Bacteria are good hosts for expressing recombinant proteins, mainly because they are easy to manipulate and grow. But their relatively simple expression systems can’t cope with every gene you throw at them so proteins will often fail to express properly.

Sometimes the protein is fully expressed but cannot fold properly. This is particularly common when the evolutionary distance between the host and the organism means that the bacterial cytoplasm is an unsuitable environment for folding. Too high a rate of protein expression, lack of a chaperone or partner protein and inability to form disulphide bonds are common examples.

These unfolded proteins will aggregate to form inclusion bodies - insoluble protein granules - in the cytoplasm. But if this happens, all is not lost. It is possible to recover the inclusion bodies, denature them and refold the proteins in vitro to recover functional protein that can be used for further study.

The problem is that every protein requires different conditions and methodologies for optimal refolding, so a lot of time consuming experiments are required to optimise the protocol for each protein.

To alleviate this problem, researchers at Australia’s Monash University have set up the REFOLD database, which contains protocols, developed by contributor to the database, for refolding hundreds* of different proteins.

If you are faced with the task of refolding an insoluble protein, this should be your first stop. If you find the protocol for your protein then you will save yourself a lot of time. And if you don’t, once you develop your protocol, uploading it to the database will save someone else the time in future - giving you some good research karma.

*Protocols for 653 proteins were in the database at the time of writing

Photo: bousinka

Regular readers will know about the advantages of T4 DNA polymerase-mediated ligation independent cloning. The fact that it is faster, more efficient and allows easier parallel cloning than conventional cloning has made it my method of choice in the lab.

But the technique does have it’s downsides - not least the requirement that existing vector multiple cloning sites be modified to convert them into ligation independent cloning vectors.

This paper by Li and Elledge recently flagged up in a comment by Max (thanks Max!) looks like it could change all that. It turns out that no sequence modification is required at all and LIC (or sLIC - sequence and ligation independent cloning as the authors call it) can be performed at any site in any vector of your choice.

If you are familiar with T4-mediated ligation independent cloning you will know that the vector sequence needs a specific LIC site containing restriction site flanked by regions that lack one of the nucleotides (e.g. adenine) for a 13-14nt stretch (read this first if you are not familiar with it). After linearising at the restriction site, the vector is incubated with dATP + T4 DNA polymerase, which chews back the 3′ end of the DNA until it stops after 13-14 nucleotides due to the presence of the dATP. This creates a single-stranded region to with a similarly treated insert can be annealed.

In this paper, Li and Elledge showed that the specific vector LIC site was not required. Treating the vector with T4 DNA polymerase and no dNTPs for a certain length of time (30 minutes was optimal for them) was sufficient for vector preparation. The single stranded stretch this creates is longer than required for annealing an insert, but single stranded gaps like these are apparently repaired very efficiently by E.coli after transformation so this is not a problem.

They also demonstrated that inserts prepared in one of three ways could be successfully annealed and transformed, which considerably increases the versatility of the process. The insert prep methods were:

1. T4 DNA polymerase treatment. Just like the vector, the insert could be subjected to T4 DNA polymerase treatment (without dNTPs) to create single stranded regions that will anneal to the prepared insert.
2. iPCR. Non-treated PCR fragments could also be annealed to prepared vectors, albeit with much lower efficiency. They showed that this is because a subset of fragments were synthesised incompletely, resulting in 5′ overhangs. Although this is was relatively in efficient, the authors found that it was robust and recommend it for routine cloning.
3. Mixed PCR. This was the most efficient method. It involves amplifying the insert using two separate reactions. In the first reaction, the forward primer has a 30nt tail homologous to the vector ss region, while in the second reaction, the reverse primer has the homologous tail. After amplification the two reactions are mixed, denatured and annealed to yield a subset of inserts that have both the forward and reverse primer single stranded tails that can be annealled into the prepared vector.

The authors showed that efficient annealing needed only 20-30 nt (single stranded) regions of homology at each end of the vector and insert. Amazingly, the homologous regions didn’t even need to be at the ends of the insert - the authors showed that non-homologous regions of up to 20 nucleotides could be tolerated as the branched products produced after annealing are efficiently trimmed and repaired by the cell.

I have not had a chance to try out this method yet, but it certainly looks very exciting. It looks to me like once this I have this protocol is up and running there will be no need at all for restriction enzyme-mediated cloning… which is a day I long for!

When I have some results of my own from this I’ll publish them, and my protocol here so watch this space.

A few months ago I mentioned about how people shouldn’t take science, on faith, but instead on data. Put another way, this is about post-modernism, or anti-modernism, where facts and their interpretations are all relative, at least to some degree.

I came across an outstanding essay on the subject by Daniel Dennett over on Butterflies and Wheels: Postmodernism and Truth. There are a few excerpts below the fold [Emphasis mine]. Yes, there’s still Kuhnsian sociology of science, although I’ve always read Kuhns as agreeing with the bit from the last quote that I have below the fold: “The methods of science aren’t foolproof, but they are indefinitely perfectible. Just as important: there is a tradition of criticism that enforces improvement whenever and wherever flaws are discovered.”
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Every major field has its leading thinkers, and the biology of cancer is no different. What makes their impact heard better is when one of those leaders writes a book about it. Given my interest in molecular biology of cancer, I naturally have my favorite such book on the topic - Robert Weinberg’s One Renegade Cell.

Weinberg’s focus is on what he knows best: the mechanisms that promote and regulate the proliferation of normal and malignant cells. And for that, his explanations are the best out there. These explanations take up the first half of the book, corresponds to the early events in the development of a tumor, and makes up a coherent story. For example, he covers oncogenes, tumor suppressors, apoptosis, and to a lesser extent DNA repair, in relatively easy-to-follow language.
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This week’s around the blogs focuses on lab life and impacts of science on society. That’s a big area to cover, but there are still only a handful of really noteworthy discussions in the last couple of weeks on the topic. Check ‘em out.
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Multi-tasking used to be my favourite way to get ahead.

During my PhD I saw others around me working extremely long hours in the lab and not really having much of a personal life and quite early on I made the decision that this was not for me.

Although I enjoy my work, having a good life outside the is also very important. Also, I found that if I worked very long hours then I tended to be far less efficient overall.

But, I still wanted to get through as many experiments as possible. So I also made the decision that my approach would be to work a regular 8 hour day and be as efficient as possible during that time. My basic recipe for an efficient working day was: Read more »

We have had a rush on time and money saving techniques on Bitesize Bio in the last few weeks. Ways to re-cycle electroporation cuvettes, reduce gel buffer costs, do fast restriction digests and re-cycle midiprep columns have all been suggested.

In this article I’ll add the possibility of using a microwave to sterilize or decontaminate growth media. From the outset I’d like to say that I am not too sure about this, but I’ll make the case and you can tell me what you think.

Normally, growth medium is sterilized or decontaminated using an autoclave. Autoclaves are generally expensive, energy-hungry beasts that (in my experience) break down a lot so I would be very happy to use them less if I could.

Decontamination using microwaves.

The case for using microwave ovens for decontamination of cultures or materials was made back in 1977 by Latimer and Matsen. They showed that 1-5 minutes in a conventional microwave was sufficient to decontaminate 5mL cultures or petri dishes of common clinical pathogens including E. coli, S. aureus and K. pneumoniae. B. subtilis spores proved a bit more subborn, requiring more than 10 minutes of microwaves to wipe them out.

Border and Rice-Spearman backed this up with a 1999 study that showed materials contaminated with various bacteria and yeast strains were completely decontaminated by one minute in the microwave (I guess their microwave was better). And in 2006, Silva et al, investigating the decontamination of dentures, showed that 6 minutes in the microwave sterilised S. aureus and C. albicans but only partially disinfected P. aeruginosa and B. subtilis.

Sterilisation using microwaves

A 2001 Biotechniques paper by Weiss and Galande showed that LB plates made from microwave-sterilised LB-agar were apparently sterile (control plates were no detectably contaminated by microorganisms), had a similar shelf-life to autoclaved plates and supported bacterial growth as normal. The plates were prepared from dry powders dissolved in distilled water and aliquoted into 50mL tubes.

This is a very fast way to make plates and has the added advantage that the antibiotics can be added in from the start as they are not destroyed by microwaves. Invitrogen have a product that takes advantage of this. ImMedia is LB medium provided as sachets of dried, weighed power containing all of the required media components (including antibiotics). It is designed so that you can just add the sachet contents to water, microwave and your media is ready. But Weiss and Galande’s method is just as good and much cheaper.

My view

My take-home from this is that microwaves are are reasonably good at decontamination but more stubborn microorganisms (e.g. the spore-forming B subtilis) are not effectively disinfected. So the method does not sound too reliable to me. Also, filling the lab with smelly fumes from contaminated stocks does not seem to be a good idea. For easy liquid culture decontamination I think I will stick with Virkon, and for solid media, autoclaving seems to be the only good option.

The microwave media prep method is certainly interesting. Weiss and Galande’s results seem to be pretty robust and I would consider this method for an emergency media prep - if I need to start an E. coli culture last thing at night and there’s no sterile media available. Although the decontamination results show that microwaves don’t kill everything, E. coli grows so quickly that for routine purposes, a low level of contamination by slower growing organisms can be tolerated.

But I would not use this method for anything other than routine cultures and certainly not for slow growing organisms. Maybe that’s just me being a typical scientist, reluctant to take on new methods as Liam suggested. The microwave method is also limited by the fact that only small volumes (50ml) can be sterilised so it is never going to replace the autoclave for batch media production.

That’s my view - what’s yours?

What is this thing called ‘Life?’ One popular game in the relevant area of philosophy is to provide robust counter examples, which reveal failures in operational definitions of life. Failed attempts include physiological, metabolic, biochemical, genetic and thermodynamic definitions of life, all of which face problems. For example, a metabolic definition finds it hard to exclude fire (which grows and reproduces via chemical reactions), a biochemical definition does not exclude enzymes (which are biologically functional but not living systems), while a thermodynamic definition does not exclude mineral crystals (which create and sustain local order and may reproduce).
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As per tradition, it’s time for the weekly roundup of informative blog posts outside of your regular Bite of Bio. This week, it’s striking that the posts to choose from have an extra supply of posts on the science, and light on the personal or social commentary that bloggers enjoy so much. So this week, we’re focusing on the science itself - visit the posts, and leave comments if you find them interesting.
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Staying in science - getting funding and getting peer reviewed - is tough.

That’s one of my main gripes with creationist simpletons who imply that scientists are uncritical of their peers, and that criticism is directed solely at those who refuse to take their claims at face value. They have no clue whatsoever what they’re talking about.

Every scientific claim, as it’s actually being formulated, must be paved with meticulous attention to detail. The scientist advancing some newly-considered possibility must endure a constant barrage of critiquing, on both the grant application and results publication stages.

It’s for a darn good reason - people, even scientists, are prone to error.
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