PhD – 21 months in 

Do you remember my optimistic blog post about finding my bearings in the lab after a month of the PhD? I also included pictures of a failed western blot and slightly crushed centrifuge tubes.

Well, twenty months later and I’m still making mistakes. Often they’re new and different mistakes, which could almost be exciting. But today I made the same mistake and lost a lot of plasmid-growing bacteria (bacteria I am using as work horses to produce specific DNA for me) in a centrifuge (which I subsequently cleaned!)…

Photographic evidence attached.

More Fluorescent Proteins

Although I’m approximately two years late in reporting about this discovery I still think it’s pretty cool. In 2013 Kumagai et al. for the first time discovered a fluorescent protein in a vertebrate, the Unagi eel. Until then fluorescent proteins had only been found in invertebrates, such as reef corals and the jellyfish Aequorea victoria, where the famous green fluorescent protein originally came from. My attention was drawn to this finding in a brief article in the Chemistry World journal, in which the author claims that, “Unagi’s status as a culinary delicacy means you’re more likely to encounter these eels in a restaurant than a lab”. [Picture copied directly from the article link.]

Unagi eel

Apart from (presumably) tasting good and looking pretty, the fluorescent protein – called UnaG – from Unagi eels may be able to form the basis for a diagnostic test for liver disease. UnaG only fluoresces when bound to bilirubin, which is a break-down product of haem, the molecule that carries oxygen in the blood. Livers with impaired function have difficulty further processing the bilirubin before it is excreted, leading to a build-up of bilirubin in the body, and in extreme cases to jaundice. So the intensity of UnaG fluorescence can be used as a read-out for how badly the liver is damaged.

And now for something completely different: next time I’ll be writing about what it’s like to be an intern at the journal eLife!

Reference:

Kumagai A, Ando R, Miyatake H, Greimel P, Kobayashi T, Hirabayashi Y, Shimogori T, Miyawaki A A Bilirubin-Inducible Fluorescent Protein from Eel Muscle. Cell 153: 1602-1611

The Joys of Revision #2

Exams are drawing closer and that is both a relief – at this point I really just want to be done with this taking exams business – and a consternation – they do seem to spring up on one every year rather unpleasantly and seemingly out of the blue. To deal with my mix of boredom and quiet anxiety it has been useful to seek out various different places to do revision. A friend of mine even has a blog dedicated to describing several Cambridge libraries.

To begin with there is of course my “home” library at Pembroke College. Large desks and sitting between volumes of classics (Greek, Latin, English, you name it) make for a good working environment. However, the bell in the clock tower, which strikes every half hour, is a rather unpleasant reminder of how quickly time is ticking. [Photo copied from the Pembroke College website.]

Library-Photo-resized

Just across the street is the Colman Library, the library of the Department of Biochemistry. Again this is quite an old building with large wooden desks or smaller, personal booths to sit at. It is rarely busy, often giving it a feeling of calm, but the people who do study there are often my (stressed?) colleagues, which could become a bit unpleasant the closer we get to exams.

If one is feeling particularly brave as a science student one can make one’s way to the Sidgwick site where several art/humanities and social science departments are situated. The main recollection I have of the law library is its coldness (temperature-wise). In the economics library people were generally better dressed, although I hear that this is also deteriorating as  exams approach. The Judge business school has an “information service” instead of a formal library, in which people can chat and bring in drinks, which makes for a more relaxed atmosphere, but when I heard some students (?) talking about their newest great idea in which they’ll move from the production to the service sector to make “massive profits” I started feeling out of place.

The winner of the library competition is undoubtedly the Faculty of Education: it’s bright and full of light wood, the staff are helpful and kind (there was free cake on Friday afternoon) and one can sit looking out onto a green garden. [Photo copied from their website.]

2007_library_home

CRISPR Let Loose

Most people reading this blog today will probably consider this slightly old-fashioned, but I received a newspaper clipping from my mother in the post yesterday (yes, I mean a physical piece of paper that arrived in an envelope with a postage stamp on it). The title of the article (online here), which was published in the Austrian quality daily newspaper “Die Presse”, roughly translated to, “Genetic engineering is spiralling out of control”.

The article was referring to an experiment conducted by Gantz & Bier (2015) in which they invented a “mutagenic chain reaction” (MCR) based on the CRISPR/Cas9 technology in the fruit fly Drosophila melanogaster. Their experimental rationale is illustrated in this figure, which I copied directly from the paper:

MCR scheme

The idea was to create homozygous insertional deletion mutations of a target gene, in their case the yellow gene in Drosophila. When yellow is mutated heterozygously in females (i.e. in only of the two genomic copies) the flies look normal/wild-type because mutations in this gene are recessive and the gene is located on the X chromosome; only once both copies are mutated does the phenotype manifest: albino flies. Since males only have a single X chromosome they are always albinos if they inherit the mutated yellow gene. Gantz and Bier built a DNA construct that expresses Cas9, the DNA endonuclease, and the guide RNA targeting yellow in both somatic and germ-line cells. However, the special feature of their construct was the flanking homology arms (HA; depicted in red): these facilitate the insertion of the construct at the site of cleavage by Cas9 via homology-directed repair. This means that the genome now stably expresses one copy of the Cas9/gRNA combination. Next, the second copy/allele will be targeted and again the construct is inserted in the DNA, disrupting the yellow gene. If this approach didn’t work you would expect first generation female flies, progeny from a cross between wild-type flies and flies containing the DNA depicted as a circle in panel A above, to all be phenotypically normal. However, they actually found female albino flies in these crosses. When these white flies were crossed to wild-type males they found that almost 100% of those offspring were white, although one would expect only the males to be. So their MCR approach clearly worked, and in a way it violates the age-old rules of Mendelian inheritance.

In their conclusion they stated that MCR may, “provid[e] a potent gene drive system for delivery of transgenes in disease vector or pest populations, and potentially serv[e] as a disease-specific delivery system for gene therapy strategies”. Gene drive “involves stimulating biased inheritance of particular genes to alter entire populations of organisms” (Pennisi (2014)). This approach may rapidly disseminate mutations that inactivate virulent traits in various disease vectors, such as the Anopheles mosquito, which acts as a vector for the malaria parasite Plasmodium falciparum.

Gantz and Bier also wrote that they are, “keenly aware of the substantial risks associated with this highly invasive method. Failure to take stringent precautions could lead to the unintentional release of MCR organisms into the environment”. In a comment on the paper their security precautions are mentioned: the flies are kept in three layers of tubes and boxes, locked behind five doors operated by fingerprint recognition to ensure they do not escape into the wild. However, just as Baltimore et al. (2015) called for caution and open discussions concerning CRISPR applications with regards to (human) genome-editing in general, Esvelt et al. (2014) demanded extreme caution in the use of CRISPR-based gene drive systems. They especially emphasised the need to both molecularly and ecologically contain these systems: at the molecular level, for example, the targeted gene must not be present in wild species; at the ecological level experiments should only be attempted in inhospitable environments where organisms will not easily find mating partners. Their hesitation to fully embrace this new approach is understandable. For example, what if the guide RNA becomes mutated and the nuclease starts targeting other genes as well? Furthermore, such gene drives employed by the government of a country, for instance, to combat a disease vector will almost inevitably cross political borders. If introduced into endangered species by accident they could eradicate them entirely. It seems to me that it will be quite difficult to test such approaches (in the manner of a clinical trial, say) without actually conducting the experiment in the wild. And somehow inexplicably these articles entirely slipped under my radar, whereas the recent human genome-editing experiment caused much more uproar although it is arguably a lot less prone to “spiral out of control”.

References:

Baltimore D, Berg P, Botchan M, Carroll D, Charo RA, Church G, Corn JE, Daley GQ, Doudna JA, Fenner M, Greely HT, Jinek M, Martin GS, Penhoet E, Puck J, Sternberg SH, Weissman JS, Yamamoto KR (2015) Biotechnology. A prudent path forward for genomic engineering and germline gene modification. Science 348: 36-38

Esvelt KM, Smidler AL, Catteruccia F, Church GM (2014) Concerning RNA-guided gene drives for the alteration of wild populations DOI:10.7554/eLife.03401

Gantz VM, Bier E (2015) The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science 348: 442-444

Pennisi, E (2014) U.S. researchers call for greater oversight of powerful genetic technology. Science Insider: http://news.sciencemag.org/biology/2014/07/u-s-researchers-call-greater-oversight-powerful-genetic-technology. Accessed May 3rd 2015.

The Joys of Revision #1

Please immediately note the hint of sarcasm in this title. What academic year would be complete without the harrowing prospect of end-of-year exams, which, this year, happen to also be finals? … Well, exactly. Any old academic year would be fine without. But alas, that is not how the system works. So on top of being judged on the basis of our lab project/dissertation/viva voce examination we also need to take two written exams, which are worth 50% of our mark.

To ease the transition from dissertation writing and editing into revision I thought I would start with something fun/potentially useless that can be classed as elaborate procrastination:

Landmark timeline

As mentioned previously, some of our teaching was provided in the format of seminars discussing landmark papers – papers that have shaped the way biologists think about their areas of research and that have had lasting, even if sometimes subconscious, effects. Several of the professors emphasised the importance of understanding these papers within their historical contexts, which is, I would contest, not something we scientists normally think about. Therefore I decided to look for a tool that would allow easy creation of a timeline like the one shown above and the only free app I could find in the store is simply called Timeline 3D. Right, well, and I just realised that on top of procrastinating by colour-coding different landmark papers I’ve now also spent a good fifteen minutes writing this blog post. If anyone else has well-disguised procrastination tools, please do share…

P.S.: Another way to pretend like you’re doing something useful – attend the departmental seminar given by a Nobel prize laureate, Jules Hoffmann. I was pleased to hear that he was appreciative of all of his collaborators and acknowledged his students and post-docs; something he seems to share with another Nobel prize winner, Christiane Nüsslein-Volhard, who gave a seminar a few weeks ago. Based on these examples I was about to draw the conclusion that a couple of the marks of great scientists are a) their willingness to acknowledge co-workers and b) to some extent their modesty (granted, this one doesn’t apply to other Nobel laureates I’ve encountered [e.g. James Watson]). However, as I was reading about Jules Hoffmann I very quickly came across this blog written by Bruno Lemaitre, a former research associate in Hoffmann’s lab, claiming that most, if not all, the award-winning work was conducted by him without encouragement/help from Hoffmann. Now what to believe?!