Heat Shock explained

Transformation is a method of putting DNA (either unaltered or engineered) inside bacteria. There are a couple of standard ways of doing this: Heat Shock and Electroporation.

It's funny how no one really knows how the heat shock method of transformation works. While doing my thesis, I had found this little article that some researchers from West Bengal had written. I think it's a cogent explanation for the phenomenon.

First off, you can't use any old bacteria for this process. In nature, there are some bacteria that undergo natural transformation, but-- as far as I know-- your garden variety (or maybe I should say Lab variety) E. coli cannot be used this way. You have to make the cells competent using Calcium Chloride and a little bit of black magic. My old PI told me that making competent cells was something of a dark art. Anyhow, I am digressing.

The standard protocol for heat shock (in E.coli) is as follows:
1. Mix 40 microlitres of bacteria with about 2 microlitres of DNA (the volumes vary).
2. Leave them on ice (in an eppendorf tube) for about half an hour.
3. Place the cells in a water bath at 42 degrees celsius for 45 seconds. However, this time varies for different competent cells and it ranges from 30s-1 min. I have also heard of people doing it for 2 min.
4. Get the cells out of the waterbath and incubate on ice for 2-3 min.
5. Pipet the bacteria/DNA mix into about 900 microlitres of LB or whatever you're using as culture medium. Caution: don't use the selection medium yet.
6. Place the liquid medium with the bacteria in it inside a shaker at 37 degrees Celsius.
7. After 50 min. pipet 10-200 microlitres of the liquid culture onto an agar plate with selection medium and grow overnight in a 37 degree Celsius incubator.

Now why does this process work? The Bengali researchers claim that it's because when you first cool down the bacteria on ice, they release lipids from their cell membrane in an effort to increase the fluidity of the membrane since it tends to reduce at cold temperatures. This causes pores to form on the membrane surface which can be visualised using SEM. The heat shock step apparently depolarises the membrane which tends to be a little negative on the inside. Now, I haven't read their paper. I only read the abstract. However, I know that DNA is negative and the depolarisation of the membrane may eliminate the electrostatic repulsion the DNA would have faced otherwise. I don't know if this is exactly what they're proposing, but you can find out if you want to shell out some money for their paper. (http://www.ncbi.nlm.nih.gov/pubmed/18651316).

While others were being counseled

I was captivated by the beauty of winter. I saw these awesome shaped crystals of water on the windshields of cars in the morning. They're like the snowflakes I used to see on Cartoon Network as a child. I also took a second picture of the quad in front of Earle Hall on which I saw that the part of the lawn that was in the shadow of Earle hall still had frost, but the part that the sun could reach was defrosted.
I like the picture because it tells the story and I think it might be a good cure for the aforementioned pathetic people who require peer counseling for winter-blues.

A future full of whiners

I saw a singularly distressing poster in the UC this morning. It advertised peer-counseling for winter-blues. What the hell? Is there a single thing that we don't need counseling for? Are we breeding a society of wimps who need to be cheered up by their peers for the mildest unpleasnt occurrings in their lives? Whatever happened to "Stop whining, old chap; pick up your socks and move on"? Next we'll have counseling for late trains, lost chewing gum, bad-tasting coffee, and other ridiculous trifles. Bah!

An ecosystem of my own

As I was ascending the steps to my room today, I noticed something almost...heartwarming.

I have missed two trash days in succession. Consequently, I had tonnes of rubbish at home which had begun to attract the attention of flies. In an attempt to avoid being busted by health and safety officers, I moved the rubbish bags outside and kept them on the landing of the stairs. The stairs overlook the garden. I did this three days ago.

Today, I noticed a spider web adjacent to the rubbish bags (it is indicated by the arrowin the picture to the left). It struck me that I had created an awesome bait for the spiders. Sensing the trash bags, some moronic fly would probably zoom towards it. Unfortunately for it, his velocity would be abruptly brought down to zero by the subtly woven spider web between the two bars of the railing right in front of the garbage. The aftermath needn't be mentioned.

I must say, however, that I did that old spider a bit of a favour when I put the rubbish out. To me, this is another instance of the chaos theory; I hadn't even dreamed that I'd create a sort of micro-ecosystem through my simple act of putting the garbage out. I love the complexity of our world.

Hmm...I don't suppose my actions were particularly heart warming for the moronic flies, though.

Indian Genomic Variation Database

The government of India is doing something really fantastic for once. You can see it yourself at http://www.igvdb.res.in/.

Basically, they're mapping genes of pharmacological and medical significance and keeping track of the different haplotypes and Single NucleotidePolymorphisms (SNPs) in the Indian population. They find pretty vital things about the population's response to drugs. For example, they found that 13% of the North Indian population is unresponsive to 30 of the major drugs used by doctors.

Such discoveries could pave the path to tailor-made treatments and should, probably, lower the medical expenses for the Indian people. Obviously, if they know that they're resistant to a particular expensive drug, they won't waste their time and money trying it out in ignorance.

This project shall have an impact on things ranging from Asthma to Malaria. I'm really quite excited by this. For some reason, I used to harbour the idea that molecular biology doesn't have much of an application in improving rural health in India. I suppose I'll have to rethink my beliefs now.

ICAR

ICAR. No, that’s not a new Apple product. It is the acronym for the international conference on Arabidopsis Research. I have been attending it since Wednesday (23^rd July 2008). Tomorrow (Sunday) will be the last date. It’s really been quite an experience. Anyway, I’ll try to begin from the beginning.

We drove for seven hours to Montreal. It surprisingly close. I thought it would take several days to get here from NY by car. lol! Shows you how smart I am. Anyway, the customs didn't really pose a problem and I was really delighted to be amongst signs that featured extra U''ss and re's instead of er in the word centre. Yes, Canada is part of the British commonwealth and I began to feel absolutely at home, despite the fact that the car still drove on the right side of the road. That fact disappointed me, but Dr. Hobie heaved a sigh of relief. I don't know why he did that because he was here previously and knew that already. He did it anyway. Oops, I forgot to introduce the cast of characters. There' s Dr. Hobbie, me, and this sri lankan graduate student called Mederian Kotuwale Achala Jayasen. I believe she has a fifth name as well, but it escapes me. She dearly seeks to reduce her names to a manageable two or three. I pointed out that she was bloody lucky to have only five names. A very very famous sri lankan bowler's name is Chaminda Vaas. However, that isn't his whole name. There are 46 other names in between the Chaminda and the Vass ("Hmm....what was my name again?").

When we got into montreal proper, I had to become responsible. Dr. Hobbie handed me the directions and I had to help him navigate to the Hyatt Regency Hotel where the conference is being held. I had some fun with the french names, but I was happy to see that my pronounciation wasn't as spectacularly off as it was in the german class. In fact, I think my seventh grade french teacher would be positively proud of me. Whether it be Jeanne-mance or renee- levesque boulevard. I kept my end up! Anyhow, we reached the hotel and I saw lots of chappies with banners with the letters CSN on them. I naturally assumed that they were part of some sport fan group. However, on arrival we were informed that they were, in fact, hotel staff that were on strike and were being negotiated with. Consequently, there was no parking service and we had to sort through the sorties (exits) and the entrees (entrys) to the Stationment des Jardins (parking space of the Garden mall within the hotel). I was quite happy to see that I could decipher the bloody french signs after a fashion. I suggested to Dr. Hobbie that Canada could save tonnes of money by adopting English as its sole official language and print boards only in English. However, I don't think that's happening since Canadians are like the Nepalese of North America. They're dependent on the US in many ways, but will never admit and insist on maintaining a disparate identity.

For some bloody weird reason, the hotel had 6 floors and from the 6th floor, you could take another escalator to floors 1-17, completely different from the ones we had just encountered. Anyhow, we managed to find our way around and after an ice-cream (for which we paid US dollars and got canadian dollars instead)., we went in for the first talk. This talk is by a guy who you consider to have no life since he established my weed as a model genetic organism. It was spectacular. He talked about how ethanol is not the future due to its inefficiency and how we can use other crops and processes to manage our energy requirements. It was fascinating, particularly, because if I do a Ph.D, then this is one of the fields I would consider working in. Anyway, we have set up our posters and found our way to the McGill residence hall. Honestly, this hall looks amazingly like a hotel. My room-mate is an asian post-doctoral chap called Mingje Chen. I didn't want to ask if he was from China, but I think he is. He seems to know a little bit about my work so that's good. I always thought I did very obscure work in the lab, but most of the posters I saw today are on Auxin and I actually met some of the people whose scientific papers I had read last year during my independent study. It was a great pleasure to shale their hands. However, if I meet a particular guy called Benfey, I'll make sure I wring his hand hard for all the torture his paper inflicted on my brain cells. It was horribly complex and neither Dr. Hobbie nor I could make head or tail of his work.

The highlight of the conference, for me, was a meeting with Dr. Malcolm Bennett who works in the University of Nottingham and is also working on the AXR4 gene. I had seen him speak earlier on and he actually gave one of the best presentations in the conference. At least, that’s the way it appeared to me. Having read the paper he presented might have helped my understanding and enhanced my enjoyment of his talk.We talked for a few minutes during the poster sessions and we discussed auxin transport modelling. Last year, there had been a paper, by Dr. Ben Scheres of Utrecht, in which auxin import proteins were considered of negligible importance. That paper outraged my sensibilities (no surprise because I spend a lot of time working on an auxin-import related gene) and I had protested to the author directly. Malcolm agreed with me and explained why Dr. Scheres’s model worked despite this royally flawed assumption. Apparently, physiological parameters for auxin permittivity had not been adhered to. It was heartening to be vindicated.!!!

All in all, it was a fantastic trip. I learned tonnes and it was nice to add another country to my “visited list”. I think I am also in a better position, now, to decide whether I want to do a pure Ph.D or not.

Ganga Mata (Mother Ganges)

I was quite appalled when I read the Economist's article about the crappy state of the Ganga (no pun intended). Sewage is being discharged into it without treatment and corpses are flung into the river with religious fervour. Basically, we Indians (esp. Hindus) manage to mess up the river throughout our lives and even after death. The image to the left is from an organisation called the Eco-Friends. They monitor the pollution in the Ganga and have written an admirable report about it. I applaud their efforts.

Do allow me to shock you. The coliform density (density of E.coli bacteria from human intestines) is 12000 times above the safe limit. Last year, 3000 corpses were floating around in the Ganga. This is an illegal practice. Electric crematoria have been set up to avoid this source of pollution. and there are stretches in which the river is actually Black! Millions of Indians are dependent upon this river for their domestic and their spiritual needs. Yes, the situation is indeed ghastly.

The government of India has put in 600 million dollars into the Ganga and the Yamuna, but nothing seems to have transpired yet. The irony is that the government is intent on using electric sewage treatment units. That's a joke in our country of power-cuts. Luckily, no matter how messed up a country is, its gravity usually functions well enough.

That's Mr. Veer Bhadra Mishra's plan. Units that use gravity treatment techniques are cheaper and better options for our country. The scientific Mr. Mishra drinks a glass of turbid, fetid water from the Ganga out of devotion every day. He knows that the water is dangerous and has already suffered from many water-bourne diseases. Less scientific Indians along the Ganga's coast do the same. Doctors tell them to boil the water to make it safer. Think about it, these chaps don't have enough fuel (read cow dung cakes) to make one meal a day. Do you suppose they'll waster their fuel on boiling water? This is why diarrhoea, jaundice, and cholera wreak havoc in our country. Something needs to be done.

One of the few good things that has happened is that the centre has given a directive to the Uttar Pradesh (one of the states of India through which the Ganga flows) govt. to check out Mr. Mishra's proposal. His work had been disrupted previously in 1998 when the sanctions by the US put paid to funding for his experimental gravitational sewage treatment plants. I hope India rallies and supports him. There seems to be light at the end of the tunnel and it's hopefully not the fiery breath of a dragon.



I found a few websites that you may want to look at.

http://www.abc.net.au/rn/science/earth/stories/s183185.htm

http://www.serverhostingmantra.com/eco/reports/default.htm

http://www.serverhostingmantra.com/eco/reports/05217foreigners.htm
(It's a shame when this happens. Are we Indians as powerless as the electric sewage treatment plants without electricity?)