We all know him. We all run away from him. Not that he is pleasant to hang around with; actually the farther away you are from him the better it is for you. And yet, my friends, and yet again I will say, he is your angel in disguise.
Run away, run far away
The panic in your heart is here to stay
For when he comes, no matter night or day
You know you’re trapped, you have no say.
He treads, he does, himself along
A smile upon his face so strong
Inside his mind, singing a song:
Ding-dong, ding-dong, ding-dong, ding-dong.
A single glimpse, a single glare
Keep up your pace, or you’ll have to bear
More than that, you’ll gasp for air,
Not me, not me, this is not fair!
Thus he be, unpleasant so
Don’t take away all his credit, though
For he is your angel, our angel, oh!
In ignorance, in disguise, although.
Free advice he generously gives,
Wraps it up in concern and niceties.
Yet closely you listen, you’ll realise this:
Escaped your eyes but News it is!
That smiling chap did not tell you
Nor did she, sweet girl in blue
But you know it well, yes you do
Thanks to him, he’s the angel, true.
Thank you, but sorry- you hear yourself say
Don’t hurt your conscience, sure you may
Run away, run far away
For the panic in your heart is here to stay.
Saturday, November 13, 2010
I’ll finish it by tomorrow, yaar...
“That’s cool!”
“Me too!”
“I’m up for anything new!”
These phrases happened to follow the mention of a daily game of football. Scholars all, wrapped up in blankets, fervently discussed and almost fixed the timings of the games and the teams involved, while the winds continued to ruffle the pages of books lying open on the bed.
“So we start tomorrow?”
Interestingly, all ‘good’ things in life always start tomorrow, or they will be accomplished by tomorrow. And “tomorrow” seems to have attained a broader meaning than its dictionary definition, broad in terms of the number of days included. Procrastination, the anthem of student life, is here to stay. And after putting aside my incomplete Physics record, I wonder: Why?
Why didn’t I buy a refill today? It’s alright, I’ll buy it tomorrow, and the shop is so close-by I can buy it anytime.
I should have recharged my phone today but I can’t. The shop is so damn far away!
Shall I read the chapter Sir has taught us today? Or I’ll read it tomorrow, its better I read the chapter entirely after it’s completed in the class.
Oh! I remember I have to give in an article for the magazine. I’ll do it by tomorrow, for sure.
My sister gave me some very good movies, I must watch them someday.
The people outside are calling me to play but I am too busy. I have to do some Math. I suck at Math.
Hey, I see her coming. I must ask her...
“Hi! I was looking for you. Could you lend me that book of yours? Thanks. Hey, don’t you think Natural selection is not a scientific theory because it is not falsifiable? Never mind. Tell me what exactly did that guy say to that girl that day?”
This is the sad story of a student.
Sad. Not because something tragic has happened but because nothing ever happens.
And we come back to our question: why?
When I was a kid, my grandfather used to tell me a story about a rabbit and a tortoise. I’m sure everyone has heard of it, where the rabbit quickly reaches near the finishing line of the sprinting race and then becomes lax and decides to take a short nap. The tortoise in the meantime slowly crosses the finishing line and thus unexpectedly wins the race. So have we all become overconfident like the rabbit?
Paulo Coelho, the philosopher, says that when we get too close to our goals or dreams or whatever, a fear of realising that dream arises within us. We suddenly start feeling guilty and forget all our hard work of the past. We commit a series of blunders and lose it all.
My dear friends let me tell you, this is all bullshit. Our problem is that we are just plain lazy. And the sad part is that we revel in it. It’s the best excuse: Sorry, I’m lazy. What can I do? God made me that way.
I should not ask a student to go against his nature, but if you are not counting this as a solution, there is none. Blame God or blame your fickle teenage hormones. If you are happy with your life, that’s good enough. Or else, it’s only you who can do something about it. Think again.
“Me too!”
“I’m up for anything new!”
These phrases happened to follow the mention of a daily game of football. Scholars all, wrapped up in blankets, fervently discussed and almost fixed the timings of the games and the teams involved, while the winds continued to ruffle the pages of books lying open on the bed.
“So we start tomorrow?”
Interestingly, all ‘good’ things in life always start tomorrow, or they will be accomplished by tomorrow. And “tomorrow” seems to have attained a broader meaning than its dictionary definition, broad in terms of the number of days included. Procrastination, the anthem of student life, is here to stay. And after putting aside my incomplete Physics record, I wonder: Why?
Why didn’t I buy a refill today? It’s alright, I’ll buy it tomorrow, and the shop is so close-by I can buy it anytime.
I should have recharged my phone today but I can’t. The shop is so damn far away!
Shall I read the chapter Sir has taught us today? Or I’ll read it tomorrow, its better I read the chapter entirely after it’s completed in the class.
Oh! I remember I have to give in an article for the magazine. I’ll do it by tomorrow, for sure.
My sister gave me some very good movies, I must watch them someday.
The people outside are calling me to play but I am too busy. I have to do some Math. I suck at Math.
Hey, I see her coming. I must ask her...
“Hi! I was looking for you. Could you lend me that book of yours? Thanks. Hey, don’t you think Natural selection is not a scientific theory because it is not falsifiable? Never mind. Tell me what exactly did that guy say to that girl that day?”
This is the sad story of a student.
Sad. Not because something tragic has happened but because nothing ever happens.
And we come back to our question: why?
When I was a kid, my grandfather used to tell me a story about a rabbit and a tortoise. I’m sure everyone has heard of it, where the rabbit quickly reaches near the finishing line of the sprinting race and then becomes lax and decides to take a short nap. The tortoise in the meantime slowly crosses the finishing line and thus unexpectedly wins the race. So have we all become overconfident like the rabbit?
Paulo Coelho, the philosopher, says that when we get too close to our goals or dreams or whatever, a fear of realising that dream arises within us. We suddenly start feeling guilty and forget all our hard work of the past. We commit a series of blunders and lose it all.
My dear friends let me tell you, this is all bullshit. Our problem is that we are just plain lazy. And the sad part is that we revel in it. It’s the best excuse: Sorry, I’m lazy. What can I do? God made me that way.
I should not ask a student to go against his nature, but if you are not counting this as a solution, there is none. Blame God or blame your fickle teenage hormones. If you are happy with your life, that’s good enough. Or else, it’s only you who can do something about it. Think again.
Growth Curve for E.coli
We carried out a practical demonstration of the logistic population growth we had learnt in our Ecology classes. Like always, it was our best friend E.coli who had to stay in the incubator.
November 23rd, 2010
Saturday 8:00 am
A handful of students walk into the Bio lab. Along with Nilesh Sir they prepare nutrient broth for ten side-arm flasks, 50ml in each. I don’t know how but they do prepare different kinds of the broth. Three for each of pH 6, pH 7 and pH 8. Of the three, one contains 0.5 g% of salt, another contains 1 g%, another contains 2 g%. Then they put E.coli into each of these flasks. That makes three into three: nine flasks. The tenth flask is the ‘standard’ against which all the other flasks will be compared during the course of the experiment.
It’s time=zero. The beginning of the experiment.
The colorimeter is turned on. The flask containing our standard solution is turned to its side, carefully, so that the liquid flows into the side-arm without spilling. This side arm is now lowered into the colourimeter, which is then set to zero.
Each of the other flasks are then analysed, one by one, with the help of the colourimeter and the values for “absorbance” at 545nm (normal yellow light) is noted down in the sheets of paper provided. I hope you did not miss the apostrophes. “Absorbance” here does not really mean absorbance, as with chemical compounds. We are using this just as a test for transparency of the broth. As our E.coli will expand its colony and grow in numbers, we need to be able to measure the population without having to count every individual. A good way of doing this is to use the fact that the presence of a larger number of E.coli will reduce the transparency of the broth and make it turbid. This it will do by the virtue of its opaqueness, which is not the same as absorbance. However, this hardly matters since all we are interested in is the relative amount of resistance to the passage of light in order to determine the relative number of E.coli population.
After noting down the readings, the students leave.
Saturday 8:30 am
A fresh handful of students enter. They take each of the side flasks out of the incubator and measure their ‘absorbance’ in the same way. Data is recorded.
At 9:00am, another group of students come in and repeat the process. This happens again after another half-an-hour. And then again and again.
Saturday 8:00 pm
It’s the last batch of students to enter the laboratory. They fill up the bottom-most row on the papers and disappear into the darkness outside.
Within a couple of days, we get the whole data set on our webmail. And we are to plot it.
Sitting back and smiling at the S-shaped curve on my laptop screen, I proceed to the theory. It’s easy and we have all been learning it in the class.
In the environment, in general, any sustainable food source can be increased linearly. This, however, causes the population to expand exponentially. This is known as the Malthusian Growth pattern (from the scientist Mall Thust). We shall write the following equation to represent the rate of population growth at a given instance of time.
Where ‘N’ is the population number, ‘b’ represents the birth rate and ‘d’ represents the death rate. If these two are constant, which is true in most cases, (b-d) can be replaced by another constant ‘r’, which is the intrinsic growth rate.
The dotted line represents the approximate instance of time when competition sets in. At this point, resource becomes scarce as compared to the population and a struggle for existence arises. The number of deaths increases. Only the fittest survive. So, beyond the dotted line, our graph above is incorrect. The population can’t continue to grow exponentially once competition sets in. We need to modify our equation.
We realize that the intrinsic growth rate ‘r’ is not a constant. In fact, it is a function of the population number. We can approximate this function as:
This is known as the logistic growth equation. The constant ‘K’ appearing in this equation is known as the Carrying Capacity.
The following graph shows how ‘r’ varies with the population number.
The population growth curve should now look like this.
Good news for us, our E.coli graphs look very much similar to this one.
We found that the carrying capacity of E.coli was higher in the broth with lower salt concentrations. This is, perhaps, because the possibility of bacterial death via exosmosis is higher in environments with high salt concentration.
November 23rd, 2010
Saturday 8:00 am
A handful of students walk into the Bio lab. Along with Nilesh Sir they prepare nutrient broth for ten side-arm flasks, 50ml in each. I don’t know how but they do prepare different kinds of the broth. Three for each of pH 6, pH 7 and pH 8. Of the three, one contains 0.5 g% of salt, another contains 1 g%, another contains 2 g%. Then they put E.coli into each of these flasks. That makes three into three: nine flasks. The tenth flask is the ‘standard’ against which all the other flasks will be compared during the course of the experiment.
It’s time=zero. The beginning of the experiment.
The colorimeter is turned on. The flask containing our standard solution is turned to its side, carefully, so that the liquid flows into the side-arm without spilling. This side arm is now lowered into the colourimeter, which is then set to zero.
Each of the other flasks are then analysed, one by one, with the help of the colourimeter and the values for “absorbance” at 545nm (normal yellow light) is noted down in the sheets of paper provided. I hope you did not miss the apostrophes. “Absorbance” here does not really mean absorbance, as with chemical compounds. We are using this just as a test for transparency of the broth. As our E.coli will expand its colony and grow in numbers, we need to be able to measure the population without having to count every individual. A good way of doing this is to use the fact that the presence of a larger number of E.coli will reduce the transparency of the broth and make it turbid. This it will do by the virtue of its opaqueness, which is not the same as absorbance. However, this hardly matters since all we are interested in is the relative amount of resistance to the passage of light in order to determine the relative number of E.coli population.
After noting down the readings, the students leave.
Saturday 8:30 am
A fresh handful of students enter. They take each of the side flasks out of the incubator and measure their ‘absorbance’ in the same way. Data is recorded.
At 9:00am, another group of students come in and repeat the process. This happens again after another half-an-hour. And then again and again.
Saturday 8:00 pm
It’s the last batch of students to enter the laboratory. They fill up the bottom-most row on the papers and disappear into the darkness outside.
Within a couple of days, we get the whole data set on our webmail. And we are to plot it.
Sitting back and smiling at the S-shaped curve on my laptop screen, I proceed to the theory. It’s easy and we have all been learning it in the class.
In the environment, in general, any sustainable food source can be increased linearly. This, however, causes the population to expand exponentially. This is known as the Malthusian Growth pattern (from the scientist Mall Thust). We shall write the following equation to represent the rate of population growth at a given instance of time.
Where ‘N’ is the population number, ‘b’ represents the birth rate and ‘d’ represents the death rate. If these two are constant, which is true in most cases, (b-d) can be replaced by another constant ‘r’, which is the intrinsic growth rate.
The dotted line represents the approximate instance of time when competition sets in. At this point, resource becomes scarce as compared to the population and a struggle for existence arises. The number of deaths increases. Only the fittest survive. So, beyond the dotted line, our graph above is incorrect. The population can’t continue to grow exponentially once competition sets in. We need to modify our equation.
We realize that the intrinsic growth rate ‘r’ is not a constant. In fact, it is a function of the population number. We can approximate this function as:
This is known as the logistic growth equation. The constant ‘K’ appearing in this equation is known as the Carrying Capacity.
The following graph shows how ‘r’ varies with the population number.
The population growth curve should now look like this.
Good news for us, our E.coli graphs look very much similar to this one.
We found that the carrying capacity of E.coli was higher in the broth with lower salt concentrations. This is, perhaps, because the possibility of bacterial death via exosmosis is higher in environments with high salt concentration.
Ethnocentrism: Strategy for winning a large scale Prisoner's Dilemma game
We decided to play the prisoner’s dilemma game on a large population scale and thus find out what is the best possible strategy for the game, given certain conditions. It is a tough task to infer any trends by observing Nature because the biological processes that we need to observe have a large time-scale. For this reason, we write certain computer programs based on theoretical models, run these, and get approximate results. We try to analyse these results and find their implications.
In NetLogo, there’s already an available library model-program on ethnocentrism that we may run. The place where all the events happen is called the playground. It has a grid-pattern, and individuals are randomly distributed among these grids. There are four kinds of individuals, each denoted by a symbol:
1.Altruists: they co-operate with everybody
2.Ethnocentrists: they co-operate only with the individuals who have a certain cognitive tag indicating that both of them belong to the same ethnic group. Or else, they defect.
3.Defectors: they defect everyone, no matter who.
4.Cosmopolitans: they have a strange behaviour- they defect individuals of the same etnic group but co-operate with those of others
Each individual interacts with each of its four neighbours based on its strategy in the prisoner’s dilemma game. So who will win this game?
After running the program several times, we concluded that ethnocentrism dominates.
Why is it so?
From our prior discussions on myxobacteria, we know that co-operation among individuals of the same species tends to increase the reproductive potential. Thus, altruists and ethnocentrists would have the best chances of dominating due to high reproductive potential. This holds, given that there are not too many defectors and cosmopolitans to take advantage of the co-operation. However, this scenario does not arise. The count of defectors in the population is low due to their inherently low reproductive potential. The number of cosmopolitans is even lower since they defect their own ethnic group and help other ethnic group(s).
In the case of altruists and ethnocentrists, there is co-operation within the ethnic group. However, when ethnocentrists come face-to-face with altruists, it is ethnocentrism that wins because ethnocentrists get extra benefit by defecting the co-operative altruists. Moreover, ethnocentrists fare better with the defectors of other ethnic groups by defecting them, unlike the altruists.
Thus, ethnocentrism is, logically, the best strategy in this game. Indeed it is a logical conclusion that the success of an ethnic group should stem from these two factors: how much co-operation there is within the group, and how much the group members can exploit individuals from other groups for their own benefit.
In NetLogo, there’s already an available library model-program on ethnocentrism that we may run. The place where all the events happen is called the playground. It has a grid-pattern, and individuals are randomly distributed among these grids. There are four kinds of individuals, each denoted by a symbol:
1.Altruists: they co-operate with everybody
2.Ethnocentrists: they co-operate only with the individuals who have a certain cognitive tag indicating that both of them belong to the same ethnic group. Or else, they defect.
3.Defectors: they defect everyone, no matter who.
4.Cosmopolitans: they have a strange behaviour- they defect individuals of the same etnic group but co-operate with those of others
Each individual interacts with each of its four neighbours based on its strategy in the prisoner’s dilemma game. So who will win this game?
After running the program several times, we concluded that ethnocentrism dominates.
Why is it so?
From our prior discussions on myxobacteria, we know that co-operation among individuals of the same species tends to increase the reproductive potential. Thus, altruists and ethnocentrists would have the best chances of dominating due to high reproductive potential. This holds, given that there are not too many defectors and cosmopolitans to take advantage of the co-operation. However, this scenario does not arise. The count of defectors in the population is low due to their inherently low reproductive potential. The number of cosmopolitans is even lower since they defect their own ethnic group and help other ethnic group(s).
In the case of altruists and ethnocentrists, there is co-operation within the ethnic group. However, when ethnocentrists come face-to-face with altruists, it is ethnocentrism that wins because ethnocentrists get extra benefit by defecting the co-operative altruists. Moreover, ethnocentrists fare better with the defectors of other ethnic groups by defecting them, unlike the altruists.
Thus, ethnocentrism is, logically, the best strategy in this game. Indeed it is a logical conclusion that the success of an ethnic group should stem from these two factors: how much co-operation there is within the group, and how much the group members can exploit individuals from other groups for their own benefit.
Isolation of Bdellovibrio bacteriovorus
I would love to start telling about it with this piece of writing I found online, by Laurel Crosby.
“A mild ocean breeze plays over the water surface, dispelling any notion that danger lurks in the murky depths. However, a gruesome event is about to occur as a silent attacker speeds forth toward an unsuspecting victim. In a furious collision, the savage meets its target and whittles its way into the body of the innocent prey. Once inside, the transformation begins - the predator ceases its frenzy and prepares to multiply. The host is reduced to a protective cocoon, supplying food and shelter for the growing parasite. Within hours, the nourishment is drained and the ghost-like shell of the host bursts open to release a new generation of deadly predators. And all the while, the waters remain still...”
This savage predator is none other than the bacteria Bdellovibrio bacteriovorus. With a length of about 1.4 microns, a comma shaped body and a corkscrew-like flagellum rotating at deadly speeds, the bdellovibrio is just one of its kind. It is the only bacteria that preys on other bacteria (bdellovibrio is gram-negative and it attacks only other gram-negative bacteria). Bdellovibrio spends most of its life between the cell membranes of its prey. It is generally found in sewage and soil. The life cycle of the bdellovibrio deserves to be elaborated upon.
The life cycle of a single bdellovibrio.
In the first step, it attacks the host cell. There is a short cognitive phase after which the bdellovibrio permanently attaches itself to the host. It then drills through the outer membrane of the host using its corkscrew flagellum. Once inside, it loses its flagellum. The hole it had drilled is now sealed. Many molecules inside the host are broken down or dissolved. The host cell assumes a roundish structure, called the bdelloplast. The bdellovibrio starts eating the host inside out, growing in size in the process. It then begins to reproduce, just like any other bacteria, via cell-division. Once the nutrients of the host get exhausted, the bdelloplast bursts open and all the new bdellovibrio produced is shot out in all directions, on their way to find new hosts. The phase of life spent inside the host cell is often referred to as the ‘growth phase’ and distinguished from the ‘attack phase’.
WHAT WE DID
Tirtashree, Pallavi, Sravani and I: We collected water samples from the sewage-rich Mula river (Pune). We mixed a bit of the water in a thick suspension of E.coli and spread it on a nutrient agar plate prepared beforehand (mix nutrient broth, agar agar,autoclave it, melt and fill the plates with requisite amount, allow it to solidify). We then kept our plates in the incubator for a day. We would come back and check the next day. If there bdellovibrio in our sample, we would be able to see plaques wherever the bdellovibrio attacked and killed E.coli.
RESULT
We observed plaques on our plates, characteristic of bdellovibrio. These were the clearings on our thick carpet of E.coli that we spread on the plates, wherever the bdellovibrio killed the E.coli. Unless there was a phage in our plates, we can say (with optimism) that we had managed to find some bdellovibrio!
Our attempts to isolate bdellovibrio by scraping out the parts around the clearings on the plate and culturing them again in other plates, along with a thick carpet of E.coli went in vain. We did not observe any plaques. We concluded that something must have gone wrong in our procedure; we might not have taken care of some crucial factors while making the plates, perhaps. Unfortunately, we could not identify our mistake. Nevertheless, we are still starting the isolation procedure afresh. There is nothing to write about it at the moment, but hopefully there will soon be.
FURTHER STUDY
We plan to extend our experiment once we succeed with the isolation process. We know it is a tough going because bdellovibrio are not easy to find. However, the fact that bdellovibrio is a species we do not know and understand much about excites me. There is always more scope for discovery. There’s one thing, for example: we know that bdellovibrio reproduces only when it is inside the host cell. However, in some experiments, bdellovibrio has reported growth even in controlled laboratory conditions in environments similar to those in host cells. What is it that the bdellovibrio actually needs in order to grow and reproduce? These are some of the mysteries that continue to elude scientists.
Who came first: Prokaryotes or Eukaryotes?
The Prokaryotes came first.
It is a commonly observed fact that complexity increases as we move on from a lower organism to a higher organism; these higher organisms appeared in the later stages of evolution. Their cells and organs became more and more specialised. We shall extend the same analogy to the evolution of cells.
A prokaryotic cell is extremely simple in structure and organisation. It is just a mass of cytoplasm surrounded by a membrane. Ribosomes and chromatin fibres are scattered inside. A eukaryotic cell is much more complex. Its chromatin fibres are enclosed in a nuclear membrane. There is a division of labour between different parts of the cell, each of the specialised parts enclosed by intracellular compartments. There is also a well-developed cytoskeleton.
We may safely deduce that in the beginning there were only prokaryotes. Ancient Fossil evidences support this assertion. Moreover, even today their descendants are found in every nook and corner of the planet, including the unimaginably cold and dark ocean depths, as well as the hot bubbling volcanoes and the poisonous sulphur springs - the conditions in the ancient earth must have been this extreme - and so ancient must be our prokaryotes.
Over the time, as the conditions settled down and the atmosphere seemed more pleasant, few of the prokaryotes decided to take some risk. The population had already increased manifold and food resources were dwindling. Many of the bacteria (prokaryotes) chose one of three ways:
Some bacteria started harnessing the solar energy to make food via photosynthesis. Others modified the way they were breaking down glucose so that they could obtain maximum energy from it. Still others started eating up each other. These bacteria became specialised as predators and they developed a strong cytoskeleton for better locomotion and engulfing motions. Their nuclear matter needed protection too and hence they developed a nuclear membrane by extending inward a part of the cell membrane, creating the endoplasmic reticulum along.
Organelles budded off this endoplasmic reticulum... and thus formed the Eukaryote.
Someone of these primitive eukaryotes ate up that prokaryote which could breakdown glucose very efficiently. This prokaryote proved to be quite strong as it continued to survive even inside the cytoplasm of the eukaryote. Today, this prokaryote is called mitochondrion. The eukaryote too lost interest in trying to digest it, as it was producing a lot more energy than it required and the eukaryote could use this up. It gave this particular eukaryote such a great evolutionary advantage that today it is hard to find a eukaryote without the mitochondria. In fact, it is more probable that that this event occurred before the development of organelles in the eukaryote because such complex developments couldn’t have proceeded without the vast amount if energy produce by the mitochondria.
On another occasion a Eukaryote might have swallowed a photosynthetic prokaryote, which was eventually known as chloroplast- thus started the story of plant cells.
So in every way that we see, and the deeper we look into the possible evolutionary development in accordance to most believable mechanisms, the more we believe that it were the prokaryotes that came first.
It is a commonly observed fact that complexity increases as we move on from a lower organism to a higher organism; these higher organisms appeared in the later stages of evolution. Their cells and organs became more and more specialised. We shall extend the same analogy to the evolution of cells.
A prokaryotic cell is extremely simple in structure and organisation. It is just a mass of cytoplasm surrounded by a membrane. Ribosomes and chromatin fibres are scattered inside. A eukaryotic cell is much more complex. Its chromatin fibres are enclosed in a nuclear membrane. There is a division of labour between different parts of the cell, each of the specialised parts enclosed by intracellular compartments. There is also a well-developed cytoskeleton.
We may safely deduce that in the beginning there were only prokaryotes. Ancient Fossil evidences support this assertion. Moreover, even today their descendants are found in every nook and corner of the planet, including the unimaginably cold and dark ocean depths, as well as the hot bubbling volcanoes and the poisonous sulphur springs - the conditions in the ancient earth must have been this extreme - and so ancient must be our prokaryotes.
Over the time, as the conditions settled down and the atmosphere seemed more pleasant, few of the prokaryotes decided to take some risk. The population had already increased manifold and food resources were dwindling. Many of the bacteria (prokaryotes) chose one of three ways:
Some bacteria started harnessing the solar energy to make food via photosynthesis. Others modified the way they were breaking down glucose so that they could obtain maximum energy from it. Still others started eating up each other. These bacteria became specialised as predators and they developed a strong cytoskeleton for better locomotion and engulfing motions. Their nuclear matter needed protection too and hence they developed a nuclear membrane by extending inward a part of the cell membrane, creating the endoplasmic reticulum along.
Organelles budded off this endoplasmic reticulum... and thus formed the Eukaryote.
Someone of these primitive eukaryotes ate up that prokaryote which could breakdown glucose very efficiently. This prokaryote proved to be quite strong as it continued to survive even inside the cytoplasm of the eukaryote. Today, this prokaryote is called mitochondrion. The eukaryote too lost interest in trying to digest it, as it was producing a lot more energy than it required and the eukaryote could use this up. It gave this particular eukaryote such a great evolutionary advantage that today it is hard to find a eukaryote without the mitochondria. In fact, it is more probable that that this event occurred before the development of organelles in the eukaryote because such complex developments couldn’t have proceeded without the vast amount if energy produce by the mitochondria.
On another occasion a Eukaryote might have swallowed a photosynthetic prokaryote, which was eventually known as chloroplast- thus started the story of plant cells.
So in every way that we see, and the deeper we look into the possible evolutionary development in accordance to most believable mechanisms, the more we believe that it were the prokaryotes that came first.
Restriction Digestion and ligation
Restriction Digestion
You might be tempted to think it’s just a ‘restricted digestion’ of DNA (DNA-what else do you imagine bio experiments to be about!). Well no! The nomenclature has an entirely different significance that we shall soon find out.
We use ‘restriction enzymes’ to cut selective parts of DNA. In our experiment we shall cut a plasmid and make it linear.
Let us know a bit more about these ‘restriction enzymes’. They seem to have been specially invented by our genes to break down harmful phage-DNA. In this way they ‘restricted’ viral infection, hence the name 'restriction enzymes’ (for enzymes which restrict)- and their corresponding enzymatic action, or ‘digestion’, is called ‘restriction digestion’ (digestion to restrict, and not restricted digestion).
Restriction enzymes recognise specific sites on the DNA, where they cut. So, restriction enzymes are highly specific. Some well-known restriction enzymes include EcoRI, RY13 and HInD III.
Restriction enzymes can cut DNA in two ways:
(1)To produce blunt ends
A sharp symmetric cut in the DNA- both the helices of the DNA are cut in the same place.
(2)To produce cohesive/sticky ends
DNA is cut asymmetrically, so the broken ends can still rejoin. There is a tendency to form bonds again at the broken end. This is very useful in cloning, and this is the sort of enzyme we are going to work with.
This is our plasmid, the common PUC18. We observe it has 3 important parts that can be demarcated.
We must make sure that the restriction enzyme we are using does not affect the origin of replication or the antibiotic marker.
The MCS is a speciality. This region contains many ‘cut-able’ sites, each of which can be recognised by some restriction enzyme. Hence, a wide variety of restriction enzymes can be used to cut this DNA at this point.
So we start the experiment. In the given centrifuge tube, we take 10µl of PUC18 plasmid DNA, 2µl of 10X array buffer, 0.8µl of EcoRI (that’s our enzyme) and 7.2µl of water to make it up to 20µl. Mix the stuff by pipetting. Now put it in the incubator at 370C for 1 hour.
That’s it! We are done with the protocol! Cool speed, innit?
We can now do the electrophoresis to check if our plasmids have really been cut.
One important thing to be noted here, before saying goodbye, is the composition of our dye:
1.Xylene Cynol ≈ corresponds to 1-2 Kbp (kilo base pairs) of DNA
2.BPB (bromo phenol blue) ≈500bp
3.Orange G ≈ 50bp
4.Glycerol/sucrose- these heavy molecules help take the contents of our sample to the bottom of the well while loading.
Ligation
How integration follows differentiation in a mathematics course, ligation must follow restriction digestion.
Here, we use another set of enzymes known as DNA ligases- these re-establish the phosphodiester bonds in DNA, thus laterally sealing the broken ends of DNA. The reaction is pH-specific (so we’ll use a buffer in our experiment) and ligases require ATP as a cofactor.
We shall start our experiment without further ado. Let’s take the DNA we cut in the previous experiment and try rejoining to get back our plasmid. In a centrifuge tube, we take 10ml of digested DNA from the previous experiment. Add 2µl of cohesive end buffer (this buffer already contains all the ATP we need), 7.5µl of distilled water and 0.5µl of T4 DNA ligase- this ligase is commonly used by bacteriophages. Incubate it overnight at 40C. That must do it.
Next evening we come back and do the electrophoresis to see how successful our experiment has been.
And it has been quite successful indeed!
A short note on RDT
RDT or Recombinant DNA Technology is a branch of biology that is rapidly growing in its popularity. Here, DNA is cut or modified using restriction enzymes and ligases. This is more popularly known as ‘cloning’ (although the general public image of cloning is quite contrary to this due to the fact that the word ‘clone’ is more often associated with the cloned sheep Dolly, and hence used to mean ‘a process of making copies of living beings’.) Directionality is very important in RDT. Sometimes we use more than one enzyme to make the reaction more specific. We can thus ‘cut and paste’ DNA fragments from one place to another, or insert more DNA into the DNA chain.
You might be tempted to think it’s just a ‘restricted digestion’ of DNA (DNA-what else do you imagine bio experiments to be about!). Well no! The nomenclature has an entirely different significance that we shall soon find out.
We use ‘restriction enzymes’ to cut selective parts of DNA. In our experiment we shall cut a plasmid and make it linear.
Let us know a bit more about these ‘restriction enzymes’. They seem to have been specially invented by our genes to break down harmful phage-DNA. In this way they ‘restricted’ viral infection, hence the name 'restriction enzymes’ (for enzymes which restrict)- and their corresponding enzymatic action, or ‘digestion’, is called ‘restriction digestion’ (digestion to restrict, and not restricted digestion).
Restriction enzymes recognise specific sites on the DNA, where they cut. So, restriction enzymes are highly specific. Some well-known restriction enzymes include EcoRI, RY13 and HInD III.
Restriction enzymes can cut DNA in two ways:
(1)To produce blunt ends
A sharp symmetric cut in the DNA- both the helices of the DNA are cut in the same place.
(2)To produce cohesive/sticky ends
DNA is cut asymmetrically, so the broken ends can still rejoin. There is a tendency to form bonds again at the broken end. This is very useful in cloning, and this is the sort of enzyme we are going to work with.
This is our plasmid, the common PUC18. We observe it has 3 important parts that can be demarcated.
We must make sure that the restriction enzyme we are using does not affect the origin of replication or the antibiotic marker.
The MCS is a speciality. This region contains many ‘cut-able’ sites, each of which can be recognised by some restriction enzyme. Hence, a wide variety of restriction enzymes can be used to cut this DNA at this point.
So we start the experiment. In the given centrifuge tube, we take 10µl of PUC18 plasmid DNA, 2µl of 10X array buffer, 0.8µl of EcoRI (that’s our enzyme) and 7.2µl of water to make it up to 20µl. Mix the stuff by pipetting. Now put it in the incubator at 370C for 1 hour.
That’s it! We are done with the protocol! Cool speed, innit?
We can now do the electrophoresis to check if our plasmids have really been cut.
One important thing to be noted here, before saying goodbye, is the composition of our dye:
1.Xylene Cynol ≈ corresponds to 1-2 Kbp (kilo base pairs) of DNA
2.BPB (bromo phenol blue) ≈500bp
3.Orange G ≈ 50bp
4.Glycerol/sucrose- these heavy molecules help take the contents of our sample to the bottom of the well while loading.
Ligation
How integration follows differentiation in a mathematics course, ligation must follow restriction digestion.
Here, we use another set of enzymes known as DNA ligases- these re-establish the phosphodiester bonds in DNA, thus laterally sealing the broken ends of DNA. The reaction is pH-specific (so we’ll use a buffer in our experiment) and ligases require ATP as a cofactor.
We shall start our experiment without further ado. Let’s take the DNA we cut in the previous experiment and try rejoining to get back our plasmid. In a centrifuge tube, we take 10ml of digested DNA from the previous experiment. Add 2µl of cohesive end buffer (this buffer already contains all the ATP we need), 7.5µl of distilled water and 0.5µl of T4 DNA ligase- this ligase is commonly used by bacteriophages. Incubate it overnight at 40C. That must do it.
Next evening we come back and do the electrophoresis to see how successful our experiment has been.
And it has been quite successful indeed!
A short note on RDT
RDT or Recombinant DNA Technology is a branch of biology that is rapidly growing in its popularity. Here, DNA is cut or modified using restriction enzymes and ligases. This is more popularly known as ‘cloning’ (although the general public image of cloning is quite contrary to this due to the fact that the word ‘clone’ is more often associated with the cloned sheep Dolly, and hence used to mean ‘a process of making copies of living beings’.) Directionality is very important in RDT. Sometimes we use more than one enzyme to make the reaction more specific. We can thus ‘cut and paste’ DNA fragments from one place to another, or insert more DNA into the DNA chain.
The Physically and Chemically Distinguishing Properties of the Lysosomal membrane
Among the many important membrane-bound organelles in the cell is the sac-like Lysosome. Its basic function is digestion. A cell ingests food or any other material into it, and stores it in a vesicle-like structure called an endosome. We do not pretty much know how this ingested substance reaches the lysosome from the endosome. However, a rough guess is that either the endosome fuses with the Lysosome, or matures and develops into it.
Lysosomes contain about 40 different types of hydrolytic enzymes, including those that degrade proteins, nucleic acids, oligosaccharides, and phospholipids. All of these enzymes are optimally active in the acidic conditions (pH~5) maintained within Lysosomes, against the basic cytosol outside. The Lysosome not only contains a unique collection of enzymes but also has a unique surrounding membrane.
The Lysosomal membrane is very special indeed. It contains transport proteins that allow the final products of the digestion of macromolecules to be transported to the cytosol, from where they can be either excreted or utilised by the cell. It also contains an ATP-driven H+ pump which pumps H+ (protons) into the lysosome, thereby maintaining its contents at an acidic pH. Most of the Lysosomal membrane proteins are unusually highly glycosylated: the sugars, which cover much of the protein surfaces facing the lumen, protect the proteins from digestion by the Lysosomal proteases.
A Lysosome may digest one of the cell’s own organelles, or even the cell itself (by rupturing the Lysosomal wall and letting the acids within to flow out)- this it does when destruction becomes necessary to protect the other cells nearby from acquiring same infection that its own cell has caught. Hence, Lysosomes are often referred to as the ‘suicide bags’ of the cell.
Lysosomes contain about 40 different types of hydrolytic enzymes, including those that degrade proteins, nucleic acids, oligosaccharides, and phospholipids. All of these enzymes are optimally active in the acidic conditions (pH~5) maintained within Lysosomes, against the basic cytosol outside. The Lysosome not only contains a unique collection of enzymes but also has a unique surrounding membrane.
The Lysosomal membrane is very special indeed. It contains transport proteins that allow the final products of the digestion of macromolecules to be transported to the cytosol, from where they can be either excreted or utilised by the cell. It also contains an ATP-driven H+ pump which pumps H+ (protons) into the lysosome, thereby maintaining its contents at an acidic pH. Most of the Lysosomal membrane proteins are unusually highly glycosylated: the sugars, which cover much of the protein surfaces facing the lumen, protect the proteins from digestion by the Lysosomal proteases.
A Lysosome may digest one of the cell’s own organelles, or even the cell itself (by rupturing the Lysosomal wall and letting the acids within to flow out)- this it does when destruction becomes necessary to protect the other cells nearby from acquiring same infection that its own cell has caught. Hence, Lysosomes are often referred to as the ‘suicide bags’ of the cell.
DNA Isolation
We’ll use our favourite E.coli yet again. Poor E.coli!
We take a pellet of it as before, and add T10E1 buffer to it. We mix up the stuff by vortexing.
We won’t do anything as elaborate as what we did while isolating the plasmid DNA- we directly come to THE POINT: 10µl of lysozyme. Using its own enzyme against itself. Sad.
Now that all its membranes are gone, we incubate it for 15 minutes at 370C for the reaction to reach a completion. Then we add 30µl of 10% SDS- that’s for dissolving all the lipid stuff. 3µl of proteinase K takes care of the protein molecules. Just give it some more time: incubate at 370C for half-an-hour.
Next, we shall use a wonderful technique to separate the cell parts- selective solvation. We add 250µl of tris saturated phenol and another 250µl of chloroform. Mix it up by inverting. Follow it up with a little centrifugation and the cell parts all separate out. Chloroform dissolves most of the cell parts while phenol dissolves the protein remains. DNA, as before, remains suspended in water forming hydro-complexes.
Carefully using a micropipette, extract as much of the supernatant as possible (that’s usually 3ml), without disturbing the phenol layer, and transfer it to a new tube. To it, add 150µl of 5M NaCl, and twice its own volume of chilled ethanol. Invert mix and then centrifuge.
A pellet of DNA is formed. Decant the ethanol. This is the familiar ‘salting out’ process that we carried out during our plasmid extraction. This is followed by ‘ethanol wash’ and another centrifugation. The ethanol is then removed by pipetting.
And our DNA has finally been isolated!
Dissolve it in 30µl of T10E1 buffer where individual molecules of DNA separate out and fold properly.
Now we can check the length of our DNA by electrophoresis.
We observe some RNA impurities too. If you really want to prevent this, you could use RNAase during the isolation process- it is an enzyme that degrades RNA.
The Mitochondrion: A story of a little cell organelle
The mitochondrion is perhaps the most fascinating organelle in the eukaryotic cell. Under the electron microscope, each mitochondrion appears sausage-shaped, or you may call it worm-shaped, from one to many micrometres long; each is enclosed in two separate membranes. The inner membrane is thrown into finger-like folds called ‘cristae’, projecting inward. Inside the double-wall, are scattered many tiny dot-like ribosomes. Remarkably, the mitochondrion even has its own DNA! And yeah, it multiplies itself.
We broke open the cell and spun the soup of cell fragments in a centrifuge- this separated the cell organelles according to their size, shape and density. We now tested the purified mitochondria to see what chemical process they could perform. And we discovered that they oxidised sugar to produce adenosine triphosphate (ATP) - the basic chemical fuel that powers most of the cell’s activities. So the mitochondria are generators of chemical energy for the cell!
And in the process, they consume oxygen and release carbon dioxide- this indeed is respiration at the cellular level. The fact is further confirmed by the observation that mitochondria are particularly concentrated in those part of the cell that use up more energy, like the contractile apparatus of the cardiac muscle cell or the tail of the sperm cell.
So how is it that the food that we eat ultimately reaches the mitochondria which release energy from it?
All that we eat primarily consists of carbohydrates and fats. Our food is broken down to these fundamental units by our digestive system before reaching each cell.
Inside the cytoplasm, glucose and other carbohydrates are oxidised to pyruvate in a process known as glycolysis, a kind of fermentation or partial oxidation, producing two ATP molecules per glucose molecule. Fats are converted into fatty acids. This is the beginning.
This pyruvate and fatty acid dissolved in the cytosol freely enters the bilipid outer membrane of the mitochondrion through wide aqueous channels in it formed by the transport protein called ‘porin’, to reach the inter-membrane space between the outer membrane and the inner membrane. Due to high permeability of the outer membrane the inter-membrane fluid is very similar to the cytosol. In addition, it contains several enzymes that use the freshly-prepared ATP to phosphorylate other nucleotides.
Our food then reaches the inner membranes whose folded walls greatly increase the surface area of the membrane. Unlike other membranes in the cell, this membrane is impermeable to the passage of ions and most small molecules.....except where a path is provided by membrane transport proteins. It also has many other important proteins that participate in the energy production process. As a consequence of the limited permeability of the inner membrane, the fluid inside, called the ‘matrix’, is highly specialised in its contents. This is where our pyruvate and fatty acids reach. And this is where the REAL STUFF takes place.
In the mitochondrial matrix, pyruvate and fatty acids- the fuel molecules- are converted to the crucial metabolic intermediate acetyl CoA by enzymes located in the matrix. In this form, our food enters the Kreb’s Cycle, more often called the Citric Acid Cycle.
Although this cycle is considered to be a part of the aerobic metabolism, it does not itself use molecular oxygen. The cycle converts the carbon atoms in acetyl CoA to carbon dioxide, which is released from the cell as a waste product. The rest of the molecule is converted to activated NADH and FADH2- the high energy electron molecules. These molecules are transferred to the inner mitochondrial membrane, where they enter the electron-transport chain- it starts off with the formation of NAD+ and a high energy electron. This electron zigzags its way through a series of electron carriers on the inner mitochondrial membrane and finally reacts with exported molecular oxygen (this is where O2 comes into the picture during respiration) to form water. The energy released by the electron during its journey through the electron transport chain is harnessed to pump protons outside, across the inner mitochondrial membrane, thus producing an electrochemical proton gradient- an uneven distribution of proton inside and outside the inner mitochondrial membrane. This configuration stores a vast amount of potential energy.
The protons are then allowed to flow back across the membrane, down their electrochemical gradient, through a protein complex called ATP synthase, which catalyzes the energy-requiring synthesis of ATP from ADP and inorganic phosphate (Pi) in the matrix. This enzyme serves the role of a turbine, permitting the proton gradient to drive the production of ATP. The linkage of electron transport, proton pumping and ATP synthesis is known as chemiosmotic coupling. This, plus the process of consumption of O2, together is known as Oxidative Phosphorylation. This process is so efficient that it produces thirty molecules of ATP for every molecule of glucose.
ATP- this is the fate of most part of the things that we eat. It is the energy currency of the cell- the form in which energy is stored in the cell. This energy can be utilised to do work anytime, converting ATP back to ADP. And the cycle goes on......
Developments in research have enabled us to have a greater insight into the mitochondrial structure, function, and evolution. Mitochondria have been found to contain their own DNA, RNA and a complete transcription and translation system including ribosomes, which allows them to synthesise some of their own proteins. Mitochondria move about in the cell along the thread-like microtubules of the cytoskeleton. They may also remain fixed in one cellular location to target ATP directly to a site of unusually high ATP consumption. They can also multiply rapidly within the cell when there arises a need for greater energy production.
The earliest cells may have produced ATP by breaking down organic molecules left by earlier geochemical process, using some form of fermentation. Over the time, some bacteria developed much more efficient methods for generating energy and synthesizing ATP, similar to the mitochondrial mechanisms that we know today. The history of evolution of such bacteria is indeed very interesting- but that is another long story. All we need to understand is that these bacteria evidently had an advantage over the other unicellular organisms which still depended on the inefficient process of glycolysis to produce energy.
It was a lucky accident when one of our single-celled eukaryotic ancestors gulped down one of these bacteria, and decided to keep it, and christened it- Mitochondrion. Ever since, the mitochondrion has become an integral part of the eukaryotic cell, producing energy in a symbiotic relationship. Today, the mitochondria in our cells have become much more specialised for energy production. Still, it has retained its individuality- its own DNA, and a double-membrane- one belonging to the bacterium, another provided by the cell.
We broke open the cell and spun the soup of cell fragments in a centrifuge- this separated the cell organelles according to their size, shape and density. We now tested the purified mitochondria to see what chemical process they could perform. And we discovered that they oxidised sugar to produce adenosine triphosphate (ATP) - the basic chemical fuel that powers most of the cell’s activities. So the mitochondria are generators of chemical energy for the cell!
And in the process, they consume oxygen and release carbon dioxide- this indeed is respiration at the cellular level. The fact is further confirmed by the observation that mitochondria are particularly concentrated in those part of the cell that use up more energy, like the contractile apparatus of the cardiac muscle cell or the tail of the sperm cell.
So how is it that the food that we eat ultimately reaches the mitochondria which release energy from it?
All that we eat primarily consists of carbohydrates and fats. Our food is broken down to these fundamental units by our digestive system before reaching each cell.
Inside the cytoplasm, glucose and other carbohydrates are oxidised to pyruvate in a process known as glycolysis, a kind of fermentation or partial oxidation, producing two ATP molecules per glucose molecule. Fats are converted into fatty acids. This is the beginning.
This pyruvate and fatty acid dissolved in the cytosol freely enters the bilipid outer membrane of the mitochondrion through wide aqueous channels in it formed by the transport protein called ‘porin’, to reach the inter-membrane space between the outer membrane and the inner membrane. Due to high permeability of the outer membrane the inter-membrane fluid is very similar to the cytosol. In addition, it contains several enzymes that use the freshly-prepared ATP to phosphorylate other nucleotides.
Our food then reaches the inner membranes whose folded walls greatly increase the surface area of the membrane. Unlike other membranes in the cell, this membrane is impermeable to the passage of ions and most small molecules.....except where a path is provided by membrane transport proteins. It also has many other important proteins that participate in the energy production process. As a consequence of the limited permeability of the inner membrane, the fluid inside, called the ‘matrix’, is highly specialised in its contents. This is where our pyruvate and fatty acids reach. And this is where the REAL STUFF takes place.
In the mitochondrial matrix, pyruvate and fatty acids- the fuel molecules- are converted to the crucial metabolic intermediate acetyl CoA by enzymes located in the matrix. In this form, our food enters the Kreb’s Cycle, more often called the Citric Acid Cycle.
Although this cycle is considered to be a part of the aerobic metabolism, it does not itself use molecular oxygen. The cycle converts the carbon atoms in acetyl CoA to carbon dioxide, which is released from the cell as a waste product. The rest of the molecule is converted to activated NADH and FADH2- the high energy electron molecules. These molecules are transferred to the inner mitochondrial membrane, where they enter the electron-transport chain- it starts off with the formation of NAD+ and a high energy electron. This electron zigzags its way through a series of electron carriers on the inner mitochondrial membrane and finally reacts with exported molecular oxygen (this is where O2 comes into the picture during respiration) to form water. The energy released by the electron during its journey through the electron transport chain is harnessed to pump protons outside, across the inner mitochondrial membrane, thus producing an electrochemical proton gradient- an uneven distribution of proton inside and outside the inner mitochondrial membrane. This configuration stores a vast amount of potential energy.
The protons are then allowed to flow back across the membrane, down their electrochemical gradient, through a protein complex called ATP synthase, which catalyzes the energy-requiring synthesis of ATP from ADP and inorganic phosphate (Pi) in the matrix. This enzyme serves the role of a turbine, permitting the proton gradient to drive the production of ATP. The linkage of electron transport, proton pumping and ATP synthesis is known as chemiosmotic coupling. This, plus the process of consumption of O2, together is known as Oxidative Phosphorylation. This process is so efficient that it produces thirty molecules of ATP for every molecule of glucose.
ATP- this is the fate of most part of the things that we eat. It is the energy currency of the cell- the form in which energy is stored in the cell. This energy can be utilised to do work anytime, converting ATP back to ADP. And the cycle goes on......
Developments in research have enabled us to have a greater insight into the mitochondrial structure, function, and evolution. Mitochondria have been found to contain their own DNA, RNA and a complete transcription and translation system including ribosomes, which allows them to synthesise some of their own proteins. Mitochondria move about in the cell along the thread-like microtubules of the cytoskeleton. They may also remain fixed in one cellular location to target ATP directly to a site of unusually high ATP consumption. They can also multiply rapidly within the cell when there arises a need for greater energy production.
The earliest cells may have produced ATP by breaking down organic molecules left by earlier geochemical process, using some form of fermentation. Over the time, some bacteria developed much more efficient methods for generating energy and synthesizing ATP, similar to the mitochondrial mechanisms that we know today. The history of evolution of such bacteria is indeed very interesting- but that is another long story. All we need to understand is that these bacteria evidently had an advantage over the other unicellular organisms which still depended on the inefficient process of glycolysis to produce energy.
It was a lucky accident when one of our single-celled eukaryotic ancestors gulped down one of these bacteria, and decided to keep it, and christened it- Mitochondrion. Ever since, the mitochondrion has become an integral part of the eukaryotic cell, producing energy in a symbiotic relationship. Today, the mitochondria in our cells have become much more specialised for energy production. Still, it has retained its individuality- its own DNA, and a double-membrane- one belonging to the bacterium, another provided by the cell.
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