What I cannot create, I do not understand
- Richard Feynman 

As of October, I switched over to an entirely new project and company at the QB3 incubator. The most difficult part of this change hasn’t been the hours of benchwork or technology, but rather the “tao” of synthetic biology – why rationally design biological systems and can human design surpass the infinite possibilities of the natural world? This unexpected turn of events has changed my view and approach to research (in a good way, of course). I’m excited for what’s in the store the next few weeks including a meeting with some folks from DARPA.

I’ll be dedicating the next few entries to a little side project I’m working on with Russell Neches. For some months Russell and I have been talking about investigating microbial communities in fermented foods, briefly chatting with cheese and salumi “microbial terroir” expert Ben Wolfe on how to go about our analysis. Russell and I are on the lookout for different varieties of miso; if you have a particular brand (or your own homemade stuff) you’d recommend please let me know!

  1. Reach for your samples in the liquid nitrogen or a hot autoclaved bucket without protective gloves – As a result of touching burning hot objects or dangerously cold tubes, my fingers fear not the cold or heat. It was a painful path indeed and I don’t recommend a similar fate for you.
  2. “Forget” to check labels – A few nights ago I was daintily razoring some two dozen bands from an agarose gel.  I just assumed the splash guard I was wearing had a UV filter…and had I checked the label I would have realized that it didn’t. My eyeballs are still hurting.
  3. Smack a machine with the intention of fixing it – I was once tasked the responsibility of fixing a non-operational magnetic stirrer. Having no background in electronics, I reasoned that the best approach was to smack the stir plate. I was lucky in that it worked the first time but has it worked since? Of course not.
  4. Leave broken equipment alone – Unfortunately, it’s incumbent upon you to at least to *try*. Case in point: Both the P.I. and post-doc in my Masters lab tried to connect a UV/Vis plate reader to the computer. After a few minutes of button clicking and software toggling, they both gave up not knowing how to troubleshoot or fix it. I stepped in, replugged all the connections (removing an unnecessary USB adapter) and the reader magically synced with the software.
  5. Leave the gas on – In the 11th grade I singed my bangs with a lit flame. In university lab my labmate tipped the Bunsen burner over while I was wiping the benchtop with ethanol. I’m not your burn victim, folks.
  6. Bring your dog/cat/animal – Bringing your dog to work has become a growing trend at many popular tech companies and I’ve occasionally seen scientists bring their dogs into a busy lab. As well trained as Fido may be, there’s always a chance that he might take a lick of the chemical coated ground or bury his face in an open container of used syringes. Just sayin’.
  7. Borrow without asking – Some are particular about it and some aren’t. Err on the side of caution and make sure to cover your tracks.
  8. Cry and punch things- You can cry when you get home, hell, you can cry a few meters outside the lab but please don’t cry at your lab bench. You become a safety hazard, you distract others who are hard at work, and you could potentially contaminate samples with the excessive snot and tears. As for the aggression – take it out during jiu-jistu practice.
  9. Record notes on a Post-it – At that point you might as well keep it all “memorized in your head.” Take the time at the end of the day (or even the week) to carefully write down what you did and keep a page in your lab notebook to jot down quick numbers or notes for the day. Post-it’s have a tendency to clutter your notebook or become misplaced.
  10. Be miserable – If this happens at any point, take a break. The worst thing you could ever do is continue working while feeling emotionally drained. I understand you just want to “get it done”, I know that if you don’t work now, you’ll fall behind later but the truth is, science stops working when you stop having fun.

For the past 3 months I’ve been working on Illumina high throughput sequencing approaches for identifying microbial communities in environmental samples using 16s rDNA.  I’ve spent the past 3 weeks troubleshooting library preparation problems and learned some interesting and possible experimental pitfalls that I thought I should share:

  • Most kits are prescribed for WGS: Most commercial kits are designed for whole genome shotgun sequencing so if you’re looking to do analysis on PCR amplicons, be prepared to Frankenstein some kits (which will require additional optimization). I wanted to multiplex 12 different samples in a lane; however, the kit only provided 10 reactions. When I asked for the next scale up, the sales rep offered to sell me a 48 reaction kit for 1,500$. In that case, you’re probably better off designing your own adapter addition strategy (be in T4 ligation or PCR)
  • Multiplexing is cheaper, but it comes at a price: If you don’t plan on normalizing your samples with qPCR or the Agilent Bioanalyzer, expect to pay a little extra for commercial labs to mix your samples. One company quoted me an additional $100 per sample in each lane. If your budget is small, you may as well normalize the DNA yourself.
  • You’ll wait longer for your data in academic labs: That’s sort-of a no brainer, but something to keep in mind. While commercial labs are marginally higher, the turnaround time is half as long and can also be a potential partnership.
  • Make sure to have enough DNA: While the sequencing reaction occurs at a pM level, if you are size selection the old school way (UV and razor blade party!) it is extremely easy to contaminate and lose yield. Most kits call for about 1 µg – give yourself a >2 µg wiggle room. Additionally, I suggest the Perkin Elmer’s LabChip device which does automated size selection for you.
  • SeqAnswers and NGS forums are your best resource: This includes troubleshooting problems, learning the technology (let’s be honest, Illumina has made it difficult for us to figure out WTF is going on), and snagging those coveted “Illumina-approved” barcoding sequences.
  • MiSeq runs are faster, slightly more expensive, and provide longer reads: Don’t automatically assume that HiSeq is the best option. The selling point with HiSeq is the 100 fold increase in coverage. It’s a bummer that the read length is stuck at 150 bp (even with the most recent upgrades) and if length is important (in this case, it is for 16s) consider using a MiSeq even though you get less reads.

Right now I am comparing and contrasting commericial adaptor ligation methods and a recently customized method published by the Knight lab. I still haven’t quite solidified the assembly pipeline, though Qiime is the analysis tool of choice.

Many thanks to Russell from the Eisen lab, the sequencing core facility staff at UCSF, and the DeRisi lab at UCSF.

I was an excellent undergraduate researcher. I gladly repeated experiments, worked very quickly at the bench, and loved learning from graduate students eager to pawn their work off. When I finally finished at Davis, I decided to give graduate school a try and enrolled in a Masters program. I was foolish to assume it’d be an easy transition, that perhaps the work would be similar but scaled up. But that’s not grad school and truthfully, you can’t ever be ready for the rigors of any higher level education. As a matter of fact, some argue that you can’t replicate the experience, that there is this “unique pain” in obtaining a Ph.D.. It’s my personal belief that one’s ability to finish graduate school is a combination of willpower and passion. So how can you test those personal limits? What situation affords a curious and doe-eyed scientist an opportunity to experience “real” science? Work at a start-up.

My definition of a “biotech” start-up: Under 10 employees with an operating budget of $5k a month. Salary equal to a post doctoral fellow or graduate student. No regimented work schedule, work hours vary dramatically depending on research goals.

Your responsibility (applicable to those with B.S.): Assigned a scientific task that you must research, implement, and conduct independently. Data is discussed as a team, but at the end of the day you are the only person actively thinking about the topic at hand. If you don’t do it, it won’t get done.

Ways a start-up is like graduate school:

  1. Resource acquisition: In graduate school you learn how to get “stuff” – be it advice/help, supplies, and even money – in order to accomplish your scientific goals. It’s no different at a start-up company and your team is relying on you to seek those resources. More often than not, it’s the relationships you build that make or break your project.
  2. No schedule: Industry jobs are regimented and I have plenty of friends who can keep a 9-5 working at larger companies. There’s no question that they work hard, but they also have control over their schedules. You learn how to operate on other people’s schedule at a start-up and in graduate school, your P.I.’s schedule.
  3. Pay rate: Generally, an industry or academic lab technician is paid more than a technician in a new company. I am paid the same as the average UCSF graduate student. It’s not a lot of money so get comfortable with microwave dinners.
  4. Perspicacity litmus: Like I mentioned before, there’s nothing like the pressure of having to be the “expert” at one topic that affords you an opportunity to actually consider how much you want to do this. If you can see a start-up through the initial growing pains, you can probably hack through the tribulations that come with true independent research.
  5. It will not work: If you are an industry technician you are typically assigned tasks which are accomplishable – there are prescribed deadlines, expected outcomes, etc.. You are intelligent hands functioning under assembly-style operations. When you are given a question to solve, that is a complete game changer. Expect things to not work and expect it to not work most of the time.

Ways a start-up is not like graduate school:

  1. Publish or perish: It doesn’t exist in start-ups. It would be ideal to publish a paper as a company, but it is not necessary and there are other avenues of acquiring funding.
  2. Equity: If you’re lucky enough to be a single digit employee, you get stock. Such monetary incentives don’t really exist in academia. In this case, your ability to accomplish your scientific goals are directly linked to money.
  3. “Ship it”: Academics dissect, critique, and beat the proverbial horse to death. The focus of academia is not the product, but the process. Companies always consider the fastest, most parsimonious, and economical route first. So as soon as something is done, even if there are still a few experimental gaps, we have to “ship it” off.
  4. Interact with non-scientists: You definitely do that more in a company and oftentimes they are the people that are giving you money to conduct the experiments.
  5. Producing a product: At the end of the day, your job is to get something to work and monetize it. Graduate school expects that and then some.

Final thoughts: The one underlying common theme to being successful in both these fields is your ability to not accept complacency. When you grow complacent, you grow lazy and in turn, stop learning. And if you’re not working hard and actively learning then you are doing something wrong. Be patient with this fact as in time, you will grow comfortable and learn to deal with such pressures and uncertainties.

Prior to graduate school, I was using Facebook, Tumblr, Twitter, and the like for personal reasons – starting music blogs, sharing cool photos and articles, keeping connected with friends, doling out clever puns/snippets of my stream of consciousness. It didn’t occur to me to use these social media platforms for research/academic purposes until my 1st year of graduate school. Within a few short months I found myself conversing with biologists over Twitter and very soon I was tweeting links to interesting research articles, posting interesting papers/commentary on blogs, and even meeting new microbiologists. I saw my number of followers start from a measly 10 people to over 300, dramatically increasing my academic/professional network. It became instinctive to tweet about laboratory triumphs and new discoveries – very quickly, my little lab bench felt a little less lonely.

Suffice it to say, social media has changed the culture of research science. Young scientists are more comfortable discussing ideas and seasoned veterans can easily furnish a response. Twitter is one of the very reasons I continued in research science and yet, last July, I deleted my Twitter account. Before I delve into a discussion about why I deleted it, I’d like to celebrate what these 140 characters have done for me:

  • Network with other microbiologists: Members of the Jonathan Eisen lab (@phylogenomics, @ryneches, @Dr_bik) as well as – to name a few – professors and graduate students from UC Berkeley, University of Puget Sound, University of Oregon, University of Chicago, University of British Columbia, Columbia, Harvard, and MIT.
  • Filtered interesting journal articles: In my database of articles, over 50 articles were I snagged from my Twitter feed. I looked back on several of them and honestly, I would never have come across them if it weren’t for my fellow Twitterers.
  • Found new experimental protocols: While most protocols can be Google’d, many contain proprietary information and are oftentimes impossible to come by through the internet.
  • Facilitated discussions of erroneous information: I oftentimes shared “#sciencefail” moments or things that are simply confusing and need clarification. Within minutes, some smarty pants will reply with either a useful response or a snarky remark (both were equally appreciated). I relied on Twitter for quick solutions and my followers were most helpful.
  • Provided a source of encouragement: When you are on your 10th hour of performing a tedious experiment, it’s nice to distill the frustrated screams down to a few words. And when you can find someone to scream with/at you, your situation suddenly becomes humorous. Instant morale booster.

So, what monster possessed me to delete my Twitter account last month? Honestly, it was a whimsical and mindless decision and I’d be lying if I told you it was planned and purposeful. As a matter of fact, I had a sinking suspicion that I would reactive my account, but it never happened. And after a few short weeks, my dependency on Twitter disappeared and I found myself with a list of things that Twitter couldn’t do and in some cases, prevented me from doing:

  • Maintain intellectual privacy: Shortly after finishing my M.S. program, I joined an incubator lab at UCSF and I currently work for a “stealth” start up company. No one in my company has issues with sharing, but I didn’t want to become an IP liability.
  • Intimacy: I’m not trying to test my relationships with other scientists, but the truth is you can’t replace a one-on-one chat with a colleague with 140 characters. I found myself using Twitter as the definitive resource for all my answers and it also became an intellectual crutch. Now I take road trips to Davis and Berkeley, chatting with graduate students I otherwise would never encounter in person. No one says Twitter is trying to replace the good ol’ meetup (in fact, people in our community have Biology “tweetups”), but I’d like to continue with the tradition of personalized invitations and conversations.
  • Time sink: I loved my Twitter feed and I spent hours scrolling through tweets. “The feed” soon replaced the New York Times, New Yorker, Discover blogs, New Scientist, Wired and all those news sources that I used to religiously keep up with.
  • Clarity: Twitter taught me the beauty of brevity, but sometimes, it’s important to have a long, drawn-out conversation about a topic. It opens up new ideas, clarifies misunderstandings, and facilitates a Q&A that is otherwise difficult to conduct over Twitter.
  • Replace Facebook: While I had well-over 300 followers on Twitter, I have 1,000+ on Facebook. Furthermore, it became hard to separate my personal and professional relationships over Twitter. I had considered starting a new personal account, but there wasn’t a way of snatching select followers and I was too impatient/lazy to encourage followers to follow me on another handle. While there may be unsavory photos of me on Facebook, I maintain my professional dialogue with scientists over LinkedIn. Now, if only someone could help me figure out these goddamn privacy settings on Facebook…

@emyrawks

Laboratory Update!

August 9, 2011

It’s 9:30 PM and I’m in the laboratory, patiently waiting for some proteins to separate. For the past year and a half I have tried numerous times to force E. coli to produce a protein responsible for oxidizing ammonia in Nitrosocaldus. While E. coli is every molecular biologist’s workhorse, my coercion has been fruitless. No matter how much I toggle with the growth conditions, it has refused to produce this protein. Having E. coli produce this protein in prodigious amounts is valuable as it will be the raw material necessary in understanding how Nitrosocaldus oxidizes ammonia. Why can’t ‘coli recognize my research needs and start producing lots of this protein?! Ugh.

Frustrations aside, there are successes. I’m approaching my final week working as a laboratory technician at Taxon Biosciences, spending the past 2 months hunting down microbes which degrade oil. In addition to successfully completing my summer internship at Taxon, I have successfully grown and harvested a 8 liter culture of Nitrosocaldus. A whopping 8 liters of cells in a single pellet:

Henry the 8 liter

Henry the concentrate

It takes about 50 mL of E. coli grown to exponential phase to produce a pellet as large as Henry. That is over a 100 fold difference in volume! So what exactly am I doing with all these cells of Nitrosocaldus?

Better than a dragon

August 5, 2011

As part of the PANACEA (for Pharmacological Augmentation of Nonspecific Anti-pathogen Cellular Enzymes and Activities) project, researchers from MIT Lincoln Laboratory have developed and demonstrated a novel broad-spectrum antiviral approach, called DRACO (for Double-stranded RNA [dsRNA] Activated Caspase Oligomerizer). DRACO selectively induces apoptosis, or cell suicide, in cells containing any viral dsRNA, rapidly killing infected cells without harming uninfected cells. As a result, DRACO should be effective against virtually all viruses, rapidly terminating a viral infection while minimizing the impact on the patient.

Source:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0022572

http://www.ll.mit.edu/news/DRACO.html

Last September I saw this headline in Nature and thought to myself, “I’m okay with bootleg DVDs and Gucci watches but they’ve really crossed the line.” This recent NPR sound bite suggests that this ethical problem is rooted in culture – the lack of innovation and original thought stems from politics and tradition.

It surprises me that we’ve made a discrepancy between American and Chinese science. American science is original, thought provoking, and critically analyzed. Chinese science is unoriginal and prolifically empty. Given that science is (or at least ought to be) a universal language, there really shouldn’t be differences in how it is practiced in any given country. The standards should stay true regardless of race or creed – Scientific theory governs every principle and practice. Theory has already failed us a few times in the past few years (arsenic bacteria and autism linked to vaccinations) and I’m beginning to wonder what it means to conduct science. For some science is executing a set of experiments that answers an original question; for others it can be cherry picking experiments to answer a question or relying on other people’s data to answer an original question. While we can accuse the Chinese for lacking original thought, it appears that the rules of the game may have never been clearly established.

Introducing Nitrosocaldus

August 3, 2011

Since I started this blog with a brief introduction of myself, I figured I’d briefly introduce our study organism, Nitrosocaldus yellowstonii.

Our study organism is a thermophilic ammonia oxidizing archaea isolated from a hot spring in Yellowstone National Park. Since its cultivation in 2008, we have kept it continuously in culture. Every seven to ten days we take an aliquot of the culture and “passage” it into fresh media. While it sounds straightforward, there are a few factors to consider when growing these organisms. Transfer too late and you run the risk of passaging dead cells; too soon and you risk passaging a fraction of the culture with no cells.

Heart Lake region - Home sweet home

In addition to Nitrosocaldus, our culture contains a few other bacteria. This paper goes into further detail about the bacteria in our culture. I am frequently asked by other scientists how we conduct experiments when our culture is mixed. The bacteria represent <10% of the total population. Despite the heterogeneity, Nitrosocaldus is the dominant organism in our culture. While several have attempted to remove or kill the bacteria, it appears that Nitrosocaldus can’t survive without the bacteria. This has left us all wondering – what could our archaea possibly need from these bacteria?

Me diligently preparing their feed. Those red and green gas tanks in the background contain the nitrogen/carbon dioxide/oxygen gas mixture.

Nitrosocaldus is kept in gas-tight glass tubes that are filled with a proprietary blend of  UV treated water, vitamins, and salts. They prefer to munch on dissolved carbon dioxide present in the water (we add research-grade baking soda) and seem to hate organic carbon. Unlike atmospheric air (78% nitrogen, 21% oxygen, 1% argon) these archaea prefer to respire a mixture of gas that consists of 70% nitrogen, 20% carbon dioxide, and 10% oxygen. Once prepared, the cells are transferred and incubated at 72 C. Their optimal growth temperature (the temperature at which they will divide the fastest) is supposedly higher, but it’s difficult enough trying to handle these bottles at 72 C.  According to my lab mates, the optimal growth temperature is actually lower. If they grow better at lower temperatures, why are we growing them at a higher temperature?! That’s for me to know and you to think about.

I hope to discover a couple of things about this organism before I part ways with the lab:

  1. I’d like to know where ammonia oxidation is occurring in the cell. The annotated genome helped us determine models for how it occurs, but I am hoping to localize the proteins involved in ammonia oxidation.
  2. These ammonia oxidizing archaea are ubiquitous. They’ve been identified in exotic (sediment from the Gulf of Mexico), modest (greenhouse soil), and lowly (wastewater sewage) locales. Furthermore, Nitrosocaldus’ marine and soil brethren appear to greatly impact the global nitrogen cycle and I hope to learn a little more about how this particular class of archaea might collectively impact the environment.

Stay warm, kids!

August 2, 2011

My background

Prior to graduate school I worked as an undergraduate research assistant in numerous research laboratories spanning a wide range of topics – from aquatic toxicology to nematology to microbiology. While most of my formal training is in microbiology, I’ve always made it a point to stay up-to-date with popular science and remain well informed within the field of biology. And while I secretly wish I were a mathematician or computer programmer, I am thrilled to be conducting research on this hidden biota of life.

Why “Treehouse Science”?  

My love affair with microbiology began with a series of well-told stories by my undergraduate microbial physiology professor. Every Thursday afternoon I would pay him a visit during his office hours where he would recount tales of endosymbiosis theory, heterocyst and hormogonium differentiation in cyanobacteria, and the progress he’s made with his vintage Ford pick-up. While most stories were bogged in jargon, his enthusiasm was infectious. There was this certain je nais sais quoi about the way he taught microbiology, a child-like spirit that was endearing and relatable. Every great scientist I have encountered channels a similar enthusiasm for research and I suppose their laboratory is like their treehouse.

Goals for this blog

  1. Share my personal research with a larger audience.
  2. Summarize interesting research articles.
  3. Express my thoughts and opinions on trending topics in popular science.
  4. Improve my writing skills and become a more effective communicator of science.
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