Posts

Posted on

An experiment in open notebook science – Sortase mediated protein-DNA ligation

In a recent post I extolled the possible virtues of Open Notebook Science in avoiding or ameliorating the risk of being scooped. I also made a virtue of the fact that being open encourages you to take a more open approach; that there is a virtuous circle or positive feedback. However much of this is very theoretical. We don’t have good case studies to point at that show that Open Notebook Science generates positive outcomes in practice. To take a more cynical perspective where is the evidence that I am willing to take risks with valuable data? My aim with this post is to do exactly that, put something out there that is (as far as I know) new and exciting, and kick off a process that may help us to generate a positive example.

I mentioned in the previous post that we have been scooped not once, but twice, on this project. I will come back to the second scooping later but my object here is to try and avoid getting scooped a third time. As I mentioned in the previous post we are using the S. aureus Sortase enzyme to attach a range of molecules to proteins. We have found that this provides a clean, easy, and most importantly general method for attaching things to proteins. Labelling of proteins, attaching proteins to solid supports, and generating various hybrid-protein molecules has a very wide range of applications and new and easy to use methods are desperately needed. We have recently published[1] the use of this to attach proteins to solid supports and others have described the attachment of small molecules[2], peptides[3], PNA[4], PEG[5] and a range of other things.

One type of protein-conjugate that is challenging to generate is one in which a protein is linked to a DNA molecule. Such conjugates have a wide range of potential applications particularly as analytical tools where the very strong and selective binding that can often be found in a protein is linked to the wide range of extremely sensitive techniques available for DNA detection and identification[6]. Such techniques have been limited because it is difficult to find a general and straightforward technique for making such conjugates.

We have used our Sortase mediated ligation to successfully attach oligonucleotides to proteins and I have put up the data we have that supports this in my lab book (see here for an overview of what we have and here for some more specific examples with conditions). I should note that some of this is not strictly open notebook science because this is data from a student which I have put up after the event.

We are confident that it is possible to get reasonable yields of these conjugates and that the method is robust and easy to apply. This is an exciting result with some potentially exciting applications. However to publish we need to generate some data on applications of these conjugates. One obvious target here is to use a DNA array and differently coloured fluorescent proteins attached to different oligonucleotides to form an image on the array. The problem is that we are not well set up to do this in my lab and don’t have the expertise or resources to do this experiment efficiently. We could do it but it seems to me that it would be quicker and more efficient for someone else with the expertise and experience to do this. In return they obviously get an authorship on the paper.

Other experiments we are interested in doing:

  • Analytical experiment using the binding of a protein-DNA conjugate that utilises the DNA part for detection.
  • Pull down of peptide-DNA conjugates onto an array after exposure of the peptides to a protease
  • Attachment of proteins to a full length PCR product containing the gene for the protein. Select one of the protein and then re-amplifity the desired gene. (I had a quick go at this but it didn’t work)

So what I am asking is this:

  • If any reader of this blog is interested in doing these (or any other) experiments to aid us in getting the published paper then get in touch
  • If you feel so inclined then publicise this call wider on your own blog and let’s see whether using the blogosphere to make contacts can really aid the science

We will send the reagents to anyone who would like to do the experiments along with any further information required. In principle people ought to be able to figure out everything they need from the lab book but this will probably not be the case in practise. The idea here is to see whether this notion of a loose collaboration of groups with different resources and expertise that is driven by the science can work and whether it is a competitive way of doing science.

My criteria in accepting collaborators will be as follows:

  1. Willingness to adopt an Open Notebook Science approach for this experiment (ideally using our lab book system but not necessarily)
  2. Interest in and willingness to engage in the development of the published paper (including proposing and/or carrying out any new experiments that would be cool to include)
  3. Ability to actually carry out the experiment in reasonable time (ideally looking for a couple of months here)

So this is notionally a win-win situation for me. We will be getting on and doing our own thing as well but by working with other groups we may be able to get this paper out more efficiently and effectively. Maybe others will come up with clever experiments that would add to the value of the paper. The worst case scenario is that someone comes along and sees this, copies the results, and publishes ahead of us. The best case scenario is that someone else already working in a similar direction may come across this and propose working together on this.

In any case, the results promise to be interesting…

References:

[1] Chan et al, 2007, Covalent attachment of proteins to solid supports via Sortase-mediated ligation, PLoS ONE, e1164

[2] Popp et al, 2007, Sortagging: a versatile method for protein labelling, Nat Chem Biol, 3:707

[3] Mao et al, 2004, Sortase-mediated protein ligation: a new method for protein engineering, J Am Chem Soc, 126:2670

[4] Pritz et al, 2007, Synthesis of biologically active peptide nucleic acid-peptide conjugates by sortase-mediated ligation, J Org Chem, 72:3909

[5] Parasarathy et al, 2007, Sortase A as a novel molecular “stapler” for sequence specific protein conjugation, Bioconj Chem, 18:469

[6] Barbulis et al, 2005, Using protein-DNA chimeras to detect and count small numbers of molecules, Nature Methods, 2:31

Posted on

Increasing the persistence of online Open Notebooks

Weird. I came across WebCite this morning while having a quick scan through the Eysenbach paper on Open Access increasing number of citations in PLoS Biology. At the bottom was the comment that all the web pages have been archived on WebCite. Going across to WebCite I find the following:

What is WebCite®?
WebCite® is an archiving system for webreferences (cited webpages and websites), which can be used by authors, editors, and publishers of scholarly papers and books, to ensure that cited webmaterial will remain available to readers in the future. If cited webreferences in journal articles, books etc. are not archived, future readers may encounter a “404 File Not Found” error when clicking on a cited URL.

A WebCite® reference is an archived webcitation, and rather than linking to the live website (which can and probably will disappear in the future), authors of scholarly works will link to the archived WebCite® copy on webcitation.org.

Now this is interesting in its own right but my thoughts went back to a question I was asked several times during the talks I’ve given recently, ‘How do you know it will still be there in X years time?’ So might this be a good secondary archive for pages of electronic lab books, particularly when we wish to refer to specific pages in a peer reviewed paper.

Part of the answer to this may be to have many independent copies in different forms. However another answer is explicit archiving, whether that be in institutional repositories, general online repositories, embedded in supplementary information, or just trusting in Google. WebCite or something similar could have a useful role to play here. It could also provide a method of getting third party datestamps on the notebook.

That’s not the weird bit:

The wierd thing is that Deepak Singh just blogged about WebCite a few hours ago, which he picked up on from Jon Udell’s blog. Perhaps it is just morphic resonance :)

Some of the comments on Jon’s blog mirror my own concerns. The business model for this doesn’t seem to be well thought out. I can’t see many publishers buying into a system which is essentially trying to bridge the gap between their collapsing business model and a future of online publishing. In this context it is interesting that the list of members does not for instance include Nature Publishing Group or any PLoS journals who you would think wouldbe at the forefront of this kind of initiative. I would support the comment by Jon Galloway that this would be a good case for contextual advertising.

So I can see that this could well be useful as an archive for cited web pages, and I will use it when citing material from the web in future in my own papers. In terms of using it more generally as an archive for the notebooks there are two major issues. Firstly, will there be objections to us dumping large quantities of material into the system? The FAQ explicitly excludes charging authors but currently only asks people to be considerate in terms of what they archive. Secondly I would want to see a more clearly sustainable business model.

Posted on

Getting scooped…

I have been waiting to write this post for a while. The biggest concern expressed when people consider taking on an Open Notebook Science approach is that of being ‘scooped’. I wanted to talk about this potential risk using a personal example where my group was scooped but I didn’t want to talk about someone else’s published paper until the paper on our work was available for people to compare. Our paper has just gone live at PLoS ONE so you will be able to compare the two sets of results.

Attaching proteins in a site selective manner to solid supports is a challenging problem. A general approach to attaching proteins to resin beads or planar surfaces while retaining function would have applications in chemical catalysis, analytical devices, and the generation of protein microarrays.

We established in my laboratory that the Sortase enzyme of S. aureus was an effective way of attaching functional proteins to solid supports in about March 2006. This was before I started taking up ONS and as the student is finishing up the project has not been moved onto an ONS basis so the data was not made available when we had it. We delayed publishing this as we attempted to generate a ‘pretty picture’ in which we would create the Southampton University logo in fluorescent protein on a glass surface. The idea of this was to make it more likely that we would get the paper into a higher ranked journal but ultimately we were unsuccessful.

In March 2007 we were scooped by a paper in Bioconjugate Chemistry (1). This paper, amongst other things, included an experiment that was very similar to the core experiment in our data (2). I should emphasise that there is absolutely no suggestion that this group ‘stole’ our data. They were working independently and were probably doing their experiments at about the same time as we did ours.

The first point here is that in the vast majority of cases being scooped is not about theft but about the fact that a good idea is an idea that is likely to occur to more than one person. It is essentially about not being first to get to publication. I can argue that I had the idea some years ago – but we didn’t get on to the work until 2006 and we’ve only just managed to get it published.

The second point is that our work is clearly different enough from Parthasarathy et al to be published. This is often the case. Indeed we have recently been scooped again on a different aspect of this project (3) but I expect we will still be able to publish as our data is again complementary to that reported.

So, from the perspective of traditional publication we were scooped because we didn’t publish fast enough. We can’t claim any precedence because we weren’t taking an ONS approach that would support this claim. But let us consider what would have happened if we had taken an ONS approach. I think there are a series of possible outcomes;

  1. It is possible, or even likely, that the other group may not have noticed our results at all. Under these circumstances we would at least be able to claim precedence.
  2. The other group may have seen our results and been spurred into more rapid publication. Again we would have been able to claim precedence but also there would be a record of the visit. I suspect this is the most common route to being scooped. In most cases results are not ‘copied’ from e.g. conference presentations but much more often the fact that someone is close to publication spurs another group to get their work published first.
  3. The most positive outcome is that, having seen we had some similar results, the other group may have got in contact and we could have put the results together to make a better paper.

Outcome 3) may seem unlikely but it really is the best outcome for everyone. Pathasarathy et al published in Bioconjugate Chemistry and we will publish in PLoS ONE after chasing around a number of other journals. If we had combined the results and, possibly more importantly, the resources to hand we probably could have put together a much better paper. This could possibly have gone to a significantly higher ranked journal. Apart from possible arguments over first and corresponding authorship everyone would have been better off.

This is the promise of being open as well as practising Open Notebook Science. By cooperating we can do a lot better. Being open has its risks but equally there are significant potential benefits including doing better science, better publications, and better career prospects as a result.

But let us now put the shoe on the other foot. What if the other group had made their data available? Would I have rushed out our paper to prevent them getting in first? It is one thing to advocate openness but would I really have gotten in touch with them myself? The answer is that 12 months ago I probably wouldn’t have got in contact. I would have pushed the student to work 24 hours a day and got our own paper out as fast as possible with whatever data we had to hand. I probably would not have contacted the other group. And we may have cut corners to get the data together, missed out controls that we know would work but didn’t have time to do and glossed over any possible issues.

But today, faced with the same dilemma I would get in touch with them and propose combining our data. Why the change? Partly because I have spent the past 12 months considering the issues around being open. But a strong contributor is that if I didn’t I would be exposing myself to criticism as a hypocrite. I have come to think that one of the real benefits of ‘being open’ is that being exposed means you hold yourself to higher standards precisely because being out in the open means that people have the evidence to judge you on.

I find that as I do my experiments and record them I take more care, I describe them more clearly, and I take more care to preserve and index the data properly. More generally I feel more inclined to share my ideas and preliminary results with others. And part of this is because I am aware that double standards will be obvious to anyone who is looking. Standards and discipline in maintaining them make for better science and for better people. Anyone who is honest with themselves knows that sometimes, somewhere, there is a temptation to cut corners. We all need help in maintaining discipline and being open is a very effective way of doing it.

It may sound a bit over the top but I actually feel like a better person for taking this approach. So for all the sceptics out there, and particularly for those academics with blood pressure issues, I recommend you try throwing the doors open. The fresh air is a bit bracing but it will do you the world of good.

  1. Parthasarathy R, Subramanian S, Boder ET (2007) Sortase A as a novel molecular “stapler” for sequence-specific protein conjugation. Bioconjug Chem 18:469-76
  2. Chan L, Cross HF, She JK, Cavalli G, Martins HFP, Neylon C (2007) Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation, PLoS ONE 2(11): e1164 doi:10.1371/journal.pone.0001164
  3. Popp et al., (2007) Sortagging: a versatile method for protein labelling. Nat Chem Biol Sep 23 (Epub ahead of print)
Posted on

Sourceforge for science

I got to meet Jeremiah Faith this morning and we had an excellent wide ranging discussion which I will try to capture in more detail later. However I wanted to get down some thoughts we had at the end of the discussion. We were talking about how to publicise and generate more interest and activity for Open Notebook Science. Jeremiah suggested the idea of a Sourceforge for science; a central clearing house somewhere on the web where projects could be described and people could opt in to contribute. There have been some ideas in this direction such as Totally retrosynthetic but I don’t think there has been a lot of uptake there.

This was all tied into the idea of making lab books findable and indexed in places where people might look for them. I have been taken with the way PostGenomic and ChemicalBlogSpace aggregate blogs, particularly blog posts on the peer reviewed literature and in the case of ChemicalBlogSpace aggregate comments on molecules, based on trawling for InChi Keys (I think). So can we propose that one of (both of?) these sites start aggregating online notebook posts? If we could make these point at peer reviewed papers online it would also be possible to use a modified version of the Blue Obelisk Grease Monkey that would popup whenever you were looking at a paper for which there was raw data online.

It wouldn’t be necessary, or perhaps even advisable, to limit these to people strictly practising Open Notebook Science. People could put up data once a paper was published or after a delay. Perhaps we could not even require that all the raw data be put up. If the barriers are lowered more people may do it. A range of appropriate tags (‘Partial Raw Data is available for this paper’, ‘Full raw data is available for this paper’, ‘Full raw data and associated data is available as an open notebook’,) would distinguish between what people are making available. Data could be dropped anywhere online and by aggregation it gains more visibility encouraging people to move from making specific data available towards making all their data available.

Any thoughts?

Posted on

Discussion with OpenWetWare people

This morning I got to sit down with Bill Flanagan, Barry Canton, Austin Che, and Jason Kelly and throw some ideas around about electronic notebooks. This is an approximate summary of some of the points that came out of this. This may be a bit of brain dump so I might re-edit later.

  1. Neither Wikis nor Blogs provide all the functionality required. Wikis are good at providing a framework that within which to organise information where as blogs are good at logging information and providing it in a journal format. Barry showed me a hack that he uses in his Wiki based notebook that essentially provides a means of organising his lab book into experiments and projects but also provides a date style view. In the Southampton system we would achieve this through creating categories for different experiments, possibly independent blogs for different projects.
  2. Feature requests at Southampton has been driven largely by me which means that system is being driven by the needs of the PI. At OpenWetWare the development has been driven by grad students which means it has focussed on their issues. The question was raised of where the best place to ‘promote’ these systems was. Is it the PI’s who, at least at the moment, will get the greatest tangible benefits from the system. Or is it better to persuade grad students to take this up as they are the end users. Both have very different needs.
  3. Development based on the needs of a single person is unlikely to take us forward as the needs of a specific person are probably not general enough to be useful. Development should focus on enabling the interactions between people, therefore the minimum size ‘user unit’ is two (PI plus researcher, or group of researchers).
  4. The biggest wins for these systems are where collaboration is required and is enabled by a shared space to work in. This is shown by the IGEM lab books and by uptake by my collaborators in the UK. This will be the best place to take development forward.

I need to add links to this post but will do so later.

Posted on

Talks on Open Notebook Science – some initial thoughts

So I have given three talks in ten days or so, one at the CanSAS meeting at NIST,  one at Drexel University and one at MIT last night. Jean-Claude Bradley was kind enough to help me record the talk at Drexel as a screencast and you can see this in various formats here. He has also made some comments on the talk on the UsefulChem Blog and Scientific Blogging site.

The talks at Drexel and MIT were interesting. I was expecting the focus of questions to be more on the issues of being open, the risks and benefits, and problems. Actually the focus of questions was on the technicalities and in particular people wanting to get under the hood and play with the underlying data. Several of the questions I was asked could be translated as ‘do you have an API?’. The answer to this is at the moment no, but we know it is a direction we need to go in.

We have two crucial things we need to address at the moment: the first is the issue of automating some of the posting. We believe this needs to be achieved through an application or script that sits outside the blog itself and that it can be linked to the process of actually labelling the stuff we make. The second issue is that of an API or web service that allows people to get at the underlying data in an automated fashion. This will be useful for us as we move towards doing analysis of our data as well. Jean-Claude said he was also looking at how to automate processes so clearly this is the next big step forward.

Another question raised at MIT was how you could retro-fit our approach into an existing blog or wiki engine. The key issues here are templates (which is next on my list to describe here in detail) which would probably require some sort of plugin. The other issue is the metadata. Our blog engine goes one step beyond tagging by providing keys with values. Presumably this could be coded into a conventional engine using RDF or microformats – perhaps we should be doing this our Blog in any case?

Incidentally a point I made in both talks, partly in response to the question ‘does anyone really look at it’, is that in many cases it is your own access you are enabling. Making it open means you can always get at your own data, which is a surprisingly helpful thing.

The CanSAS meeting was also interesting. This is traditionally a meeting where Small Angle Scattering instrument scientists, the people who maintain and support these instruments at large scale neutron and X-ray facilities, fail to agree on a standard data format. I wanted to make two points, one was the general point that making data available was a good thing, and secondly that making the instrument data available without a detailed description of the sample was pretty useless. However against all precedent they not only agreed a data format but it is also a flexible XML format allowing different tags for different ‘dialects’. So I can insert a tag into the data file that will point to our lab book, which is what I wanted.

Today I head off to talk to the OpenWetWare developers and the Simile group so that will be very interesting. More details as I have time to post.

Posted on

Growing a community – Open Notebook Science directories

As has been flagged up by Jean-Claude Bradley there are a couple of places now where people can sign up to say that they have Open Notebook Science in their laboratory, practise Open Notebook Science,or even would like to find a place where they can keep an Open Notebook.  Jean-Claude has put a list on the Nodalpoint Wiki and I have set up a database at DabbleDB. Dabbledb is a rather cool web based database system that provides free access as long as you make the database contents freely available. Because the data is completely open I am not asking for people’s email addresses.

If you want to be included in the database you can put your details in on the form here. This will allow anyone to re-use the data (which you can find here) to generate lists on appropriate web-pages, or maps or any number of other nice re-uses of the data. If you are interested in the working of the database give me a yell and I can give you admin access.

Posted on

The youth of today…

I like to think of myself as still a young person – in many ways I am stretching the definition but I still just about qualify for various schemes for ‘young scientists’ that cut out at the age of 35 or nine years post PhD. However I have to admit that I have been feeling a bit old recently. I have been struck by how many of the people driving the open science agenda forward are either current or relatively recent graduate students

It is conventional for ‘older’ people to complain about falling standards in exams, literacy, presentation, respect etc etc but I think in this field there would be a strong case for the new generation of scientists to point to falling standards amongst older scientists as people have become gradually more secretive and protective of ‘their’ work. It bodes well for the future that there are so many articulate and energetic champions of open science coming through the ranks. I only hope that it isn’t simply that people lose their drive to be open as the realities of professional research hit post-graduation.