Sunday, March 8, 2009

What's cooking and what's been koching?

I have two goals with this post: (a) to answer a question "why don't we have unzipping data yet" and (b) to explain some interesting simulation data we having related to an alternative splicing application of our shotgun DNA mapping technology.

What's been koching?

I have been getting asked the question a lot recently, in various forms and in various levels of directness: "how come you've had your new lab for two and a half years and you still don't have optical tweezers data?" This question comes about because of my mid-probationary tenure review, grant applications, collaborations, and just general behind-the-back chitter chatter. I have to stress that I find this question perfectly reasonable and totally valid. Most new professors get their lab up and running much more quickly than I have. Even I expected to be "up and running" sooner--but I am not disappointed. Rather, I am quite happy with where we are at, because my plan was to have the students involved in every aspect of our lab start-up, and to fundamentally understand optical tweezers, why we're building them, and what our impact is going to be. I've spent (or reserved) the majority of my start-up funds ($200K) on students. Those students are now poised to have a really great optical tweezers and a deep understanding of how we are going to make biological discoveries with this tool.

During my first two plus years as an assistant professor, I have failed at expressing my lab mentorship vision to my colleagues at UNM. I do think I've succeeded in privately communicating my vision to my department chair, mostly via my written annual updates and goals. But I've not communicated my plan to the vast majority of people who care about me, or at least care about me bringing in money. One of my graduate students, Andy, helped me realize yesterday this problem and I'm really happy that I now see it. Further discussion of this non-talent, I think I'll save for my other blog. My main point here is to just acknowledge that it's been my own failure to engage and communicate that has created this minor issue (only a major issue if I don't get my contract renewed, which is unlikely I think). My goal will be to much more aggressively communicate my vision to people when this issue comes up in face-to-face conversations. Maybe something like this:

"I know! I'm itching for the data to start coming out too. We've been much slower than average because my philosophy is that one of the missions of my lab is to mentor students. The students need to learn and need to be involved in the management of the construction of our instrumentation, the designing of our experiments, and even our grant writing. Because of that, we're not as far along in terms of data and publications as many people would have expected. On the other hand we are only a few weeks away from having a kick-ass optical tweezers system that the students have designed and constructed. And these students have a very solid understanding of what we're trying to achieve, and a self-driven motivation to achieve from this point onward. So, many good things are going to happen for us this year, and our rate of achievement over the next few years is going to be much, much higher than it would have been if I had simply built optical tweezers and commanded students to acquire data. Furthermore, because of their intense participation in our lab start-up, these students are going to be very strong in the next stages of their careers--particularly if they have to start-up their own lab."
I need to rehearse that. Sort of like my "30 seconds in the elevator with the CEO" kind of speech. Furthermore, when I'm asked this question by certain people, such as those that support all the extra BS and hoops that our graduate students have to jump through, I plan on going further to explain how those hoops and BS substantially impacted the progress of our research. What I have in mind here are the IGERT programs we have with ridiculous non-research requirements (including 4 extra courses) and our physics department's fixation on testing and retesting and retraining our graduate students on undergraduate quantum, classical, stat mech, and E&M.

The last thing I want to present in this section is a very clear example of the success of my student mentoring strategy: Andy, Larry, and Anthony (our three grad students) all applied for and were awarded individual research grants recently. These equipment grants were for $3,000 each, from UNM's graduate student association. Each of them drew up their own budget and individual research plan, and obviously they all succeeded in bringing in external funding for their research. They even went against my advice of combining their efforts to go for a single $3,000 award! I love that they proved me wrong, and I love even more that they have succeeded in getting their research funded at such an early stage in their careers. Each of them are purchasing specialized parts to make our optical tweezers better and they have thus succeeded in a major part of research: getting the funding so you can do the things you know you need to do. I'll end this section with a big congratulations to our grad students!

What's cooking?

Optical tweezers

We have a lot of things cooking right now, and like I said above, I think 2009 is going to be a really big year for our lab. Of course, optical tweezers construction has been a major focus. Anthony, Larry, and Linh have been working on this for over a year and achieved a number of milestones. Recently, Andy Maloney has brought his amazing optics and design skills to the project. He's also making a transition to open science, and has started publishing a lot of his work on OpenWetWare, Google 3D Warehouse, and elsewhere. Just one cool example is the current condenser / detection optics design, you can see a snapshot of Andy's sketchup design here.

Molecular biology and yeast genetics

In addition to constructing optical tweezers and control software, we've had a lot of things going on. One major area has been with our collaborator, Mary Ann Osley, working to create site-specific chromatin tethers and other biological constructs. Diego Ramallo Pardo (now at Stanford biophysics graduate school) initiated this work for us and made a lot of progress towards our goal of creating "unzippable" native chromatin fragments from a specific locus in yeast. Diego succeeded in transfecting yeast with a PHO5 plasmid having a unique I-SceI restriction site and he also succeeded in making dig/biotin DNA tethers. Further, he worked on our protocol for creating the versatile unzipping construct. This is a construct I designed during my graduate work. The beauty of this construct is that it allows for unzipping of virtually any DNA fragment that has a sticky end (see the figure). The versatility and power of this design has been demonstrated by students in the Wang lab who used the design to study helicase (Dan Johnson, preliminary work by Richard Yeh), mismatch repair protein (Lucy Bai), and mononucleosomes (Alla Shundrovsky, now Michael Hall), and of course my own work demonstrating the ability to map and probe protein-DNA interactions by unzipping.

Before he left for graduate school, Diego taught another student, Brandon Beck, what he had learned, and Brandon succeeded in creating unzipping constructs from an XhoI-digested plasmid DNA. The torch has now been passed to Anthony, who also has a strong natural talent for molecular biology. Furthermore, he's added an Open Notebook Science dimension to the project. You can find his notebook on openwetware. Anthony is focusing on creating shotgun clones from XhoI-digested DNA. We will use this to demonstrate shotgun DNA mapping of yeast genomic DNA--because we can sequence the clones to validate our single-molecule technique.

Alternative splicing: very promising simulation results


I want to update you on one specific result that Larry achieved recently: demonstrating that single-molecule analysis of splicing by unzipping looks very promising. Like all of the above fascinating unzipping studies, this too will be enabled by the versatile unzipping construct and the ability to unzip any DNA fragment with a known sticky end. We have been thinking that structural genome mapping would be a very cool application of our shotgun DNA mapping technology. (See our preprint on Nature Precedings.) Mary Ann Osley, our collaborator on our chromatin mapping project, suggested a couple months ago that we simulate an inversion mutation, since these are tricky to detect with ensemble methods, since they are "balanced" mutations. We asked around on friendfeed and got advice to check out "inv16" for acute myeloid leukemia. Unfortunately, Larry and I didn't know enough about genetics to figure out what exactly inv16 involved, and thus we couldn't easily simulate it. However, at that time, I remembered some very intersting material I had seen Scott Ness present, related to alternative splicing. It occured to me that unzipping would be perfect for analyzing splice variants, because large deletions, insertions, or rearrangements are easily detected by single-molecule unzipping. So, Larry found a recent paper from the Ness lab, about alternative splicing of c-Myb, which is the fascinating topic I'd heard Scott Ness talk about. Larry was able to find the sequence for a couple splice variants (8 and 8-b) on Pub Med, and put them into his unzipping simulation LabVIEW application. You can see the results of this simulation in the figure. Clearly, the difference between the two splice variants is one or more exons being missing in the black (variant 8) versus the red (variant 8b) curves.

We showed this simulation to the lead author on the paper, "Johnny O" a postdoc in the Ness lab. Even without explaining what the hell we were doing, he immediately saw what we were talking about. (Man I wish he was reviewing our grants!) (Johnny O is an excellent scientist, and he even has that sweet nickname, so definitely he is destined for greatness.) Our plan is to wait until (a) we have our optical tweezers going and (b) Anthony has mastered the DNA ligation method for creating the unzipping construct. Once we get to that point, we're going to get in contact again with Johnny O and see if we can develop a collaboration with him and Ness to try some single-molecule typing of cDNA clones of splice variants (using variations of our shotgun DNA mapping algorithm). The fact that cDNA clones have known restriction sites should make this experiment quite easy to try out once we have (a) and (b) going. This will be yet another example of the versatility of the unzipping construct I developed. And the fact that splice variants have large structural changes makes it highly likely we can succeed in single-molecule typing of splice variants (supported by our simulation above). I'm in love with this idea so much that I'm thinking it should evolve into a specific aim for our upcoming R01 applications. I'll talk more about that on my research blog, which I tend to use more for discussion of funding and future research plans.

So, that's my update on what's cooking now -- hopefully I've conveyed enough for you to understand why I'm excited for our lab and for our students in the coming months!


Wednesday, March 4, 2009

The Fear of Scooping

All my hopes and dreams... gone in a flash. I wanted to be the first or at least be able to establish my thesis based on this line of work. Instead that hope has been reduced to nothing. What can I do now? How can I continue? I don't want to find a new research topic. I don't want to find a new field. I don't want to start all over again. I just don't want to...

These are the thoughts that went through my head as I glanced over a poster at the Biophysical Society Meeting. The poster described establishing a force profile of RNA Polymerase and studying how it affects nucleosome dissociation. The work was being done by a grad student in a competing lab. The lab was that of my PI's (Steve Koch) former graduate lab in Cornell, the Michelle Wang lab.

I decided to read the poster after Larry had pointed out that Dr. Wang had a student poster not too far from where I was standing. Larry didn't really care to read it. I walked over and glanced. I rapidly read the title. I don't remember gathering much information from the title, but after quickly scanning the poster an image of RNAP with the transcription bubble caught my eye and urged further reading. I walked closer and read a little more. I inched closer and read more. Each reading prodded me closer and to divulge further. Finally I decided to read the whole thing and ask the student questions.

The student (whose name escapes me as of this writing) was very friendly and nice. She discussed with me what they were doing and I told her that I would be doing the same thing (or so I thought). She was very interested in the technique I would apply and I told her it would be the same as hers. I then told her I worked for Koch and she immediately recognized the name. She shot me a smile as she realized that all her work was based on his prior work (the tweezers are courtesy of he and Richard Yeh, and their anchor construct is all Koch's doing).

In reality all the fears I had were premature. Sure they were warranted. A competing lab is working on the same topic and idea that we are. Turns out they are working with E. coli while KochLab is initially working with yeast (proof of principle stuff) and eventually will be upgrading to human genome studies.

As I gazed at every single section of the poster, I pondered in both wonder and fear. Wonder because the work done was very good and interesting. I was fearful because it was all the same work that I wanted to achieve. To feel like my project was being ripped out from right underneath me could quite possibly be the worst feeling in the world. I did have other endeavors I could follow, but the RNA Pol II studies are what I am most interested in, and I truly would like to be the first to carry out those experiments.

I have learned a very valuable lesson from that experience. The fear of scooping is a very real fear. Being an open scientist, I suppose now I could fear that even more, but I really don't have it in me. I want the whole world to know what I plan to do and share my experiences in real time instead of portraying the clean cut science at the very end of it with just a paper. I in fact would like to turn this experience into a positive. After seeing some of the work that my competitor is doing I would like to see if there is a way we could collaborate. Maybe that can never be, but at least by this offer I can put my fear aside and make it a strength.

Overview of the Biophysical Society Meeting: Boston, 2009

This is my second science conference ever with my first coming last year at the same meeting but in Long Beach, California (LBC). Last year was immensely fun both conference wise and extracurricularly. Outside the conference I had many adventures, and during there were so many talks I wanted to go to and posters I wanted to see that I couldn't keep my head on straight. I really had no chance of seeing it all.

This year I expected nothing less. I made no plans as far as venturing the city and left it all to chance. I tend to be that way. I like to go with the flow and make it up as I go. Don't think that I'm not organized or have any motivations, because that is just not true. I just don't like to make plans unless I have to and I like to keep my schedule free. As far as talks and posters go I have some experience under my belt so it shouldn't have been as tough as last year. I also know a great deal about my own research and the overall focus of the lab so I can pick appropriate talks and posters easier.

My feelings this year is that nothing really blew me away. I remember learning so much last year, but this year I am not walking away with that same feeling. There were some talks that I was really looking forward to just to find out that the research being presented had very little to do with what I expected (from the title). I was also a little disappointed in the quality of some of the posters I looked at.

With that said, it wasn't a total bust and there were some very noteworthy things as can be found here.

Some highlights:
The Block Lab showed why they are the best at what they do yet again. This year they cleverly attached DNA handles to kinesin motor heads to study the mechanics of the step. By using DNA fragments shorter than the persistence length of DNA they were able to use the DNA to manipulate the heads to gather force intel and other information about the stroke.

There was a beautiful talk detailing the truth about S-phase DNA. The group used specifically binding proteins and fluorescent labels to attach to dsDNA and ssDNA to distinguish melted DNA from the mythical s-phase.

The Wang poster was pretty good (about RNA Polymerase unzipping) and actually gave me hope for my project. We can also use their data as fuel to the fire for my own proposed experiments.

Meeting Micah and viewing the Williams Lab was awesome. We had a great night and learned a lot about aligning tweezers and setting them up from him. I look forward to meeting him again in the future.

Being in a city with a great public transportation system and plenty of things to do was amazing. The food was great and everything will be greatly missed when I return to boring old ABQ.
 
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