonic hedgehog, it sounds like some impulsive video gamer’s dream job, but the sonic hedgehog gene name actually has a logical origin.

Hedgehog proteins, in general, are best known for their role in regulating development by creating concentration gradients. The first hedgehog gene was discovered through a mutation in Drosophila melanogaster, the fruit fly. The mutation caused a bunchy, spiky appearance in the embryo and so researchers gave it the name “hedgehog.” Soon more and more hedgehog analogues were found in other species. These hedgehog-like proteins were first given the names of actual known hedgehog species, such as desert and Indian hedgehog, but when those ran thin all that seemed left was that notorious Sega character: Sonic the Hedgehog.

Sonic hedgehog, as it turns out, is one of the best studied morphogenic genes around. The sonic hedgehog gene produces the sonic hedgehog protein (note: genes are italicized and proteins of the same name are not), which binds to cellular receptors as a ligand and plays key roles in vertebrate organogenesis. The developmental patterning of many systems is dependent upon hedgehog proteins, including the limbs, the midline of the central nervous system, and the thalamus.

Sonic hedgehog (shh) proteins work to create correct developmental patterning through diffusion gradients. In the limb, shh is secreted at the zone of polarizing activity, creating higher concentrations of shh protein in the fifth digit and none in the first, with a gradient in between. This gradient is responsible for distinguishing the posterior and anterior ends of the limb. In the absence of shh, the digits will not differentiate correctly resulting in a symmetrical, deformed hand, paw, or flipper (depending on who you are).

Similarly, shh signaling is required for proper patterning in the central nervous system. In the neural tube, shh proteins bind to patched (ptc) and smoothened (smo) to begin the hedgehog signal transduction pathway, below:



In humans, mutations in the sonic hedgehog gene cause holoprosencephaly type 3 due to the loss of the ventral midline. As a result, the cerebral hemispheres of the brain do not divide, nor does the eye. Usually, death occurs in utero, but less severe cases may allow the organism to survive for some time after birth, such as in the disturbing case of the sad little cyclopic kitty below:


In zebrafish, a deletion of the sonic hedgehog gene will also result in gross morphogenic differences during development. The embryo has a truncated body type, shrunken head and eyes, poor (if any) lens and photoreceptor development, and often dies around three days post fertilization. This is the mutant I am primarily investigating in the Stenkamp laboratory.

Sonic hedgehog also plays a role in the adult organism. It is known to signal hair follicles into the active phase (geeze, maybe I could use some shh protein for my sluggish follicles!). Procter & Gamble are currently developing a hedgehog agonist to treat certain types of baldness. Aside from hair growth, shh controls cell division in stem cells and is currently under investigation for its use in maximizing the potential of stem-cell research. It is also implicated in certain types of cancer, as in some instances its over expression may result in uninhibited cell division.

Sources:
http://www.futurepundit.com/archives/002462.html

http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Watson/Sonichedgehog.htm

Bumcrot, Takada and McMahon. “Proteolytic processing yields two secreted forms of sonic hedgehog.” Mol. Cell. Biol. 1995

Currie and Ingham. “Induction of a specific muscle cell type by a hedgehog-like protein in zebrafish.” Nature. 1996.

Kolpak, Zhang, and Bao. “Sonic hedgehog has a dual effect on the growth of retinal ganglion axons depending on its concentration.” J Neurosci. 2005

Perron, Boy, Amato, et. al. “A novel function for Hedgehog signalling in retinal pigment epithelium differentiation.” Development. 2003

Scholpp, Wolf, Brand, and Lumsden. “Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon.” Development. 2006

Stenkamp, Frey, and Mallory. Developmental Dynamics. “Embryonic Retinal Gene Expression in Sonic-You Mutant Zebrafish.” Developmental Dynamics.

All images from wikipedia, where, although the information can be unreliable and should be double checked elsewhere, the pictures are absolutely free!

On Consciousness


One of the most interesting and notorious questions in the field of neuroscience is "what is consciousness?" The question itself begs a definition of consciousness, as debates over the subject can revolve around syntax alone.

Dictionary.com defines it as: "...awareness of one's own existence, sensations, thoughts, surroundings, etc."

In the colloquial sense, people often mean being aware of their own thought processes, or "thinking about thinking" when they say they are conscious. When people say "I just did it without thinking," they usually mean that they weren’t conscious of their thoughts at the time. For example, of course I was thinking, when I locked myself out of the house, I’m thinking all of the time, but some of my thought processes are just not conscious (and certainly not at that fumbling moment!).

This is similar to Freud’s definition of consciousness. The processes in our mind which occur "without our awareness" are referred to as subconscious thought, while those that have our awareness (being able to "think about thinking") are considered conscious. For this article, I would like to stick with the psychological and colloquial meaning of consciousness: the ability to "think about thinking."

Most of the brain does not hold the internal monologue that many of us perceive as consciousness. The constant "chatter" of thought over every detail would be too overwhelming. Rather, many of our thoughts go without saying; there is simply too much going on in the world to convey it all at once in the talkative regions of our cerebral cortex we call our conscious mind. (Imagination, too can be considered an internal monologue, just of the nonverbal variety)

Among the brain functions that we readily consider to be unconscious (or subconscious) include the reflexes and autonomic functions that keep us safe and alive. These functions sometimes by-pass the cerebral cortex completely, so it is easy to consider them "unconscious;" after all, they reside primarily in more primitive regions of our central nervous system.

Harder to grasp is the realization that there are also thoughts in our cerebral cortex being produced constantly without our awareness. Being the part of the brain where intellect comes from, it is hard to imagine the cerebral cortex as largely subconscious, but those regions responsible for conscious thoughts are relatively few.

One of my favorite examples of conscious versus subconscious thought processing in the cerebral cortex comes from studies of the human visual system. The condition of “blind sight” arises when patients cannot name any object before them and have no conscious sensation of sight, yet their reflexes to “seen” objects (such as a ball coming toward their head) and their ability to reach out, grab, and mechanically maneuver “seen” objects remains intact.

A common test for the condition of blind sight as described by Dr. Ramachandran in his book Phantoms in the Brain is to ask the patient to mail a letter. Patients will often become exasperated, saying they see no letter, yet they can reach out and grab the object without the aid of any auditory or tactile clues. They are then able to turn the letter at the correct angle and insert it into a mail slot without ever having touched the slot!

One needs many parts of the brain functioning to see correctly, and blind sight often occurs in individuals who either have a portion their temporal lobe that deciphers what one is seeing destroyed or in individuals who have parts of their visual cortex damaged. According to a study by Stoerig and Cowey, even in monkeys where the entire visual cortex was experimentally removed blind sight was still found to occur. This means that seeing is not entirely a conscious act, rather only understanding what it is you’re looking at involves the conscious mind. Below is a diagram of the portions of the brain required for full conscious interpretation of a visual object:

Regions of the parietal lobe where the physical location of seen objects in space is interpreted are not shown as they are considered largely unconscious. The visual cortex is needed to make a conscious interpretation of a visual object, but is not necessarily the conscious element. It seems that the inferior temporal cortex and lateral prefrontal cortex are where consciousness resides with regards to sight, as without these regions an individual cannot make heads or tails of what a seen object is.

Another conscious versus subconscious mystery I’d be interested in looking into goes right back to Terry Armstrong’s project and the five intelligences. I believe that many people who find tasks like mathematical computations or drawing easy perform these functions on a primarily subconscious level. For example, when a baseball player hits a home run they might say, "I don’t know how I did it, I just let it happen." Would you say this is because the thought processes behind reflexes and hand-eye coordination are largely subconscious? Many neurologists would say so.

A good example of the same idea is the way in which my older brother, Tony, approaches math versus my way. For me, math is something I can do but not without effort. I rely heavily upon my conscious mind--likely the highly organized systematic methods of my temporal lobe--to perform mathematical functions as I walk myself through the process step by step. My older brother, on the other hand, thinks math is a breeze. He flew through calculus in high school like it was nothing and scored a perfect on the math portion of the SATs. As it turns out though, I am possibly the better math teacher because I can explain the processes involved in completing a problem as I am very consciously aware of the steps I go through. Tony has a difficult time explaining math though, as it is something he "just does," like a painter just has a knack for putting form to paper.

I theorize that when people are more skilled in areas like math (or for me, my intrinsic area that I can’t explain is art), they are utilizing parts of their mind that perform these functions subconsciously, without the need for wasting time and brain space on internal monologue.

So how can we maximize the potential of our subconscious and bring it to surface in our conscious mind? According to studies using EEGs to record brain wave activity, the prime time for surfacing of these subconscious thoughts is during trance, meditation, right before sleep, or right after. These are times when the brain produces alpha waves; a characteristic pattern made when the subconscious, unconscious, and conscious patterns are all high and seem to be communicating best with each other.

This may account for why common wisdom tells us to “sleep on it,” before you make a big decision. There are often observations you’ve made and elements to consider regarding a decision that you already technically “know,” but they must be given time to surface into the conscious mind. Sometimes, giving your chatty conscious mind a rest seems to do the trick. According to Dijksterhuis in a study publish in Science, more satisfying decisions were made by subjects who did not undergo conscious deliberation on highly detailed decisions. The subconscious appears to be the real genius in these instances, as it can deliver the best results while the conscious mind takes a rain check.

Sources:
Ap Dijksterhuis et al. "On Making the Right Choice: The Deliberation-Without-Attention Effect." Science. 2006
V.S. Ramachandran. Phantoms in the Brain. 1998
http://www.crossroadsinstitute.org/eeg.html
http://brain.oxfordjournals.org/cgi/content/abstract/120/3/535

Images:
http://www.altered-states.net/barry/newsletter193/index.htm
http://www.web-us.com/brain/PhysiologyOrdinaryConsciousness.htm
http://pr.caltech.edu/periodicals/eands/articles/LXVII2/koch.html

Out-of-Body Experience: All in the Mind

As a person with an interest in neurology, I often get asked questions about the brain. They are usually not simple questions like, “tell me about the visual pathway” or “why are some people left-handed,” rather; the more popular questions are often spiritual or paranormal in nature. I am often asked:
“What is consciousness?”
“What is the soul?” and
“How do you explain an out-of-body experience?”

The first two are delightful topics for a heated discussion, but to be honest they are probably better suited for a Rabi or Priest than a scientist.
The last one was a real stumper, and I could only theorize about what was going on. Now there is research that may be leading us in the right direction toward unraveling this mystery.


I myself have had an out-of-body experience. I was in the hospital by my friend’s side after he had been in a car accident. He was not hurt too badly, but a combination of late night sleepiness, dehydration, and possibly some chemicals in the air of the ER caused my consciousness to seem to “float” above myself, until I fell backward and hit my head in an embarrassing display of girlish fainting.

Now scientists can replicate the experience, sans late-nigh ER visit. They have discovered the area of the brain that seems to be responsible for the out of body experience, an all time favorite of mine, the angular gyrus.


Lying just caudal to the auditory centers, rostral to the vision centers, and below the kinesthetic sensory cortex, the angular gyrus (both in the left and right hemispheres) is a sort of integration ground for sensation. It is no surprise that stimulation of this tissue creates a disjunction between the senses, causing patients to have an odd sensation of body “shadowing” or a full out-of-body experience.

In Geneva, a wealth of knowledge has come from two epilepsy patients who have had dozens of electrodes implanted in their brains to pin-point the cause of their epilepsy. The women both experienced out-of-body sensations when their angular gyrus was stimulated.

The neurologist, Dr. Olaf Blanke, reported that his patients had normal psychiatric history and good mental health before the experiment.
One patient reported feeling as though she were at the ceiling and gazing down on her legs when a current flowed through the electrode near her angular gyrus. The sensation subsided when the current was off. The other woman reported a shadowy presence that seemed to loom beneath her and mimic her every move. When he turned the current off, the sensations again subsided.

Dr. Blanke’s published discoveries regarding the angular gyrus can be found here: http://www.nature.com/nature/journal/v443/n7109/abs/443287a.html, and here: http://intl-brain.oxfordjournals.org/cgi/content/abstract/127/2/243

Source:
From the article “One Body When the Brain Says Two”
Forwarded to me via Karen Cassil
Related articles: http://www.nature.com/nature/journal/v443/n7109/abs/443287a.html http://intl-brain.oxfordjournals.org/cgi/content/abstract/127/2/243
Image sources:
http://www.snibbe.com/scott/body_image/shadow_bag/index.html
http://www.answers.com/topic/angular-gyrus