Meeting report: "The Architecture of Life"

A few weeks ago, the Institute for Research in Biomedicine (IRB) Barcelona held its first ever PhD student symposium and I had the pleasure of being part of its organizing committee, along with a few other graduate students. In this post I want to present part of the exciting science discussed there, but first I’d like to briefly highlight the importance of science communication in the life of a scientist

Science, for me, is not just sitting at the bench performing experiments and generating data. An important part of the life of a scientist is the actual communication of his research, not only to the rest of the scientific community, but also, and perhaps just as important, to the non-scientific population.

Regarding the former, I had the chance of getting firsthand experience in organizing a scientific meeting, something for which scientists rarely get any instruction during their careers. This was an exciting experience and I highly recommend it, despite all the hard work involved: in this case, the pros definitely outweighed the costs.

The concept behind this symposium was straightforward: let PhD students organize a meeting for other PhD students. We had to handle every detail, like coming up with the meeting’s theme, contacting scientists of different fields and putting together its website.

Since this was our first time being a part of such a project, we decided to give it a broad orientation, such that would encompass most of the fields studied in our institute. In the end, we booked 8 speakers with very different scientific backgrounds divided in 4 sessions: DNA and RNA, Proteins, Cells and Tissues/Organisms (as you can see, the categories cannot be broader).

We decided on “The Architecture of Life” for the meeting’s theme and so, we intended to start of from the basic building blocks and, throughout the meeting, work our way to whole organisms. I won´t go into much detail regarding the talks, but I do want to give you a brief idea of the wide array of interesting topics addressed.

Starting everything off was Gene Myers and Eric Miska.

As some of you may know, Gene Myers pioneered the BLAST algorithm (which most of us use on a daily basis), and during his talk he gave an overview of its inner workings. He then went on to discuss how he developed the shotgun sequencing strategy (which nowadays is pretty much standard) and he concluded his talk by presenting some of his current work regarding high-throughput image analysis software.

Eric Miska talked about non coding RNAs, with a particular focus in miRNA studies. He works on C elegans, and he showed some very interesting projects aimed at studying the complex biology of miRNAs in this organism. Notably, he showed that the repressive function of certain miRNAs can be stably inherited. If you are interested in this area I recommend you keep an eye on Eric’s work.

The second session focused on protein biology, and we had the opportunity to listen to this year´s Noble laureate Ada Yonath, as well as to the cutting edge science of Tanja Kortemme.

Ada´s talk focused on the ribosome, and she gave an extensive structural overview on how this amazing macromolecular machine works. Furthermore, she talked about how structural insights have led her group to hypothesize about the evolutionary history of the ribosome.

Tanja Kortemme´s talk focused on “protein engineering”. By protein engineering I mean that her group is actually designing and testing new interactions between proteins through a combination of computational structure predictions and old-school classic chemistry, which is mainly used for validation purposes. I think she gave a wonderful talk I consider her work to be very impressive.

The third session combined talks given by two scientists working in very different areas: Wolfgang Baumeister and Magdalena Zernicka-Goetz.

In his talk, Wolfgang discussed how the application of EM tomography can help elucidate very complex biological processes. For example, by applying the methodology developed in his lab, they can literally count the number of ribosomes present in a particular subcellular compartment (he showed some beautiful results of studies done in neuron synapses in culture). This can also be used to count other macromolecular complexes present in a particular subcellular location.

Next up was Magdalena Zernicka-Goetz, who, in my opinion, gave the best talk in the symposium. Her lab is studying how the first cell-fate decisions are made in the mouse embryo and their question is quite straightforward: how do identical cells end up with such different phenotypes? In her talk, Magdalena summarized the major breakthroughs her lab has made in the last few years. For those who are more interested in the subject I recommend that you read her recent review in Nature Reviews Genetics (doi:10.1038/nrg2564).

The final session, centered on developmental biology, also had two great speakers: Darren Gilmour and Steve Cohen.

Darren´s work focuses on cell migration using zebrafish as a model. His group aims to understand the molecular and biophysical properties underlying the development of the zebrafish lateral line system, an attractive model system for studying cell migration during organogenesis. Interestingly (and I’m leaving out a lot of details), it appears that the leading cells have distinct signaling pathways activated (compared to the lagging ones), that when disturbed inhibit the movement of the entire group of cells.

The final talk was given by Steve Cohen, who is currently studying miRNAs in Drosophila. One of his most attractive projects involves knocking out every single miRNA from the Drosophila genome. Shall this area of research pique your interest, keep your eyes open for his upcoming work and also check out some of his already published research (for example, see PLoS One 2007;2(11):e1265)

That’s it for this brief overview of the symposium we organized, “The Architecture of Life”. This was a great experience and I sincerely hope these sorts of initiatives are imitated in other institutions worldwide.

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A few great biologists...

Here's the latest cover from Genetics (Nov 2009 issue):

From the journal:

About the Cover
Some members of the band of geneticists who established and extended the guiding principle of biology.

Here's the key in case there are some you don't recognize:

1 James F. Crow
2 Theodosius Dobzhansky
3 Ronald Aylmer Fisher
4 Motoo Kimura
5 John Burdon Sanderson Haldane
6 Hermann Joseph Muller
7 Sewall Green Wright
8 Carl Erich Correns
9 William Bateson
10 Carl Linnaeus
11 Frederick Sanger
12 Gregor Johann Mendel
13 Alfred Russel Wallace
14 middle-aged Charles Robert Darwin
15 Erasmus Darwin
16 young Charles Robert Darwin
17 old Charles Robert Darwin
18 Alfred Day Hershey
19 Francis Harry Compton Crick
20 James Dewey Watson
21 Oswald Theodore Avery
22 Rosalind Elsie Franklin
23 Max Delbrück
24 Salvador Edward Luria
25 Joshua Lederberg
26 Margaret Oakley Dayhoff
27 Linus Carl Pauling
28 Emile Zuckerkandl
29 Calvin Blackman Bridges
30 Alfred Henry Sturtevant
31 Thomas Hunt Morgan
32 Sydney Brenner
33 Seymour Benzer
34 Barbara McClintock
35 Thomas Henry Huxley
36 George Wells Beadle
37 Edward Lawrie Tatum
38 Edward B. Lewis
39 A bulldog

[H/T: Sandwalk]

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An alternative cloning strategy: yeast recombinational cloning

As part of my Ph.D thesis, I have to generate a lot of transcriptional fusions (constructs in which a promoter of choice is cloned in front of a reporter gene in order to evaluate transcriptional regulation. Such plasmids can then be transformed into your model organism to study this regulation in vivo).

Traditionally, this involves amplifying the region of interest (in my case a promoter region) by PCR using primers that harbor the recognition site of particular restriction enzymes. Sometimes you can use the PCR product directly in a digestion reaction with the proper enzymes (although it’s recommended that you column purify your PCR product first) in order to clone it into the vector of interest, but many prefer to clone it first into pGEMT. In order to do the latter, you’ll have to do a TA tailing, as high fidelity enzymes (used for cloning) typically produce blunt ends. Once again, it’s recommended that you column purify your PCR product before tailing.

Once tailed, you can clone the PCR product into pGEMT and transform E. coli in order to propagate the plasmid, which can now be sent out for sequencing. After everything checks out, you would have to set up a digestion reaction in order to get the segment out of pGEMT and clone it into the final plasmid. After that digestion, and in order to get this segment into the final plasmid, you would digest the target plasmid with the same enzymes, set up a ligation reaction and then use an aliquot of the reaction to transform E. coli.

Ideally you’ll use two different enzymes for target plasmid linearization, which would avoid plasmid re-circularization, but sometimes things are not that easy. If you can only use one enzyme for linearization (due to plasmid design, enzyme availability in your lab, etc.) then you are going to have to dephosphorylate the linearized plasmid (typically using Calf intestinal phosphatase) after digestion to avoid re-circularization. Some phosphatases can be heat inactivated, but others can’t. In any case, after digesting and dephosphorylating your plasmid, you’ll have to do a phenol:chlorophorm extraction followed by ethanol precipitation to get your dephosphorylated plasmid, which can now be used for ligation with the segment of interest. After setting up this ligation, an aliquot is used for transformation into E. coli.
You can then check your plasmid by colony PCR of the antibiotic-resistant colonies resulting from the transformation.

At this point you’ll realize that generating several constructs following this protocol can take some time. This strategy, then, does not fit my cloning needs.

We’ve taken a different approach to satisfy most of our cloning requirements, which makes use of my favorite organism: Saccharomyces cerevisiae.

The cloning strategy we use routinely in our lab is called Yeast Recombinational Cloning (YRC), a strategy which has been around for some time (Ma et al., 1987) and has been refined over the last ~13 years (Oldenburg et al., 1997; Gibson, 2009).

The concept behind this strategy is simple: if the segment you’d like to clone into a particular plasmid bears homology to defined plasmid sequences, you can directly “ligate” it into the linearized vector by in vivo recombination: yeast machinery will take care of it. This alleviates the need for an in vitro ligation reaction.

Initially, it was shown that a DNA restriction fragment containing appropriate sequence homology could serve as a substrate for such “recombinational repair” (it’s called repair, as you’ll repair the gap in the linearized plasmid). However, an important advance in the use of such recombination-based methods for gene cloning in yeast, involved the use of PCR (rather than restriction assays) to generate the DNA fragment to be used.

As it was shown that the length of sequence homology needed to promote efficient recombination between the segment of interest and the plasmid was small (~20-40 bp), it was quickly realized that these sequences could be included as part of PCR primers used to amplify a segment of interest. This “recombination-mediated PCR targeting” is very efficient and I consider it now one of my favorite techniques.

So, how do you generate your plasmid through YRC? The linearized target plasmid containing a selectable marker (e.g. URA3 ) is co-transformed into yeast with the PCR fragment of interest: this fragment has 20-40 nt of homology at each end to the region of the plasmid at which recombination is to occur. These nucleotides were added to the fragment as part of the primers. You don’t even have to purify the PCR product before transformation; an aliquot taken directly from the PCR reaction tube works fine. By homologous recombination, some of the cut plasmids are recircularized (due to the integration of the segment of interest into the plasmid) and the plasmid can now be propagated in yeast. Recombinants are then selected as Ura+ transformants (See figure 1).

FIGURE 1. Basic outline of yeast recombinational cloning. Figure based on the one at the “Yeast Model Systems Genomics Group” website.

You can then do yeast colony PCR on the Ura+ transformants to check for your plasmid. Note that the linearized plasmid will not lead to Ura+ transformants, as such a plasmid cannot be propagated.

This protocol may take longer (in days) than the traditional approach outlined at the beginning of this post (as you'll have to wait for the transformed yeast to grow in selective media), but it has significantly less steps, uses less restriction enzymes (as you'll only use them to linearize the target plasmid), uses no ligase, does not require any sort of purification and the only methods involved are digestion, PCR and yeast transformation (the latter is very simple, efficient and requires reagents typically found in a molbio lab. We use the Liac/SS Carrier DNA/PEG method with excellent results).

Further, this approach is more amenable to high-throughput cloning than the traditional strategy (See Colot et al., 2006).

In addition, homologous recombination in yeast can be used to build complex constructs from multiple overlapping constituent parts (see figure 2). Generating such constructs through traditional approaches would take considerably longer. Note that the target vector doesn’t necessarily have to be the final vector. You can use YRC to assembly this complex construct and then the DNA insert can be cut out of the yeast vector and placed into any other vector.

FIGURE 2. Multiple overlapping segments can be cloned into a target plasmid (in this case a shuttle plasmid) through homologous recombination in yeast.

OK, enough about the methodology. How is this strategy used for the generation of my transcriptional fusion constructs? The target plasmid we use is linearized just upstream of the coding region of a reporter gene. Due to proper primer design, the PCR amplicon of the promoter of interest bears homology to particular plasmid sequences that allow its integration upstream of this reporter gene through homologous recombination. The particular target plasmid that we use can be propagated both in yeast and bacteria (i.e it’s a shuttle plasmid) and can also be transformed into our model organism, where by homologous recombination, will integrate in a particular locus in the genome so that we can study the promoter’s biology in vivo.

So, in summary, we have generated a yeast strain which contains a plasmid with our promoter of interest controlling the reporter gene. Now, it’s time to obtain that plasmid in working concentrations, sequence it and then transform it into our model organism.

How do we do this? How do we get the resulting plasmid out of yeast? That will be the matter of a follow-up post, so stay tuned!

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Some of the articles discussed in this post:

MA, H., KUNES, S., SCHATZ, P., & BOTSTEIN, D. (1987). Plasmid construction by homologous recombination in yeast Gene, 58 (2-3), 201-216 DOI: 10.1016/0378-1119(87)90376-3

Oldenburg KR, Vo KT, Michaelis S, & Paddon C (1997). Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic acids research, 25 (2), 451-2 PMID: 9016579

Gibson, D. (2009). Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides Nucleic Acids Research, 37 (20), 6984-6990 DOI: 10.1093/nar/gkp687

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“Bacterial Fortresses” and “gene-trafficking phages”, in my picks of the week from RB

Another week has gone by and some very interesting molbio blog posts have been aggregated to Researchblogging.org. Every week [see my opening post on the matter], I'll select some blog posts I consider particularly interesting in the field of molecular biology [see here to get a sense of the criteria that will be used], briefly describe them and list them here for you to check out.

Note that I'm only taking into consideration the molbio-related blog posts aggregated under "Biology".

Congratulations to everyone who got their post selected.

1) As I’ve commented before, in a previous issue of my Picks of the Week, under certain conditions bacteria can cooperate as a population to create biofilms, which consist of “sessile aggregates of bacteria embedded in a self-made extracellular polymeric matrix”.
Interestingly, this matrix can act as “tough physical barriers that are immune to attacks by many antibiotics and other bacteriocidal agents”, including the action of the host defence system.

Pseudomonas aeruginosa, an opportunistic pathogen, is capable of forming such biofilms, and Lab Rat discusses an interesting new article reporting that biofilm P. aeruginosa cells can react to the presence of polymorphonuclear neutrophilic leukocytes (phagocytic cells which are important players in the innate immune response since they produce a range of antimicrobial molecules able to kill pathogens) by producing a “shield” made of a particular glycolipid biosurfactant called rhamnolipid, which can cause lysis of these immune cells. Interestingly, the synthesis of these lipids is under control of the quorum-sensing system.

2) Hundreds of bacterial genomes have been sequenced and are now available for comparative genomics. Perhaps one of the most fascinating discoveries has been that the genomic diversity, even among the genomes of closely related species, can be enormous.
Bacteriophages have played an important role in shaping bacterial genomes, particularly by contributing to lateral gene transfer through transduction.

From the article:

“Bacteriophages have the ability to manipulate the life histories and evolution of their hosts and evolved many adaptation and defence mechanisms for efficient survival and multiplication. Most of these involve manipulation of the host DNA, as well as the incorporation, into the phage genomes, of bacterial genes that encode proteins with a potential to facilitate bacteriophage reproduction” (my emphasis)

Apparently, the latter is the case with cyanophages and cyanobacteria, as discussed by Iddo Friedberg at Byte Size Bio. He comments on a recent (and purely bioinformatic) Nature article reporting “the presence of photosystem I (PSI) genes in the genomes of viruses that infect marine cyanobacteria, [by] using pre-existing metagenomic data from a global ocean sampling expedition as well as from viral biomes”. Further, they show (by modeling) that apparently, the use of one of these proteins encoded in the phage genome can make the bacterial PSI function more efficiently.


That's it for this week. Stay tuned for more MolBio Research Highlights!

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Some of the articles discussed in this week's selected posts:

Alhede M, Bjarnsholt T, Jensen PØ, Phipps RK, Moser C, Christophersen L, Christensen LD, van Gennip M, Parsek M, Høiby N, Rasmussen TB, & Givskov M (2009). Pseudomonas aeruginosa recognizes and responds aggressively to the presence of polymorphonuclear leukocytes. Microbiology (Reading, England), 155 (Pt 11), 3500-8 PMID: 19643762


Sharon, I., Alperovitch, A., Rohwer, F., Haynes, M., Glaser, F., Atamna-Ismaeel, N., Pinter, R., Partensky, F., Koonin, E., Wolf, Y., Nelson, N., & Béjà, O. (2009). Photosystem I gene cassettes are present in marine virus genomes Nature, 461 (7261), 258-262 DOI: 10.1038/nature08284

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Peer review during WWII: HILARIOUS

Even the Führer has to deal with some annoying reviewers....

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“Hey! Where’s that cancer cell going?” and “M-cells, gateways to the mucosal immune system”, in my picks of the week from RB

Another week has gone by and some very interesting molbio blog posts have been aggregated to Researchblogging.org. Every week [see my opening post on the matter], I'll select some blog posts I consider particularly interesting in the field of molecular biology [see here to get a sense of the criteria that will be used], briefly describe them and list them here for you to check out.

Note that I'm only taking into consideration the molbio-related blog posts aggregated under "Biology".

Congratulations to everyone who got their post selected.

1) As we’ve discussed before, tissue invasion and metastasis are hallmarks of cancer. Cells from primary tumor masses can travel and colonize the same or different organ sites leading to the formation of secondary tumors. Importantly, these “metastases” are the cause of 90% of human cancer-related deaths.
The first step towards metastasis involves the “movement of cancer cells into tissue surrounding the tumour, and the vasculature”. This generates lots of questions: What makes cancer cells motile? How do these cells move?

Lab Rat discusses a review commenting on the mechanisms underlying the motility of cancer cells.

2. In the gastrointestinal tract, the largest mucosal membrane surface in the human body, the highly specialized M-cells play an important sentinel role by sampling and transporting antigens from the lumen of the small intestine, to underlying mucosal lymphoid tissues (in a process called antigen transcytosis), where antigen-specific immune responses are evoked. Despite the importance of this process, the molecular mechanisms underlying this antigen uptake are largely unknown.

Geek discusses a recent article in Nature reporting “that glycoprotein 2, specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens” by recognizing a component of type I pili on bacteria.


That's it for this week. Stay tuned for more MolBio Research Highlights!

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Some of the articles discussed in this week's selected posts:

SAHAI, E. (2005). Mechanisms of cancer cell invasion Current Opinion in Genetics & Development, 15 (1), 87-96 DOI: 10.1016/j.gde.2004.12.002

Hase, K., Kawano, K., Nochi, T., Pontes, G., Fukuda, S., Ebisawa, M., Kadokura, K., Tobe, T., Fujimura, Y., Kawano, S., Yabashi, A., Waguri, S., Nakato, G., Kimura, S., Murakami, T., Iimura, M., Hamura, K., Fukuoka, S., Lowe, A., Itoh, K., Kiyono, H., & Ohno, H. (2009). Uptake through glycoprotein 2 of FimH+ bacteria by M cells initiates mucosal immune response Nature, 462 (7270), 226-230 DOI: 10.1038/nature08529

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Science Laughs: Science Comedian Brian Malow

This is just a preview. You can watch the full video here.

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