The Molecular Biology Code

“You’re pirates. Hang the code. Hang the rules. They’re more like guidelines anyway.”              Elizabeth, The Pirates of the Caribbean: The Curse of the Black Pearl


Molecular biology, the overall practice of manipulating DNA sequences using biological scissors and glue, is a major component of any modern research lab. Its techniques have been revolutionary with respect to expanding the types of questions scientists can answer. While it may be grounded in the fundamentals of biology’s central dogma, molecular biology is often practiced with rituals and superstitions characteristic of pirates and other Caribbean stereotypes. And, like the pirate code, molecular biology contains as many exceptions as rules. What one molecular biologist may swear by is probably not so strictly enforced by another. After all, with most molecular biology construction projects, you only need it to work once and hope the sequence is right. So what I write here today are more along the lines of guidelines for cloning success.

  1. The enzyme is the last thing to add to any reaction.
  2. Do not vortex enzyme reactions.
  3. Use a new tip for your pipettor each time.
  4. Prepare a master mix for multiple reactions.
  5. Avoid gel purification of DNA fragments if at all possible.
  6. I’m not convinced that the enzyme ligase does anything, but add it anyway.
  7. Use restriction enzymes until you have evidence they don’t work anymore.
  8. Do the controls.
  9. Phosphatase treat your vector or insert but not both.
  10. Selection > Screen > Hope
  11. Sequence your finished construct to verify it is error free.
  12. Make freezer stocks.

See below for more explanations…

The enzyme is the last thing to add to any reaction.

This is basically another way of using Biochemistry Rule #4 (Use a buffer). Your expensive enzyme will likely be ruined if you put it in a tube of water or other dilute solution with no buffering capacity. So, pipet everything else first (DNA, nucleotides, water, concentrated buffer solution etc) and add the enzyme last. I would even argue that you shouldn’t even take the enzyme out of the freezer until you are ready to pipet it. Always keep it on ice or a cold block container. NEVER let it sit around on your lab bench at room temperature. If you were thinking of taking your reactions right away to the necessary water bath or instrument, think again. PUT THE ENZYME BACK IN THE FREEZER FIRST!!! That way, there’s no chance of you moving onto the next experiment without your precious enzyme safely stored away. Now, whether or not you choose to dilute your reactions with Holy Water or otherwise blessed dH2O is up to the individual scientist.

Do not vortex enzyme reactions.

After you’ve taken the care to ensure your enzyme is gently pipetted into an appropriately buffered reaction, don’t kill it by mechanically ruining it. You will need to physically mix the enzyme into the reaction because the enzyme exists in a glycerol solution that will sink to the bottom of your reaction. Do this by gently pipetting up and down or flicking the tube with your fingers. You may develop your own distinct style of mixing akin to a secret handshake. This routine is usually the superstitious result of a single instance that ‘the experiment worked when I did it this way.’

Use a new tip for your pipettor each time.

Reagents are precious and must not be contaminated. Use a fresh tip every time for each reagent and sample. Contaminated reagents may not ruin the experiment you are doing today, but they will ruin yours tomorrow and all of your labmates’ experiments. Be mindful when pipetting in general. If you can’t remember whether you added a reagent or not, just start over. Soon enough you will have your own Rain Man-like process for pipetting your reactions. On top of this, you may also develop your own style for using tips out of the box. Some of you may be a strict left-to-right or right-to-left row user, others prefer a diagonal strategy, and others may prefer to introduce designs in the tip boxes (initials or emoticons) with their tip use. Just go with whatever you find the most comforting or the technique with the highest rate of success on your reactions.

Prepare a master mix for multiple reactions.

The more times you have to pipet anything, the more potential error you introduce. Reactions become inconsistent across different samples. The way to avoid wearing out your opposable thumb doing tedious, pointless and downright erroneous pipetting is to prepare a master mix. This is just a scaled-up version of your reaction to accommodate as many samples as you have. Multiply the reagents in a single reaction by (x + 1), where x is the number of reactions you really need. Again, there is always pipetting error and even with the master mix, you will end up short on volume if you only use ‘just enough’. Once the master mix is prepared and gently mixed (very important in this scenario or some of your reactions to do not get enzyme), pipet out equal amounts of your master mix into individual reactions. In this case it is sort of OK to break rule #1. Usually the only difference among your many samples is the DNA or other reagent that’s not the enzyme. In this case, add the enzyme second-to-last, divide the master mix among individual reactions and then add the variable reagent. This variable reagent is usually such a small fraction of the total reaction volume that your enzyme is still under safe buffer conditions. Feel free to come up with your own ‘lucky number’ for scaling up your reactions with more than enough volume to accommodate the reactions you really need.

Avoid gel purification of DNA fragments if at all possible.

Sometimes you may need to isolate a DNA fragment for molecular cloning purposes. Agarose gel purification is a way of doing that. Companies will sell you easy-to-use kits to do this. In my experience, the sample losses are so great that it’s not even worth it. It is very difficult to obtain the quantities of DNA you need for subsequent steps from gel purification. At that point, you can either resort to faith-based cloning, in which, you can’t see your fragments on analytical gels with the human eye nor with help from the imaging camera, but you use it for ligation any way. Some times this works, but usually you are just disappointed when you check your transformation results. There are other tricks to avoiding this technique and if you are a clever cloner, you can get around having to gel purify anything. I swore it off several years ago and have never looked back. Sometimes, gel purification is unavoidable and I would recommend invoking some kind of Voodoo incantation to help success along.

I’m not convinced that the enzyme ligase does anything, but add it anyway.

Ligase is the enzyme responsible for gluing two desired DNA fragments together. The stitching together of these molecules in vitro is not the most efficient process, but we’ve been told that ligase ultimately seals the deal between our pieces of interest. Let’s just say, I’ve done enough positive and negative control reactions (more on that below) and I’m not sure ligase really does anything. However, I add it anyway because… protocols. Really, at this point in the cloning process, you’ve done so many purely superstitious acts, it doesn’t matter if you do one more.

Use restriction enzymes until you have evidence they don’t work anymore.

These enzymes are the molecular scissors that cut DNA. They expensive, but they can last decades past their expiration dates when they are properly taken care of. Y’know, when you keep them cold, don’t vortex them or contaminate them with other reagents. You will also have to keep them in a frost-free freezer so they do not endure the temperature fluctuations of a self-defrosting freezer. So until you have evidence confirming a failed digest, keep using the enzyme.

Do the controls.

This rule is true across all scientific disciplines, but in the case of molecular biology work, it can save you lots of time, headaches and wasted reagents. For anything that you are doing, make sure you do a negative control that you know shouldn’t work and a positive control that should work. If both of these types of reactions give the expected result, then you know how to interpret all of your other samples. If only one or the other or neither of these controls work, then you will have difficulty saying what is going on with your experiment. It usually means there is a problem with user error or some other fatal flaw in your construction plan. You may try to interpret faulty experiments and even hope against hope that your experiment worked, but this relies more on faith and superstition than scientific probabilities.

Phosphatase treat your vector or insert but not both.

In many molecular cloning experiments, you are trying to combine two separate pieces of DNA with one another (a vector and an insert) in a useful way. However, sometimes there’s nothing or not much to stop the ligase enzyme from gluing together the vector with itself or the insert with itself, giving you useless byproducts. This is what the phosphatase enzyme does. It can remove the reactive chemical group from a piece of DNA such that ligase can’t use it. So if you treat one piece of DNA but not the other, it eliminates the possibility that ligase will glue any piece to itself, but instead glue the two pieces together. However, if you ligate both pieces, they all become useless to ligase. Sure, I didn’t think it was doing anything anyway, but now those reactions are guaranteed not to work.

Selection > Screen > Hope

When you are trying to get organisms to produce the DNA construct that you have engineered, it is better to select than to screen and better to screen than hope with blind faith. When you are selecting for a construct, all of the cells with the wrong thing will die and only cells with the right DNA will live. Thus, anything living at the end of that experiment is likely to be correct. When you can’t do this, there are ways of screening either based on color or replica plating onto a special medium. The most widely-used example is blue/white screening. If your bacterial colonies are white, they have the correct DNA. If they are blue, they do not. This color gives you a visual clue as to which colonies are most likely to give a positive result based on another experiment. If you can do neither of these things and can only hope to find the correct clone in a plate with hundreds of colonies to choose from, then you have more work ahead of you. In any event, doing the controls is still important. In the case where neither selection nor screening is possible, it may not even be worth looking through the colonies with subsequent experiments to verify a positive DNA sequence. There may just be too many false clones to sort through. Nevertheless, you may try it anyway. 99% of the time you will just end up wasting reagents and time. There does exist a possibility that the correct one can be found, and if you find it, you should probably buy a lottery ticket on the way home.

Sequence your finished construct to verify it is error free.

Once you think you are done with piecing together the DNA sequences you need, you will want to perform experiments to verify that the pieces have come together as you intended. Sure, you’ll cut them again with enzymes and run them on a gel to make sure it looks as expected, but you need to sequence the DNA to make sure that no point mutations have been introduced somewhere along the line. The enzymes responsible for copying the DNA pieces along the way have error-checking features to maintain sequence integrity, but the course of a typical molecular biology project will involve such a length as to make a sequencing error a formal possibility. To assume correctness is just hubris. Sequence it to make sure. Data always trumps assumptions, and that’s no superstition no matter what your scientific discipline.

Make freezer stocks.

Once you have your precious construct and are sure that the sequence is error free, you will want to get to work on your exciting new experiments so you can get groundbreaking results, publish a paper in a high impact journal, secure your own funding, get a job offer at a top research institution, win the Nobel Prize and ride off into the sunset on a unicorn.* But first, YOU MUST MAKE A FREEZER STOCK OF THE CELLS CONTAINING YOUR PRECIOUS DNA CONSTRUCT. Seriously, if you don’t, I’ll make you walk the plank. Make several tubes; label them with an identifiable name (pNobel2015), include the date and any antibiotic resistances. Then write all that shit down in your notebook. For extra credit, generate a graphic map which notes the important features of the sequence. Put the freezer stock tubes in your own freezer box as well as the lab repository. If you don’t do it now, there is a high probability you will forget to do it. You may have to kiss your Nobel prize goodbye if your peer reviewers ask for additional experiments that may require you to go back and use the DNA and your cells are too old to resurrect. Well, maybe not, but I guarantee there will be some poor graduate student or postdoc will carry on the torch of your research several years after you made the construct and it is nowhere to be found. Thanks to you slacker, they’ll have to remake the whole thing from scratch. You did write down your primer sequences, right?** Just save the world a lot of trouble and make the freezer stock.

I’m sure there are many more guidelines that other cloners could add. Maybe we should all just resort to Gibson cloning methods now anyway. Feel free to add your own guidelines in the comments section.


*Just me? OK then.

**So help me God, if you didn’t… Chickens are being sacrificed to empower Voodoo dolls of you to exact vengeance.


Today is the first day in my new role as Biochemistry Instructor for my department. It’s a change I’m very excited about. I’ve inherited a great lab course along with some experienced TAs. Of course I’ll be teaching my students the rules of biochemistry. However, the lab course also incorporates a great deal of molecular biology as well. Stay tuned on the blog for the rules of molecular biology. As for this blog, I’ll keep posting as much as time allows. There may be some topic deviations, but I plan on keeping the primary subject matter the same. After all, somebody has to speak for the trees.



Superhero PhD: Nike!

We last left Superhero PhD and her labmates in a state of noncompliance with the system that is supposed to support them. Fortunately, their misdeeds did not attract the attention of accountants higher up in the matrix. Even the recently purchased equipment from e-bay works just fine. These small triumphs make the days in the lab pass easier, which was good since several more days passed beyond the funding agency’s deadline for divulging the fate of tenured PI’s latest grant renewal. Finally, the e-mail from the Program Manager arrives… victory! The grant is renewed for another three years. Tenured PI must send a flurry of additional e-mails as slight adjustments to budgets must be made, but these are a welcome burden given the possible alternatives.

Superhero PhD takes a break from benchwork to battle with the peers who reviewed her latest manuscript submission. She has spent the last months painstakingly repeating experiments to satisfy their demands for MOAR DATA! Now is the time to revise, remake figures and resubmit. Tenured PI submits a more appropriately censored version of the responses to reviewers’ comments to the editor than the first draft provided by Superhero PhD. It’s probably for the best. It will be another few weeks before the next battle with reviewers over this manuscript.

Back to the routine of research Superhero PhD focuses on projects that have been on the sidelines. A new mutant is giving unexpected results, but our heroine knows better than to get too excited. DNA samples are submitted for sequencing analysis to check for possible errors in the intended sequence. It seems like an easy task, but the new on-line system for submitting the samples and payment information requires more steps and approvals than acts of congress to approve federal budgets. Tenured PI even gets automated and unrecognizable e-mails from the system requesting approvals. Finally, the analysis is complete and Superhero PhD receives the results. “Nooooooo!” she cries, shaking her fist at the screen. The construct has a point mutation in addition to the complex construct she has pieced together. The mutant is unusable because it runs afoul of a central tenet of scientific experimentation- only change one variable at a time. These results sentence Superhero PhD to weeks of molecular biology work to remake a clean version of the mutant. In all truthfulness, Superhero PhD already had a stint of molecular biology ahead of her with other constructs and mutants to be made for her new project. For biochemists, molecular biology is a necessary evil.

Despite the occasional sequencing error, Superhero PhD also has molecular biology superpowers as well. She sets out to spend her days tediously pipetting PCR reactions, restriction enzyme digests and running agarose gels. To visualize these gels, she must rely on departmental common equipment whose other users can be less than meticulous. Today, she finds something even more unusual. An unexpected power outage overnight has forced the computer to restart, a computer still running WindowsXP that probably hasn’t restarted in three years. Sigh. Login required. The password is not written anywhere in the area of the computer. Superhero PhD summons her powers of clairvoyance to divine the password, and she is correct.* She captures the image of her gel on the screen. Print. But something again is wrong. “Damn, the printer is out of the special thermal paper. Chances of other users reporting it to the Departmental Coordinator in charge of ordering the replacement- zero.” But Superhero PhD is prepared for such instances; she keeps a secret stash in her lab for just these occasions. “Incomplete lab notebook, I think not.” she chuckles. “However, I will have to step up my notes describing the proper procedures for acquiring more paper from passive-aggressive to full-on aggressive.” Superhero PhD muses.

Another consequence of molecular biology work is defrosting the frost-free freezer. For those of you wondering why a sophisticated modern research lab would have a frost-free freezer, it is because the precious enzymes used for these projects must not endure the temperature cycles of a typical modern freezer. They must remain cold at all times and fluctuations too warm will diminish their activity. Superhero PhD’s lab has still-active molecular biology enzymes whose purchases pre-date the births of many undergrads working there. However, after about a year of opening and closing the door of a frost-free freezer, the ice build-up inside necessitates the removal of all contents and a manual defrost. Superhero PhD uses her skills of organization to temporarily store all of the contents of the full size frost-free freezer, taking special note to remember where each item must be returned. It would be nice to say that Superhero PhD then uses her laser vision to melt the excess ice on all of the shelves and coils, but alas, that is one superpower she lacks. Instead, she and Graduate Student use a hair dryer** and spatula to melt and hack away at the ice for about an hour.

Elsewhere in the departmental common equipment room, Research Technician is battling with the ultracentrifuge, an expensive and formidable foe. “The imbalance error light is on, but there is no imbalance. I cannot get it to start.” he says. Superhero PhD gives him a hard, disbelieving look. “No, really. It’s balanced.” he retorts. Instances of imbalanced ultracentrifuges are the stuff of scientific research legends. The instruments turn rotors at such high speeds that failures in components or user error like imbalanced samples can turn these metal rotors into dangerous projectiles that penetrate walls and kill unsuspecting graduate students down the hall. “Did you turn it off and turn it back on?” she asks. “Yes, the error is still there.” Research Technician says exasperated. Superhero PhD then uses her superpower of pressing seemingly random buttons on the control panel (SET, IMBALACE, CLEAR, ENTER) and the imbalance error is cleared. The instrument will now be able to start the run. “What buttons did you push? I’ve been pushing buttons too!” he says even more exasperated. Superhero PhD’s fingers fly too fast in that mode, “I’m not sure.” is all she can answer. She shrugs, “But wait until I leave the room to start the run.” She’s not completely convinced there may be an imbalance. For the record, there wasn’t.

There are other problems in the lab. She senses trouble immediately because Research Technician and Undergraduate Researcher bombard her with their pleas as soon as she opens the door. “Something is wrong with the cooler! The blotter won’t work! What do we do?” they cry.*** These things only happen when Tenured PI and Lab Manager are out of the lab for the day. Superhero PhD assesses the situation. Indeed, the deli cooler is a mess. It seems to have cycled much cooler than normal at some point overnight causing the coils to ice up. This caused the case to stop trying to keep the temperature low and the ice build-up was currently dripping all over the contents inside. “What a mess.” she thinks. However, experiments must be salvaged. “Remove the blotter from the cooler. We need to get it to work or borrow a replacement.” she decides. The blotter screen wasn’t giving a reading of the output voltage, but the electrodes appeared to be responsive to the controlling knob. Superhero PhD estimates the correct setting based on her perception of the rate of bubbling along the wires (and the transient flashing of a reading on the LED screen). “Take it to the cold room, and run it for two hours. Turn off the cooler. Take everything out of it. Turn it back on in the morning and monitor the temperature.” she orders. The blots are saved. The blotter screen miraculously starts working during the run. The cooler works normally after the reset. Another crisis averted.

Later that week, Tenured PI receives an e-mail from the editor regarding Superhero PhD’s latest manuscript. The verdict is favorable. Finally, acceptance! We can put that research behind us. He comes to share the news with Superhero PhD. He says only one word, “Victory!” She is having her lunch at the moment, so she only gives him a puzzled look. “What are you talking about?” she asks. He responds, “When the Greek messenger Pheidippides ran from Marathon to Athens to announce the victory over the Persians, he exclaims ‘Victory!’ Well, ‘Nike!’, in the Greek language. Your paper was accepted, so I said ‘Victory.’” Superhero PhD smiles to herself at the triumph, then says, “But in the legend, Pheidippides falls over dead after that exclamation.” Tenured PI answers, “Yes, that’s how I feel!” Superhero PhD agrees. So she announces, “Me too. I’ll be taking that Biochemistry Instructor teaching position in the Department.” At that moment, Tenured PI fell over dead.

NOT REALLY, but his heart probably stopped for a few beats to mourn the death of Superhero PhD’s research career.****

What will happen to the research projects that were on Superhero PhD’s to-do list? Will Tenured PI find a replacement SuperPostDoc to work on the recently renewed federal grant? Does Superhero PhD have instructional super powers? Stay tuned next time for the answers to these questions in the continued adventures of Superhero PhD: The Instructor Chronicles.



*It’s the same as the login ID BTW.

**Yes, our lab has a deluxe model hair-dryer with a very powerful temperature and speed output. If your lab doesn’t have one for this purpose, you should get one, but good luck justifying the purchase with accounting.

***If you read the footnotes of my last post carefully enough, you know how critical the blotter instrument is for our research.

****He really should have written his reference letter more carefully.

Two Tales of a Manuscript

It has been a shamefully long time since I’ve done a post for the Journal Club category. So today’s will be a deluxe edition of Dickens-proportions. Normally, you only get the science tale as presented in any journal article, neatly fit to the scientific method. However, for every scientific publication, there is another tale, a more elaborate backstory with twists, turns and subplots. While these secondary tales may be more dramatic, the traditional publication process relegates them to the shelf locked inside lab notebooks. Well today you will be getting both tales, because I’ll be breaking down my latest accepted manuscript. Read the science version (Tale 1), the behind-the-science version (Tale 2) or both.

I’ll be enlisting the help of Charles Dickens because many of the quotes from A Tale of Two Cities are as true for the practice of science as they are for complicated human struggles with relationships, sacrifice and revolution.

Tale 1

“It was the best of times, it was the worst of times.” Charles Dickens

All science projects seem this way. They can begin so full of promise, then change direction down a pathway you were not expecting and perhaps did not want to follow. You may think you know what you are doing, but some results case doubts. You have come to a conclusion, but then do one experiment too many and it shatters.

The PsbP-Domain Protein 1 (PPD1) Functions in the Assembly of Lumenal Domains in Photosystem I

Hypothesis: The lumenal protein PPD1 plays a critical role in photosynthesis, specifically in the accumulation of Photosystem I (PSI)

Experiments: In the model plant Arabidopsis, RNAi mutants of the PPD1 gene were characterized with respect to photosynthetic activity. The RNAi technique allows researchers to target a specific gene and knockdown its expression quickly and easily. These mutants can show a range of phenotypes that are useful in teasing apart the functions of genes whose complete elimination causes the death of the organism. The PSI activity in the PPD1 RNAi plants was extensively characterized as was the accumulation of many thylakoid membrane proteins (including PSI subunits). Native gel electrophoresis was also used to characterize the state of thylakoid membrane protein complexes in wild-type and PPD1 RNAi mutant plants.

PSI activity in PPD1 RNAi plants and representative plants from each group I-IV

PSI activity in PPD1 RNAi plants and representative plants from each group I-IV

Results: The PPD1 RNAi mutants with the lowest PPD1 expression were extremely small and pale green plants. Analysis of chlorophyll fluorescence showed that the mutants had much higher levels of fluorescence, indicative of an over-reduced plastoquinone pool and problems on the PSI-side of the photosynthetic electron transfer chain. Specific measurements of PSI activity showed that the PPD1 RNAi mutants had reduced amounts of active PSI reaction centers. However, energy could be transferred to these reaction centers by an alternative antenna system (LHCII). Moreover, the function of the PSI centers which did accumulate was not normal. Further analysis of protein accumulation in the thylakoids of the PPD1 RNAi mutants revealed there were specific problems in the accumulation of proteins on the lumenal side of PSI. In wild-type plants, the PPD1 protein was found to be associated with a thylakoid protein complex of ~300 kDa, which is smaller than any PSI complex.

2D gels showing thylakoid protein complexes in WT and PPD1 RNAi mutant. 1,2,4 complexes are forms of PSI; 3 is ATP synthase

2D gels showing thylakoid protein complexes in WT and PPD1 RNAi mutant. 1,2,4 complexes are forms of PSI; 3 is ATP synthase

Conclusions: The PPD1 functions in the proper assembly of PSI components on the lumenal side of the complex. In this area, PSI contains an extrinsically associated protein, PsaN, as well as extensive loop regions of the membrane proteins PsaA, PsaB and PsaF. All of these components create the binding environment for the soluble electron carrier, plastocyanin, which delivers electrons from upstream in the transfer chain. Reduced amounts of PPD1 affect the accumulation and assembly of these components. The mutant plants try to compensate for this loss of functional PSI by shifting some of the LHCII antenna such that it can funnel energy into PSI (the default for LHCII is to drive energy into PSII). The PPD1 protein is not considered a subunit because it was not found to be associated with fully assembled PSI complexes, but a smaller protein complex.

Think Ahead: The assembly of PSI is not a well-characterized biological process because, unlike PSII, PSI is an extremely stable enzyme. Thus, once it is assembled, the complexes can function properly for very long periods of time. Because it is a rare process, it is difficult to study. The original characterizations of PSI subunit mutants were performed many years ago, and it may be interesting to give them a fresh look with respect to PPD1 and some of the antenna effects we described. Identification of the other proteins in the complex with PPD1 (other PSI subunits perhaps) may help to define a PSI assembly intermediate. The secondary effect that the PPD1 mutation had on the antenna system will also be interesting to follow-up on because we don’t really know all of the details governing how plants allocate light energy between the photosystems. It is a sophisticated system with multiple layers of control.


Tale 2

“Nothing that we do, is done in vain. I believe, with all my soul, that we shall see triumph.” Charles Dickens

While scientific endeavors may have their dark moments, scientists tend to think that ultimately their research will see triumph. In the world of academia, this means publication in a peer-reviewed journal. Thus, all of the experiments that were done leading up to that publication but not included in it are not done in vain. They helped to work out the procedures necessary for acquiring the data that did appear in the figures. They were experiments that yielded negative data which eliminated hypotheses. Alas, those are never published.* It may be useful for scientists to know what wasn’t, but publishers only want to tell the stories of what was. (Hey, that almost sounds like Dickens too.)

The PPD1 story started with a blanket search for functions of the PPD family of proteins in the thylakoid lumen. They must be doing something to help plants photosynthesize, right? I was hopeful that maybe one of them had something to do with my favorite enzyme PSII. The way I chose to attack this problem was to characterize mutants of each of these proteins in Arabidopsis.

The easiest way to acquire Arabidopsis mutants is to order T-DNA lines (insertion mutants) for your gene of interest from the ABRC stock center. They send you seeds and you check to see if the mutants are useful or show a phenotype. There were two available lines (independent insertions) for the PPD1 gene and both of them were less than helpful to me. One line that another postdoc had been working with that had been passed on to me, which may have shown a phenotype, turned out to be heterozygous (not a complete mutant; still contains one wild-type PPD1 gene and should be normal). I couldn’t replicate any subtle phenotype, but neither could I find any homozygous (complete mutant) plants. Ever. I spent a lot of time verifying that that particular T-DNA mutant was embryonic lethal for homozygotes. Strange, but possibly extremely interesting. However, before taking this as a fact, it had to be confirmed that the homozygous lethal phenotype was because of the mutation in the PPD1 gene and not some other random mutation elsewhere in the genome. This can be sorted out by backcrossing heterozygote mutants to wild-type plants a few times and trying to recover the mutants. Ultimately, my experiments showed that the link between the PPD1 mutation and the embryonic lethal phenotype was not so absolute. At the same time, I was growing the other T-DNA mutant and it was proving to be equally unstable. Some plants would show a variegated phenotype, streaked leaves with patches of green and pale yellow. Other plants looked normal. I could never consistently link the phenotype to the ppd1 mutant genotype. With all of these inconsistencies in results, I decided that the T-DNA mutants were not useful in telling me anything about PPD1 function.

The alternative approach to Arabidopsis mutants is to use the RNAi technique to selectively suppress the expression of your favorite gene. While I was wrestling with the PPD1 T-DNA lines, I began the process of generating my own PPD1 RNAi lines. These plants turned out to be the most useful for figuring out what PPD1 does. When screening through these mutant individuals, there was a range in what the individual plants looked like- some looked almost normal while others were very small and pale. This is the good thing about RNAi because this range among individuals gives so a sort of picture of what is happening when the expression of the gene of interest is turned up or down over a gradient, like tuning up a brightness or volume knob on an old TV.

There was very little material to work with for the most severely affected PPD1 RNAi plants, but the biophysical measurements I could do on the tiny leaves indicated there was no problem with PSII. The defect was further downstream, probably in PSI. When using an instrument to specifically measure PSI function it was clear that was where the problem was. I would have to learn more about the PSI complex to say enough to turn my results into a publication, but at least I knew where this was going.


“There is prodigious strength in sorrow and despair.” Charles Dickens

The same week as my PSI results, I received an after-hours e-mail from my PI with the link to the following journal article: PsbP-domain protein1, a nuclear-encoded thylakoid lumenal protein, is essential for photosystem I assembly in Arabidopsis, Liu et al 2012 Plant Cell. When I quickly skimmed the abstract, my heart sank. My response was $%&^!, $%^@!, #$%@!, *&$%! I think I even drowned my sorrow in a pint of Hagen-Daz. There was only the slightest glimmer of satisfaction from the validation that researchers on the other side of the globe had come to the same conclusion as me.

Validation is not the name of the game. You see there is no prize for second place in scientific publishing. When you are the first group to publish a new idea, you have more control over the limits of the tale are. When you are second place, you cannot merely confirm what has been done (PPD1 has something to do with PSI). You must take it further, press on to unravel more details. Pressing on into the details of PSI territory was not really what I wanted to do.

However, after carefully reading what Liu et al had done, I reassessed my data and found a way to move forward. They had managed to characterize a clean T-DNA line, and the homozygous mutant plants they worked with were completely devoid of PSI. The small pale plants had to be grown on sucrose-containing medium since they could not support themselves photosynthetically. In my work, the RNAi lines allowed me to characterize plants that were very sick, but could still grow on soil. They accumulated some PSI, which could be analyzed more closely. Of course, that meant that I had to do a lot of experiments on precious little material. These experiments meant using a lot of brute strength just to get enough material for the experiments (spectroscopy measurements and blots, oh the blots!), investing time in fine-tuning protocols and money in antibodies for our second-favorite thylakoid complex.

“Vengeance and retribution require a long time; it is the rule.” Charles Dickens

Pushing forward with the experiments was difficult and took quite a bit of time. The sickest of the PPD1 RNAi lines were very small and would not set seed. Getting enough material meant screening for primary transformants every time. Learning the literature for a different enzyme complex was challenging. The papers describing the original characterizations of PSI subunit mutants were at least a decade old and often lacked data I would have liked to have seen. Not really flaws with that work; it’s just that what was not necessary for that work would have been extremely helpful to me.

Eventually, all of my data was written up in manuscript form and submitted away for peer review. I dabbled in other projects waiting the weeks until reviews came back. It took longer than usual, which meant only one thing- it was sent to a third reviewer. Yes, the two reviewers that initially evaluated my work had such differing views as to what my manuscript’s fate should be, a third reviewer was enlisted to help the editor in making the appropriate decision. Please revise with additional experimentation and there were specific concerns about how we went about doing some of our experiments.

Yes, a long time is the rule. I spent the next months painstakingly addressing the reviewer’s points with new experiments. One issue was how we estimated the amount of PPD1 protein in the mutants. With our antibody and a number of variations of our gel system, the PPD1 protein ran at the same molecular weight as the LHC proteins- the most abundant membrane protein on earth. These proteins obscured the signal for PPD1 such that we could never reliably estimate its amounts on denaturing gels. It either could not be seen or samples would require too much handling and treatment to consistently give a signal. However, PPD1 could be perfectly detected on native gels because the LHCs were nowhere near it in that system. Finally, I had point-by-point addressed all of the issues, revised the manuscript and created new figures.

We were ready to try again, but the tone of one of the reviewer’s comments gave us pause about resubmitting. Sure we had responses, but the original comments seemed like they would never invite satisfaction. There were some things about our results that would just not change. Experiments were done properly and yes, the results were still slightly unexpected. We would not be making up data because it would be easier for reviewers to accept. That is a cardinal sin in science, and a separate rule that should never be broken. For the revised manuscript, we took the chance on submitting to a separate journal with different reviewers for the chance of a favorable decision. This wager did not pay off because the two new reviewers had a completely separate set of comments to be addressed, many of which seemed impossible to satisfy with our sample limitations. We declined the invitation to revise and resubmit and ultimately resubmitted to the original journal. It went back to the original reviewers who seemed mostly satisfied with the improvements. Of course, there were some new comments by the reviewers that we could just not accommodate; not because the concerns were invalid, but because the questions went well beyond the scope of our work. There will always be more experiments to be done, but we firmly and politely stated we would not be addressing the new questions our latest experimental results sparked. We could only speculate as to future possibilities in the discussion section.

“A multitude of people and yet solitude.” Charles Dickens

In the case of this particular project, it was a multitude of data, yet not figures. I have numerous notebooks filled with raw data from experiments related to PPD1. In science, you go through a lot of preliminary work to get the answer, but when you show your work it must be much neater (certain colors, intensities, certain samples in certain orders). It’s like taking a math test and having a separate scratch paper (the lab notebook), but your answer is only a single circled number or neat graph (the manuscript figures). In this particular case, it was a multitude of blots. I cannot tell you how many blots I developed for this project. I practically lived in the dark room for months, eagerly waiting for films to emerge from the developer, praying the signals would be beautiful enough for figures.**

“I wish you to know that you have been the last dream of my soul.” Charles Dickens

Appeasing the reviewers in this final round felt a lot like the emotions in this quote. I had started forming the publication framework haphazardly because it wasn’t on a topic that I found exciting. Admittedly, I was only trying to do just enough to get acceptance. Even though my sentiment for some of my reviewers was more akin to a different saga, their requests did make the story better and forced me to expand my general knowledge on PSI and technical expertise in new protocols. All research can continue ever and on, but lines must be drawn somewhere because of the universal limits of effort, time and finances. I felt that the story was finally new and good with enough potential tangents to drive future research by possibly myself and others in the field. I had finally come to the point that I didn’t just want it done for the sake of adding another publication to the tally, but I wanted it published because the results deserved to be part of our body of photosynthesis knowledge.***

“It is a far, far better thing that I do, than I have ever done; it is a far, far better rest that I go to than I have ever known.” Charles Dickens

My PPD1 manuscript was eventually accepted at the Journal of Biological Chemistry this summer after a very long road of experimental struggles and research-related drama. Of all of my publications, this was definitely the most difficult to get to the point of publication. It is probably at the bottom of the list of my works if I had to rank them my favoritism based on any scale. This post was actually quite difficult to write; I so long to leave it in the past. However, I can now recognize that it is one of the better things I have done just to not give up on it. And after all the co-authors thought that everyone who would ever be interested in the PPD1 protein would have written the paper or reviewed it, we get an e-mail from another research group requesting our PPD1 antibody because their work may have a link to PPD1. “We read your paper with great interest,” they said in an e-mail sent a mere two days after our accepted manuscript appeared on-line. “They did!” I laughed. I sent them a sample, some behind-the-science instructions and well wishes. Apparently, my perseverance wasn’t just a better thing in terms of racking up publication numbers on my CV, but also for some other researchers embroiled in their own scientific epic. The best of times and the worst of times indeed.

As for me, it is a far, far, better rest that I go to than I have ever known as well. It’s not just a project change and definitely not the guillotine. Announcement coming soon to the blog.



*However, Elsevier is launching a new journal where you can publish those results. Introducing the new journal New Negatives in Plant Science.

**There is a popular song this summer by Lil Jon and LMFAO “Shots”. There are not many lyrics. The rapper mostly just says shots over and over the backbeat track. To stave off insanity, or perhaps the opposite, I would sing my own version of the song “Blots.” All my biochemists, where y’at? Let’s go. When I walk in the lab, gloves on me, with the antibodies, I love chemiluminescence, I came to develop, lights off, it’s on! Blots, blots, blots, blots, blots, blots, blots, blots, blots, PPD1, blots, blots, blots, blots, blots, PsaB, blots, blots, blots, blots, blots, blots, LDS-PAGE, blots, blots, blots, blots, blots, Blue native, blots, blots, blots, blots, blots… If I don’t do these blots, I can’t resubmit! You get the idea. For visual effect, you can also picture me making it rain with x-ray films. Hey, what do you know another parody for the blog.

***Although if it would not have been accepted, I had threatened to just dump all of my results on this blog anyway and be done with it. Not sure how it ranks in terms of impact factor though.

References and Links:

Dangerous Photosynthesizers

Because photosynthetic organisms are the energetic foundation of our biosphere, we always tend to think of them as allies, organisms with a positive connotation. Their trademark color green is universally linked with goodness, growth, and life. However, there are some bad apples in the bunch that just seem to have it out for us heterotrophs. Well, maybe not apples (though I’m sure there are some poisonous apples out there somewhere*), but nature is filled with examples of poisonous plants. Many toxins and pharmaceuticals have botanical origins.

The focus of today’s blog post sinks even lower- algae, pond scum, cyanobacteria. Most of these aquatic photosynthesizers quietly convert sunlight to biochemical energy without any ill effects to anyone. I’ve written previously, that under the right conditions (warm and nutrient-rich waters) these otherwise inconspicuous organisms bloom in great numbers and overwhelm their environments. Like all life on earth, algae are programmed to capitalize on favorable conditions for reproduction. The ultimate crashes of these blooms can result in aquatic dead zones, areas with dissolved oxygen levels too low to support life.**

Algal bloom in Lake Erie 2011 from the NASA Earth Observatory Credit: Jesse Allen and Robert Simmon via Wikimedia

However, in some cases, the effects of these algal ‘blooms’ go beyond sheer numbers. Some algae produce toxins which cause serious health problems for those of us heterotrophs sharing their environment. The individual constituents of these ‘harmful algal blooms’ (HABs) measure in at ~1-2 µm, but they can wreak all kinds of havoc on the scale of large cities. HABs can stop your summer fun by forcing beaches to close or eliminating certain shellfish from your diet, but this summer the problems went beyond recreation to something much more fundamental- potable water.

Clean, safe drinking water is a fundamental service of human civilization. In our modern society, just turn on the tap, cook, clean, bathe, drink. It has been such a staple of American cities that it is taken for granted. That is, until it’s no longer available. That was the exact situation in Toledo earlier this month. A large American city, in the year 2014, was without safe drinking water for a whole weekend. Approximately, 500,000 citizens were affected. All because of toxic algae.

Microcystin-LR chemical structure Credit: cacycle via Wikimedia

In this case, the culprit was a bloom of cyanobacteria which produce the toxin microcystin. This molecule is harmful to the health of humans, pets and wildlife by acting as a liver toxin. It also has neurotoxic effects. The toxicity of microcystins has been extensively characterized and long-known to be associated with certain cyanobacterial species. Because of the potential adverse effects of microcystin-producing cyanobacteria on modern water supplies, treatment facilities routinely check the levels of this toxin. Only one part per billion of this molecule is considered acceptable. When a microcystin-producing algal bloom occurs near the intake of a municipal water supply (as it did for Toledo this month) the facilities can quickly be overwhelmed causing the water supply to exceed acceptable microcystin levels. The situation is compounded by the fact that microcystins are resistant to boiling. While boiling water may destroy other toxins or contaminating bacteria, it only concentrates microcystins. In order to bring the toxin levels down, the problem must be addressed at the water treatment facility (using methods like activated carbon, ozone treatment and membrane filtration). By adjusting the normal procedures to account for increased microcystins, the water supply can be treated to once again safe levels. All of this is accompanied by exhaustive analysis of microcystin levels and vigilant monitoring after the incident.

Scientists and government agencies are always working to monitor our water systems for HABs. Check out some of the links below for descriptions of continuous efforts to monitor our environment for HABs. How do we get to the point of 500,000 people without water for a weekend with everyone watching out for it? I mean, we’re watching it from space! Even with all of these sophisticated tools and models, nature can be surprisingly swift. Check out these images and reports from the NOAA Great Lakes Environmental Research Lab on Aug 1, 2014 and Aug 4, 2014 showing false-colored images tracking the algal growth.

Simultaneously in the news cycle with the Toledo water crisis, two Ebola patients were being treated in premier isolation facilities on American soil. The nation’s attention was rapt with the details of their treatment and speculation was rampant as to the possibility of an outbreak in America (an infinitesimally small probability not worth talking seriously about among scientists and epidemiological experts, but a great ratings-driver). Worldwide, the Ebola death-toll numbers in the thousands, not just from this year, but ever. Based on pure body count, there are many deadlier infectious diseases, which we as the public dismiss more easily. Beyond those numbers, the lack of clean water for drinking, food preparation and sanitation results in the deaths of ~3 million people every year across the globe. A safe and reliable water supply, as a basic right, continues to elude human civilization.

Water, water everywhere, but not a drop to drink… Credit: de:Benutzer:Alex Anlicker

HABs are only a part of the world’s water problem. However, the disruption of Toledo’s water supply should have been an event that caught our attention and held it for a while longer. It may be easy to turn on the tap, but getting the clean water to that point takes a significant amount of effort with infrastructure maintenance, monitoring and treatment. All of these things are largely invisible to us in modern society. Unfortunately, all of these things are affected by other societal choices like economics, aesthetics, environmental regulations, and the practices of our agricultural systems and other industries. As a society, we should start having the longer, difficult conversations necessary to attack this complicated problem rather than the transient chats that occur when we are in crisis mode. Find out about your community’s water situation and the issues related to your supply. Talk to your community leaders today to ensure that safe water is part of your future.




*Hey, it’s hard to transition away from the summer’s Disney theme completely in a single post. However, I’m not just talking about the evil queen’s poisoned apple from the fairy tale. Apples have a huge amount of genetic diversity and I’m sure there are some varieties out there that are poisonous or so foul-tasting that you would think they are. After all, apples concentrate toxic substances in their seeds.

**In case you’re wondering, this year’s Gulf of Mexico hypoxic zone measured ~5500 square miles. That’s not breaking any records for size, but still about as large as the state of Connecticut. Read more about it here.


References and Links:

PSII is a Fixer-Upper


The final song from our Frozen parody reminds us that just because something is damaged or broken, doesn’t mean it can’t become whole again with a few repairs. This sentiment makes it the perfect theme song for my favorite enzyme, Photosystem II (PSII). Here’s the Disney version…



Is it the slower QB reduction?

Or lack of water oxidation?

No use trying state transitions,

Although we know it transfers well, PSII ends up photodamaged

Such photoinhibition is such an imposition

So it’s a bit of a fixer upper

So it falls behind

Its peculiar mechanism

Is a one-of-a-kind

So it’s a bit of a fixer upper, but we’re certain of this one

You can fix this fixer upper with a newly made D1

Is it the oxygen singlets?

In chlorophyll protein ringlets?

Is it the way the water splits?

Electrons zipping towards quinones

Causing D1’s aches and groans?

Now permanently on the fritz.

PSII’s just a fixer upper

It needs a protein exchange

Its phosphorylation is confirmation

That something is strange

So it’s a bit of a fixer upper

Plant cells know what to do

The way to fix this fixer upper

Is to make D1 anew

Damaged PSII is a bit of a fixer upper

That’s a minor thing

Just disassemble then reassemble

Voila just like recycling!

So PSII’s a fixer upper

It’s function is nixed

Get the damaged protein out of the way

And the whole thing will be fixed

We aren’t saying to remake the whole thing

‘Cause that’s just too much work

We’re only saying that light’s a force that drives PSII berserk

Electrons just excite the wrong things if they go off their normal path

Changing out the D1 protein erases all their wrath

New D1 clears the path!

All PSII’s are fixer uppers

They’re made to fizzle out

DegP, phosphorylation

FtsH, degradation

Get them on the repair route

All PSII’s are fixer uppers

When the electrons start to move

The only fixer upper fixer

That can fix a fixer upper is

To get the damage removed



Photosystem II performs the unique reaction of splitting water to form oxygen and extract electrons used to fuel photosynthesis. Not all of this energy goes in the direction that it should. When energy gets backed up in the system or electrons venture off of the designated path, irreversible damage to the proteins can occur. This damage means that PSII doesn’t work anymore. Because this photodamage is an unavoidable hazard, photosynthetic organisms have an efficient way of dealing with this problem.

For one thing, the D1 protein at the heart of the PSII complex bears the brunt of the irreversible damage. This makes sense because the D1 protein also coordinates many of the cofactors that comprise the electron transfer pathway through the system. On the one hand, damage to this one protein means function gets knocked out as well; on the other hand, it means that the damage is concentrated on just one protein. So, to fix it and restore function to the complex means photosynthesizers mainly focus on replacing one protein, not twenty. That’s what has to happen. The damaged protein must be replaced by a newly synthesized copy.

It sounds simple enough, but anyone who’s done any fixer-upper work knows there’s more to it than that. Repairing the damage starts with recognition; there must be systems in place to differentiate functional PSII from damaged PSII. Phosphorylation of certain residues on PSII subunits labels those complexes as targets for repair. These labels are interpreted by specific proteases, which then remove and chop up the damaged D1 protein. Next, a newly made D1 protein is inserted into the complex to restore function.

The proteases involved in removal of the damaged D1 protein are DegP and FtsH. Researchers still debate over which one is more important, but it is likely to be a combined effort by both. Also, because the D1 protein is located within the middle of the PSII complex, many other subunits and cofactors must partially or transiently disassemble as a result of D1 protein removal. Exactly how this works and what additional proteins are involved in this process are active areas of research in the photosynthesis community.

Photosynthesizers don’t give up on their PSII complexes just because they get a few dings. The constant recycling of PSII complexes through this repair process ensures that the light reactions of photosynthesis will continue to churn away, even in bright light (more energy, higher rates of damage). It may seem like a lot of trouble to run this elaborate repair shop, but it’s still easier than starting from scratch each time PSII is damaged.




References and Links:

PSII damage and repair

Let it Go: Abscission


Up next in the Frozen series: Plants let it go! Here’s the Disney version in case you’ve been living under a rock for the past several months.


The cell walls grow thin in the zone tonight

No chlorophyll to be seen

The leaf is about to fall

Thanks to all the ethylene

In the winter weather those cells would have died

Wouldn’t have mattered how many genes I transcribed!

Turn them red, set them free,

I can’t move I’m just a tree

Breakdown, recycle, not just a show

But, here’s your show!

Let it go, let it go

Not holding on any more

Let it go, let it go

Away they fall, away they soar!

I don’t care

What they’re going to say

Let my limbs lay bare,

The cold never bothered me anyway!

It’s funny how abscission

Makes mincemeat of cell walls

And the genes that once controlled me

Can’t get to my leaves at all!

It’s time to see what I can do

Other organs can shed too

No petals, no flowers for me, I set them free!

Let them go, let them go

Already pollinated, that’s why!

Let them go, let them go

Useless now to me, so let them fly!

Here I stand

And here I’ll stay

I can’t move on!

My fruit flutters through the air onto the ground

My seeds spiraling on the wind all around

The next generation moving forward at last

But I’m never leaving

My roots hold me fast.

Let it go, let it go

It’s really better for me and my spawn

Let it go, let it go

That springtime form is gone!

Here I stand

In the light of day

I can’t move on,

I’m better at biochemistry anyway!



It’s hard to tell if plants have any inner emotional turmoil like the one Elsa had to fuel this song. However, it is safe to say that no matter how stoic they appear, there’s a lot going on inside them. Since they can’t move, plants have to roll with environmental changes using biochemistry and a flexible developmental program. Plants may not run away to the North Mountain, but they know how to ‘let it go’ when necessary. They do this in a process called abscission, a regulated way of shedding parts of themselves for the good of the plant or the next generation.

One of the most spectacular shows of abscission is the autumnal color change and shedding of leaves in deciduous trees. We call it ‘Fall’ because of the dramatic leaf drop before winter. Even though plants can take some measures to protect themselves from the cold, keeping some tissues over the winter requires too much energy of the plant. The trees do what they can to recycle the valuable contents of the leaf cells, which results in their colorful display. Then they coordinate a way to drop their leaves that doesn’t leave open wounds on the plant.

It’s not just letting loose of leaves before winter, abscission is an important plant process for other organs as well. Plants don’t need to waste their energy on maintaining flower petal tissue after they’ve already been pollinated. If seeds are on their way to forming, no need to keep the lush colorful tissues that can’t photosynthesize enough to support themselves. Further down the line, once fruit has formed to encapsulate the seeds, it must also be let go from the mother plant in order for the next generation to find a hospitable place to germinate. These examples represent normal developmental progessions for plant tissues, but sometimes plants have to improvise. When plant tissues become infected with bacteria, viruses, or fungi, they can kick the abscission process into high gear in the hopes that shedding the infected or damaged parts will prevent the death of the whole plant.

The abscission process creates an area of tissue designated the abscission zone, in which the cells take on distinctive characteristics. These cells are smaller and have an extensive network of endoplasmic reticulum and Golgi membranes with connections to the plasma membrane. The specialized cells of the abscission zone acquire the ability to respond to certain triggers to induce cell separation. These triggers can be a combination of environmental (ex. defense proteins induced upon infection) and internal (ex. the plant hormone ethylene) stimuli. When these triggers are perceived, the cells of the abscission zone up-regulate enzymes that breakdown the cell walls making cell separation easier. Ultimately, the abscission zone cells that remain on the main body of the plant differentiate into a protective layer so there is no open wound on the plant. The abscission process requires the coordinated activity of a large number of genes that must straddle the intersections of developmental pathways and environmental sensory integration.

Plant scientists are still working out the details, but the confluence of so many processes during abscission makes it a difficult problem to attack. However, understanding the abscission process remains a high-priority pursuit in the world of plant science. Agriculturally and horticulturally important plants have been heavily selected with respect to their abscission properties over many generations. In some cases, preventing early abscission may increase yields of certain crops (think- use those leaves longer, don’t drop them). Slowing down or halting the abscission process is also important for aesthetic reasons (keep those flowers and leaves around longer). Facilitating the abscission process may be equally useful and aesthetically pleasing. If plants could be engineered to abscise sooner or more completely, harvests would be easier because fruit would require less force to remove from the plant.

Consider the case of the gardenia- a striking example of a plant that needs to learn to let it go. It is perhaps the most perfect garden shrub- beautiful white blooms that coordinate with any background palette and a lovely scent. However, the blooms past their prime are truly one of the most pitiful sights in the botanical world- shriveled and brown and hanging on for dear life many days longer than anyone would care to look upon them. I’m not sure what inner turmoil is raging within this species, but biochemically, the processes of programmed cell death within the petals and their abscission from the plant are not coordinated in a way that is pleasing to the eye. If there were a gardenia variety that timed these processes more closely with one another, then gardeners would not be forced to look at the crumpled brown blooms.




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