There are few things that tickle biologists’ fancies as much as cheekily naming plasmids, although devising unnecessary acronyms is a close runner-up. Here is a rundown of the dankest vectors in existence.
All placements are scientific fact; dispute at the risk of being a science denier.
Plasmids are rated on these factors to determine their rank:
Dankness
Applicability of the name to the vector function
S Tier BABY
pDawn / pDusk – Coming in hot from the Moeglich lab is a set of blue light (470 nm) inducible/repressible protein expression vectors. The names are fitting, and they come in a set. Moreover, these vector names take into account the aesthetic qualities of a clean SDS-PAGE full of expressed protein. S+ Tier name.
“Shit, I left my transformed cells outside overnight and now they’re producing Botulism toxin again…”
pCold II – Simple, elegant, and self-descriptive, the pCold family of plasmids are cold-shock inducible expression vectors that also encode chaperones for your folding pleasure. S tier + a haiku:
In cold I delight,
yet should no expression come,
you should have sequenced.
pSHAG-MAGIC2 – You’ll find this vector hiding under the diminutive alias of pSM2C, but alas, there is no hiding the glory that is SHAG MAGIC. This vector fucks. S tier.
A Tier
pLOVE – LOVE is apparently an acronym for “Lentiviral OVErexpression”. The name isn’t clever and doesn’t offer any insight into the plasmids function but, hey man, I had to put it next on the list because what is SHAG/MAGIC without LOVE? Carried to A tier.
pSi – This under-rated vector name is truly a sleeper act of genius. Brace yourself, this vector is exactly the same as pSiM24, but the author just removed the M24 promoter making it pSi. Enlightened author from Maiti lab. A Tier.
pET28-Snoopligase / pET28-SnoopTagJr / pET28-DogTag– This comprehensive dual-epitope tagging and linking system is a certified hood classic named for the one and only patron saint of 2 AM cell culture- Snoop Dogg. A tier is for ayy, smoke weed every day.
B Tier
pMULE – Now this is what I’m talking about. A mule is a work animal derived from crossing a donkey and horse; this vector is a work plasmid derived from a godless cross between multiple gateway vectors. Sinful, clever, and memorable. High B tier.
pBigT – What does the T stand for? Why is it big, specifically? Some say transgenic, but personally I think it stands for “tears”, as this is a vector for making Cre-lox mice T_T. Either way, any “Big” vector is a B tier in my book.
pBABE– I think this vector is probably familiar to a lot of you as it’s quite common. The name serves as a stark reminder that your scientific labors will always be in service of a fickle and cruel mistress.
C Tier AKA Resentful PhD Student Tier
pcDNA3.2 – The vector literally does exactly what it says. It’s a gateway vector for cloning in your cDNA, but it has both a T7 and CMV promoter for expression in mammalian cells or in vitro transcription. Lacks creativity. C tier for cDNA.
D Tier AKA Undergraduate Tier
pmStrawberry – It expresses a mutagenized form of RFP and makes me hungry, which I resent. D tier.
pNgamma1.12 – Couldn’t google to find out the alt code for gamma- D tier.
γγγγγγγγγγγγγγγγγγγγγγγγγγγγγγγγγ
F Tier AKA Forgot to upload vector map Tier
pMpGWB220, 221, 222, 223, … 406 – What kind of degenerate masochist created this set of over 100 expression vectors? I had to scroll through 8 pages of Addgene to escape the pMpGWB wasteland. Moreover, the sequences aren’t even available. Who commits such atrocities? I think it goes without saying that it was a plant biologist. If the person who made these was in charge of writing hieroglyphics in King Tut’s tomb he would have just drawn a stick figure with a different number of tallies next to it for every single word- F tier, fuck you.
In Conclusion
Naming our creations is one of the few creative outlets available to the humble molecular biologist. Where else can you inject your identity and have it live on forever in the hands of a bumbling PhD student who just contaminated their stocks with GFP?
As a rule of thumb, however much time it takes to make the vector, twice as much time should be spent coming up with a dank name.
The industrious individual is a golden tool: rare, highly valued, and gleaming with potential.
The average graduate student is a 200 pound gorilla with a micropipette duct-taped to its hand: expedient, irritable, and lacking fine motor control.
Confident, bold, and daring. Like it or not, this is the ideal graduate student.
That said, we should not forget that even the most brutish lab wildlife gleams with the potential of an experiment well done and a question elegantly answered.
So where is the line between lab gorilla and Gautama buddha: prince of pipettes? To put it simply, the difference between the two lies in their adeptness in theart of corner cutting.
Obviously, every individual has the capability to do careful, thoughtful work and expedient, shit-tier work. As humans we often like to assign moral values such as good and bad to the quicksilver and quaint respectively, but let us for a moment consider that the entire spectrum of output quality has its corresponding spectrum of appropriate niches.
What wily and clever lab rats know, consciously or unconsciously, is that to maximize efficiency is to employ the correct quality depending on the demands of the situation.
Why did I spend an hour on Friday night making this?
Serial Dilutions of Skill
Now when we talk about quality, and in particular the quality of one’s work, we must first define standards to compare to. Taking that into account, I’ve gone ahead and split the quality spectrum into tiers and given examples below of the kind of work and worker you can expect at each.
“I’m not actually a shitty scientist I was just pretending haha” Tier
At rock bottom we find the “laser eyeball colorimetric readout” your mentor hastily taught you when he was too lazy to walk to the plate spectrophotometer. This type of work is all corners and cuts, no actual product.
“Yup that look’s like 2mg/mL to me.” – That one grad student who leaves at 3pm every day
Sadly, this is usually a grad student who knows better but has completely lost any passion for science and discipline.
Leaves samples in the thermocycler for weeks-on-end Tier
Increasing in quality a bit we find the average undergraduate’s typical output. This consists of assays where directions were technically followed but without any real forethought or care. Examples include the no longer supercoiled DNA preps that have been left on the bench for 3 weeks and an unfathomable number of unlabeled tubes that may or may not be important.
This tier also encompasses the work of those who lack motor, technical, and intellectual skill. That guy who always contaminates the LB after taking 20 mL from the 1 L bottle comes to mind but he’s far from the most dastardly criminal who resides in this cursed domain. Case in point: RNAse guy.
Ancient representations of RNAse guy. Note the elongated head that allows for more surface area to shed RNAse from. Disgusting.
RNAse guy is the extrovert that, no matter when or where you start your RNA prep, will come up and start talking and breathing in your personal space, spewing all his disgusting RNAses into your precious sample. I’m non-ironically mad writing this because it happened yesterday. Fuck RNAse guy.
Side note: leaving your DNA at room temp really will cause loss of supercoiling and downstream losses in efficiency when used for transfections or in vitro assays.
Avoids talking to the PI / Time Wizard Tier
Another increase in quality brings us to what may be considered passable work, sometimes.
This is the dude who cruises Instagram in between flow cytometry samples but won’t take the time to use the autosampler. Experiments and corner cutting at this level resemble a gambling addiction more than science and while failure is common, occasionally this high risk-high reward strategy pays off big.
“Gloves are the only PPE I need!”
This tier is unique in that it has a very recognizable aesthetic. Contrary to the norm, lab coats and aseptic technique are typically not employed at this level due to the student’s own perceived expertise and “secret breath holding technique” (Side note the breath holding totally does work though).
This student has been around for awhile, at least 1 lab generation and could be considered a journeyman corner cutter. When not catastrophic, the shortcuts this student takes do not have an immediately noticeable effect on the work produced. The student, however, will constantly complain that assays are, “inconsistent” and that, “science is a joke”.
To be fair, this student is right to complain that science will always be contaminated with unknown variables due to the unfathomable complexity of the universe, but still foolish because he discounts his own role in increasing the unknowns in every experiment through expedient action and insufficient planning.
Overall the most fun strategy. Highly recommended.
Lab Tech Tier
There’s not much to say about this tier. These people are actually paid to do their research and they generally complete all necessary work in an organized and orderly fashion. Sure they leave at 5pm but that’s their prize for not procrastinating until the last minute to pass their cell cultures.
Corners are not cut in this sacred domain and as a result quality is high, but this consistency comes at the cost of time and adventure.
Enlightened Buddha Tier
The enlightened student is the paragon of corner-cutting optimization: quick, fluid, and with a fairly high rate of success. This student empathizes so intensely with the mistakes of his colleagues that he learns more from their errors than they do.
This student never contaminates their sample with RNAse because they don’t ever breath, they simply allow oxygen to diffuse into their blood by coating their body in DMSO every morning. Mmm, garlicy.
The cultured cells of the Buddha student are cared for as one cares for a friend, or for themselves, because the student recognizes that all living beings have a nature tied to his own- not of the same birth or the same blood, but of the same living essence and possessing a share of the divine. This student holds funeral processions when they bleach their high passage number cultures.
This student is an artificer of the highest quality and to explain why some corners are cut and others not would be comparable to explaining mans’ place in the universe.
The Sound of Water…
Why is this article titled “The art of corner cutting” and not, “5 TIPS YOU DIDN’T KNOW FOR CORNER CUTTING FAST | UPDATED 2020 | IMPRESS YOUR ADVISOR | FALSIFY DATA ™ | ‘Free… your work… from the British Empire” -Ghandi“? Well, it’s because learning when to focus on speed and when to be meticulous is too complex an equation to compute from any set of written instructions.
Arts are arts because their performance defies the conceptual boxes of language. The explanation of how to dance can’t give me the skill of dancing any more than the statement, “The tea is hot”, can give me the feeling of hot tea on my tongue. Only dancing and drinking tea can do those things.
Luckily, humans possess the often underestimated tool of metaphor, whereby I can transfer a conceptual image (a myth) to you that conveys the performance of an art. Additionally, I can give you examples of good and bad corner cutting so that the complex patterns that underlie successful skimping can be transferred to you.
What follows is an attempt to condense the sound of water to a, “plop!”
Types of Corner Cutting
I categorize the types of corner cutting into 2 main categories: insufficient planning and improper execution. As we will see, while the implementation of an idea can (and almost always will) diverge from the plan, the lack of a plan entirely cascades into exponentially more trouble than is worth.
Improper Execution
Reducing & Omitting Procedures
The decision to omit a step should be made based on these criteria: predicted influence on outcome (perceived importance) and cost-benefit of skipping.
When designing an experiment your brain will unconsciously assign an “importance value” to each step. Calculating such a value empirically is not feasible so this is a contributing factor to the whole art concept.
As an example, skipping the addition of IL-4 to a B cell culture is a bad idea because survival cytokines are extremely important for survival (obviously) and the time cost of the procedure is about 30 seconds. The risk is a crashed culture and 2+ weeks lost.
On the other hand, if you go to your fridge to find you only have half the amount of IL-4 necessary for the culture volume and have to decide between bleaching half the cells or taking the risk on halving the cytokine concentration, a reasonable argument can be made for skimping.
If you go down this path, err on the side of reducing rather than eliminating steps and always document your modifications. Sometimes less turns out to be more!
Saving Timeor “Lust”
Now I get it, graduate students have the schedule density of world leaders without any support and sometimes time needs to be cut from somewhere. Let’s approach protocol condensation with harm reduction in mind and with the disclaimer to use your best judgement based on the downstream application. Here’s how to do it:
Do
Reduce incubation times by up to 50% when working with nucleic acid when a purification step follows
Use fewer higher volume washes over more lesser volume washes (ensure >2000-fold dilution factor)
Skip incubations when transforming bacteria with high quality plasmid
Solidify agarose gels in the refrigerator instead of at room temp
Dirty passage cancer cell lines (Spin down, resuspend, plate w/ out wash)
Estimate PCR annealing temps instead of doing a gradient (Annealing temp usually = half way between melting temps of both primers)
Dont
Deviate from protocol on any primary cell culture
Omit addition of any reaction components in in vitro assays
Skip ANY step or precaution when working with RNA
Fail to record protocol modifications
Freeze/Thaw protein stocks any more than necessary
Treat main stocks of reagents without care (you will regret it later, trust me)
Pausing a Protocol Overnight or “Sloth”
This lesser sin is one of the most tempting ways to cut corners. When a protocol is 12 hours long it’s reasonable to try and space it over 2 days to reduce fatigue.
What really matters here is what kind of biomolecule or assay you are working with. Remember that average stability is as follows: DNA >>> Cell Culture > Protein >>> RNA
DNA is basically bomb-proof but improper storage may result in conformational change (i.e. loss of supercoiling) or degradation if nucleases are present.
In cell culture assays there isn’t usually any good pause points and you will see the ramifications of your sloth in the results. The best place to call it is just prior to any kind of nucleic acid or protein extraction from a pellet, in which case you can just flash-freeze the cell pellet and keep it in the -80C overnight.
Purified protein is the chill guy from California who takes it easy under favorable circumstances but loses his shit when things get a bit warm. Clarified lysate and purified protein / columns can be stored at 4C overnight for most proteins with the addition of a protease inhibitor, but occasionally our polypeptidic friends will take the easy way out and degrade, or more likely, precipitate. Don’t expose to room temperature.
RNA does not want to exist in this world. Water hydrolyzes it at every temperature including -80C. Don’t skimp here or you’re in for a world of RNAse activity.
Add lots of murine RNAse inhibitor to your stocks if you wish to store them overnight at 4C but if you’re wise you will do an ethanol preciptation and store RNA as a pellet under ethanol at -80 C. Yes it’s a pain in the ass. No it will not phase into the void under these conditions. Ever.
Insufficient Planning
Winging Itor “The Free Bird”
The “Winging It” strategy is the 9th circle of Hell (Treachery) in Dante’s Inferno. You will find yourself frozen in place by gusts of chaos and uncertainty generated by what will seem to be a literal demon.
A few months back I decided to check my activated primary B cell cultures for the presence of exosomes with no prior knowledge about exosome isolation or characterization. How is it then that I ended up sitting at the flow cytometer at 2AM alone, afraid, and exosomeless? Well, after 6 hours of culture supernatant ultracentrifugation the effects of my lack of premeditation became apparent. It turns out my instrument can’t detect particles as small as exosomes without a suite of modifications and degassed, 0.2 um filtered buffers. Oops.
My undergrad and I call these types of experiments “fuck it expeditions”. Crass? Yes. Accurate? Absolutely.
Deciding to completely wing an experiment, going in with only a hypothesis and no plan, is analogous to trying to talk your way out a speeding ticket when the cop is deaf: you better be quick on your feet. The key in this type of experiment is to make like Lewis and Clark and map the territory effectively.
Recognize from the beginning that your chances of a reproducible success are nil and that the gold to be gathered here is in how what you learn will inform your future experiments. Focus on taking detailed notes and when problems arise, which they will, write them down so they don’t take you by surprise in the future.
Don’t spend more than a day on this type of experiment, and definitely do it on a day when your PI is not present.
In my case I learned that any exosome investigation will require a team of dedicated undergrads to do all those ultracentrifugation steps for me.
Insufficient Planning – The Partial Wing or “Mad Libs for Scientists”
Partially winging an experiment means plunging into the depths with the mere outline of a battle plan. They say the difference between madness and genius is the outcome of the ideas and this strategy is the same.
Success here is dependent on the length of incubation times associated with the experiment. This is because as you proceed through the steps you will need to be constantly readjusting the strategy to account for the uncertainty in the unplanned steps, and this takes time.
As progress is made and experience gained the correct course of action will become obvious, but if you have insufficient time to consider your options or gather reagents you will pay the price in delays. Remember this: note carefully all the alterations in protocol you make.
When I started working in a lab, I thought being clever was enough. “Each variable in an experiment is a Chess piece,” I told myself, “and the successful experiment is nothing more than moving the correct piece, in the correct way, ”
And so I tried to play science like one plays Chess. But what I didn’t realize is that in Chess your worst enemy is not the person sitting across the board from you, but actually your own blindness. Under the illusion of competition with another, your vision becomes tunneled on capturing his Queen, or putting him in check, and in so doing your mind becomes restricted. You blunder- you find yourself in checkmate.
In exactly the same way, the most dangerous enemy of scientific investigation is not any particular experimental detail but the 200 pound gorilla in the lab coat.
If you strive to cleverly checkmate your experiment you will eventually encounter big problems. The difficulty will appear to be external- in the fine details of what you endeavor to do, in the arrangement of the “Chess pieces”- but if you continue to investigate long enough you will find the source of the confusion is actually some wrong idea that you’ve stuck yourself to.
“This part of the assay works, the problem must be in this other part.” Sometimes this is troubleshooting, sometimes it is blindness.
You have some experimental result that indicates a method is working as intended, but you may expand that confidence to all of your assumptions about the “working” step. You’re liable to confuse your idealization of the experiment with reality. This is what causes problems down the road, but because the problem manifests in a later part of the experiment the connection is not obvious.
So the “problem” is not a misbehaving protocol; the “problem” is that you have a wrong idea that’s causing you confusion. You can’t see it or fix it because the cause and effect occur too far apart in time.
When I investigated the source of such misperceptions for myself, I found that ultimately my mistake was my answer to the question, “What is the job of the scientist?” I thought the job of the scientist was to be clever- to solve problems creatively in ways that other people couldn’t. While this may be how outsiders see a scientist, it turns out there is a big mismatch between what a scientist looks to be doing and what they’re actually doing.
The scientist is someone who observes some phenomena under some experimental circumstances in an effort to answer a question. From an external perspective they appear to be solving problems, but the baseline activity of a scientist is nothing more than unadulterated observation.
In the realm of pure science there isn’t even a single problem to solve– only dispassionate observation of some phenomena in some circumstances. The “problem” arises when you fail to maintain an open mind.
In other words, when you think you have a problem, the problem is actually that you’re not accepting your observations. If you observe your experiments with an open mind you will never be deluded and you will be able to think clearly.
“Oh this gel turned out poorly because I added too much template to my PCR reaction”, this statement is literally not even science. This is an assumption, not an observation, because you’re not seeing the data but only your surface level interpretation of it. You have instantly spun a story and added yourself to the situation, to the facts. This is not science.
“This PCR reaction appeared as a smear on this agarose gel, I should experiment further and replicate to understand why”, this is pure science. This is approaching your observations like an undergrad on their first day in lab.
When you find yourself in trouble with an experiment, you should first make sure that your science is pure. It takes effort and constant vigilance to keep your science pure and uncontaminated from the mind’s rationalizations and storytelling, but when you do you will never face a problem in your work. Your advisor will say that you have some problem to solve, but if you investigate whatever it is with a pure scientific mind then you will see it clearly, and your creative faculty will work more effectively with the extra information. You will not blind yourself to any possibility and will eventually find out why your observations differed from the expected.
What is the job of the scientist? To be an undergraduate on his first day of lab, every day.
What is the nature of the scientist? Just to watch a Chess match.
The day has finally come: you’re tired of sitting at the microscope with your Hunchback of Notre Dame lookin’ posture, haemocytometer, cell counter, and trypan blue in hand, clicking away. You look down at your ice box, unable to pull your eyes away from the source of the subconscious resentment that’s tearing apart your experiments. You keep counting, noting the mediocre cell viability and dreading the non-specific binding that is certain to ruin your day. A small brown bottle glints in the corner of your eye, its dark pigmentation conceals its vile contents but your most primal instincts, your reptilian hind-brain, is alerted and aware of what lurks inside. It’s none other than the plebeian choice of cell isolation reagent: magnetic beads.
A tear rolls down your face as you unpack the cell isolation magnet from the bottom cabinet. “I need an over-engineered solution that costs nearly a quarter of a million dollars and requires a full time skilled operator. I need something I can believe in.” You think to yourself, “…I need a flow sorter.”
What turn of fate has drafted you into being a lowly surf who uses antibody-coated magnetic beads to isolate your cell populations of interest? How can you escape the tyranny of your lower consciousness and transcend into realms of existential bliss and detachment from magnetic bead columns?
Many old buddhas, from Gautama to Bodhidharma, described such a state, and over the centuries countless eastern wisdom traditions, Zen and Mahayana Buddhism as well as the Gnostic mystics of the west, have struggled toward the experience of satori: freedom from magnetic bead purification. It was not until the late 20th century, however, that Zen reached its zenith in the form of the BD FACSAria II fluorescence-activated cell sorter.
Sorting Your Experiments Out of Saṃsāra
Saṃsāra is what the Zen Buddhist describes as the endless cycle of birth, meaningless existence, death, and rebirth, which is exactly how I would describe my various dissertation projects. Invariably, in any complex cell culture experiment I find myself suffering from impure cell populations and tedious purification procedures, and so last year I postulated that freedom from saṃsāra could only be attained via intense training in fluorescence-activated cell sorting (FACS). What follows is a highly technical decent into the workings of a typical cell sorter and an exploration of the existential questions raised by such a device. Proceed at your own risk.
A PhD student asked his adviser in all earnestness, “Does a cell have Buddha nature?”
FACS is similar to conventional flow cytometry in that cell populations of interest are first tagged with fluorochrome-conjugated antibodies specific to unique surface antigens. Internal antigens can also be tagged provided they are made accessible through a permeabilization procedure or genetically fused to fluorescent proteins such as GFP or mCherry. After washing away excess staining reagents the cells are resuspended in cold PBS or MACS buffer (PBS with some serum and EDTA mixed in for flavor), filtered to remove aggregated cells, and passed single-file (in theory) through a series of lasers that excite any fluorochromes present on the cell.
Figure 1. Simplistic FACS fluorochrome panel as depicted by the Biolegend Spectra Analyzer. Dark purple dotted/solid: Brilliant Violet 421 excitation/emission. Orange dotted/solid: PE excitation/emission. Pink dotted/solid: APC (allophycocyanin) excitation/emission. Purple, blue, green, and red vertical lines correspond to 405 violet, 488 blue, 561 yellow-green, and 633 red lasers, respectively.
Cytometers can have anywhere from 2 to 7 (or more!) lasers, flow sorters typically having more rather than less due to their already mammoth cost, that excite each fluorochrome optimally and stimulate the emission of secondary photons. The emission spectra for a given fluorochrome will always be red-shifted relative to the excitation frequency because between the moment when the exciting photon is absorbed and the secondary photon is emitted, the fluorescent molecule will lose some of its energy in the form of movement/heat. The distance between a molecule’s excitation and emission max frequencies is referred to as the Stoke’s shift [1].
Figure 2. (A) Diagram of fluorochome energetic states. (B) Excitation vs emission spectra. Source: Bio-Rad
Emitted photons are initially diffracted through a prism, passed through several filters (the complexity of which an entire article would be necessary to describe), and finally collected by photomultiplier tubes (PMTs). PMTs are essentially funnels that convert photons to electrons via scintillation (sounds titillating, no?), and allow for gain to be applied through variable voltage control of the dynodes. Each laser typically has 2 to 4 PMTs associated with it.
PMTs have several downsides, including their physical size, and it should be noted that in some new cytometers PMTs are completely omitted and replaced with smaller semiconductor detectors. In the Cytek Aurora, for instance, each laser has upwards of 8 detectors thanks to their relatively tiny size.
Figure 3. PMT diagram. The photon (red) is converted to an electron through the process of scintillation, typically by a phosphor. The electron is then amplified via its interaction with each of the dynodes, with the voltage applied to the dynodes being proportional to the gain in the electronic signal generated by the PMT.
Besides the fluorescent signals of the surface antibodies, each cell is also characterized based on its forward and side scatter pattern by 2 dedicated PMTs associated with the blue (or sometimes the violet) laser. The forward and side scatter PMTs quantify the size and granularity of the cell, respectively (figure 4).
Figure 4. Forward and side scatter are associated with size and granularity of each cell, respectively. Source: Drexel University.
Finally, all of the scatter and fluorescent data is put together as a matrix of data that represents 1 cell. Elegant, no?
The adviser replied, “μ!”
Now all of this is just the workings of your typical, everyday flow cytometer. To actually sort the cells several more tricks must be employed.
We must first realize that, as enlightened flow sorting masters, we are sorting not the cells themselves but rather the charged droplets of liquid that they are encased within. Cells are segregated into individual droplets by the action of the nozzle, a tiny ceramic aperture with a diameter of typically 70 μm, 85 μm, 100 μm, or 130 μm.
The nozzle lies directly below the interrogation point, where the tagged cells and lasers mix and mingle, and through the black magic of electronics by the time the cell is segregated the fluorescent characteristics of the cell are recorded and a decision has been made by the software to sort the cell or not.
Below the vibrating nozzle lies the sort block which contains the deflection plates. Each charged droplet will pass through the sort block and if a droplet is thought to contain a particle of interest the deflection plates will become momentarily charged and deflect the droplet to any 1 of 4 positions. There are typically up to 4 deflection angles, 2 on the left and 2 on the right where a collection tube(s) can be placed, and any uncharged or uninteresting droplets fall straight into the trash can in the middle of the sort block.
Side note: if you set the laser delay wrong then every droplet, not just the droplets without your cells of interest, will fall into the trash. While this is a truly enlightening way to end a 6 hour sort, I would not recommend it to those who have not attended at least a 7-day anger management retreat at an ashram.
Theoretically, collection is the end of the sorting process and the each sort event is quantified by the software. Droplets of interest can be defined in any number of ways, and so the capabilities of a sorter are nearly endless.
Furthermore, sorts can be optimized for either yield or purity, and collection vessels range from 1.5 mL Eppendorf tubes to 96-well plates.
Meditations on Cell Sorting (So you don’t have to!)
Larger nozzle size = less pressure applied to your cells but slower sorting
Pre-filter your resuspended cells through a 0.2 um mesh immediately prior to sorting to prevent clogging of the nozzle. This is particularly important with 70 and 85 um nozzles.
Keep samples on ice and protected from light before sorting to prevent photo-bleaching of your fluorochromes
Use a fixable live/dead stain to exclude dead cells from your analysis. They stick to viable cells and bind non-specifically to antibodies and can form convincing, but unreal, populations of cells during analysis
Use compensation beads over live cells for compensation controls, they are more consistent. Be aware that there are antibodies that don’t bind well to them, though.
Beware of dyes that conjugate multiple fluorochomes such as PE/Cy5 and PerCP/Cy5.5. While useful, if not cared for correctly they will begin to dissociate and give signals in both component channels- messy!
Be aware of the brightness of each fluorochrome you use. Put the brightest fluorochromes on the dimmest (least expressed) antigens.
Source: Biolegend
Pre-add around 200 uL of serum-containing PBS to your collection tubes and vortex with the cap on. The static charge on the plastic of the tube will repel your tiny charged cell droplets and cause them to bounce out of the tube. Serum proteins will coat the plastic and neutralize the charge. Also, the volume gives them a nice little pool to land in.
A Zen poem for the road: “The old Eppendorf tube, a cell sorted in; plop!”
Final Thoughts
A helper T cell and a memory B cell are traveling through the lymphatic system when they come upon a soluble viral coat protein coated with iC3b. The B cell pinocytoses the viral protein and presents it via MHC II. The T cell, knowing that B cells should not pinocytose antigen unless it binds specifically to the B cell receptor thinks about it disapprovingly as they continue along. Eventually, they reach the draining lymph node. The B cell hands off the viral protein to a subcapsular macrophage and the T and B cell again continue on their journey. The T cell continues to ruminate on the odd behavior of his companion. A few hours later as they approach the thoracic duct the T cell can no longer contain himself and says to the B cell, “You know as a memory B cell you are not supposed to pick up non-specific viral proteins.” The B cell replies, “I put down the viral protein hours ago. Why are you still carrying it?”
References
Banwell C.N. and McCash E.M. Fundamentals of Molecular Spectroscopy (4th ed., McGraw-Hill 1994) p.101 and p.113 ISBN0-07-707976-0
Don’t get me wrong, I work and think about my vocation constantly- without end. However, when it comes down to implementing abstract ideas in the form of bench work my mind makes like Wile E. Coyote and chases conceptual road runners that have absolutely no relation to my intended goal. It’s absolutely impressive the lengths to which my mind will go to avoid a task with the conceptual label, “work” by pursuing equally difficult tasks labeled equally as arbitrarily, “not work”. Bad brain! Or perhaps good brain, bad brain controller.
Creative procrastination is an example of exactly the kind of sustained output that characterizes productivity geniuses, only it is fired in the form of cleaning your apartment or researching the peculiar qualities of shark antibodies (they are monovalent, curiously) instead of at the intended task. This mental hip fire represents a source of massive potential power should it be harnessed, or at least its targets constrained appropriately. So how might we go about directing this latent psychic energy?
Good Brain, Bad Brain
Now first I must say that I hear you highly industrious and organized individuals and your calls for self-discipline. Would it not just be easier to adhere to the schedule I made? Would it not be fulfilling to beat every ADD-addled neuron into submission with intravenous Ritalin? Is it not possible to, over time, shift my neural wiring into a state that favors responsibility and being a productive member of society? Well, for me it turns out that while those things are great I just really dig reading about shark antibodies. The exhilarating, creative nature of an irresponsibly-directed attention span, I believe, is tainted by all this talk of discipline. Why not compromise and gently guide the firehose of dopaminergic fervor from the gutter to the garden, a much easier task than forcing its incredible pressure through the sink faucet where it poses no risk of splashing on your clothes.
And so my attempts to guide the Rambo of my orienting reflex began.
The You Who Opposes You
It’s important to realize that the power of the misdirected consciousness lies in, to borrow the language of Alan Watts, the mind’s element ofirreducible rascality. This is to say that when we unintentionally procrastinate by fixating on some problem in lieu of the original that it is not the inherent appeal of the distraction that imbues it with such psychic glitter. Rather, it is the fact that the substitute thoughtform is conceived of in different terms than the intended goal. The substance of the task to be detested is secondary, while the psychological frame that surrounds it primary. To put it more elegantly, “the enemy [distraction] of my enemy [intended goal] is my friend”.
Luckily, the stubbornness and rascality of the human ego is predictable. As social animals refined through the forces of natural selection, humans exhibit oppositional and tribal behaviors and we all know it. Humans (like you) tend to:
Undervalue things, people, favorable situations when they are abundant
Overvalue things, people, favorable situations as soon as they are taken away or made scarce
Bandwagon in hopes of social acceptance
Pursue the familiar and avoid the unfamiliar
Avoid exerting more energy than absolutely necessary
Bias evidence based on how recently it was encountered
Bias evidence based on personal experience
Clearly, the human mind has an inherent propensity towards being kind of an asshole. So without going into the existential aspects of the ego, the assumption can be made that the conscious mind may be able to exploit the tendency of the unconscious to entertain fallacious reasoning.
The Greatest Scheme Inside Your Skull
Would you believe me if I told you that we could scam your unconscious mind, a.k.a. you, into directing its psychic hip fire to a narrow and non-arbitrary direction? Well, regardless of what you believe here’s the way to do it.
Before starting we must prepare for this most devious of scams; every great heist has great planning and to fool your own unconscious mind right under its own nose requires that respect be paid in the form of a list. The list is the 2nd-5th most important things you need to do today. Scribble it down and put it away. Take a short walk and think about something else- forget the list, it’s really not that important.
Now sit down and identify the most important task of the day. Consider its importance, how much your colleagues are relying on you to finish it. Imagine the disappointed look on your face in the mirror when you fail to complete it; imagine the steely, disapproving gaze on your superior’s face when you are confronted. Feel free to catastrophize the consequences of not completing the task- you have to really live it.
Now do the task.
Immediately, notice that internal resistance is established on the setting of and carrying out of your intended task. The more resistance and dread you feel, the better.
Stop.
Pull out your list and look at it without any intention. Notice where your attention is drawn, what item on that list sparkles with procrastinative potential! Give in to your impulse and procrastinate, it’s either that or facing the dark reality of the primary task. As each task is completed the other tasks on the list will become more palatable and begin to race with electric energy- the potential of putting off your primary task for even longer imbuing them with unholy energy. Before long you’ll have completed 5 out of the top 6 things you had to do today. Congratulations.
Why It Works
Now, any level-headed person would be right to be skeptical of the practicality of an approach to self control based on self-deceit but the reality is that the unconscious mind is completely vulnerable to many logical and psychological fallacies. These flaws in subconscious structure are not easily steeled through explicit learning of the structure of fallacy, and even those with intimate knowledge of the nature of thought can easily fall victim to a heavily emotionally biased decision.
All we have done is presented your conscious mind with 2 options: face the primary task under the conceptual label of, “important work”, or procrastinate in comfort with another high priority task masquerading under the label, “opportunity to get out of work”. The emotional biasing of the decision makes doing even 4 other high priority tasks much easier than the single top priority task.
Getting Away With It
This manipulation of my own psyche has proved to be extremely valuable on hard days when in the past I would have accomplished nothing. I have developed this over some time of observing my own affinity for lazy behaviors and look forward to fresh ideas from others.
Out of Coomassie Blue 2 Hours Before Lab Meeting? Probably Not.
Coomassie blue, or SimplyBlue Safe Stain as we use in my lab, is a nonspecific protein dye used to allow visualization of SDS-PAGE bands. If you need some in a hurry there’s a quick and easy substitute that you probably have on hand: Bradford reagent. The normally red or rust colored Bradford reagent is the same exact dye as SimplyBlue, Coomassie Brilliant Blue G-250, differing only in solution pH.
Forbidden Technique: Bootleg Coomassie
To make bootleg SimplyBlue (probably not Safe Stain), aliquot around 40 mL of Bradford reagent and slowly add 3M NaOH (around 5 mL) until it becomes “brilliant blue”. Incubate this dye with your gel on a shaker for an hour or more and your bands will appear as normal, however you may notice some minor frothing if shaken vigorously. Proof below:
Figure 1. Lane 1: Dual protein standards. Lane 2-4: dCas9 (~169kDa) eluate from my most recent purification. These two gels are actually 1 gel cut in half and the only difference between them was the stain, SimplyBlue or Bradford pH 8. Gels were stained for 1 hour and are unwashed.
Do keep in mind that Bradford reagent is usually going to be more expensive than the bulk dye purchased for SDS-PAGE staining, but hey if you need results fast then price becomes subjectively irrelevant very quickly.
Additional note: sodium ions are noted in the SimplyBlue Safe Stain protocol to slow the staining process and thus overshooting with the pHing may be detrimental to your data. Take it easy, cowboy, you’re already going off-label so you may as well do it carefully!
I hope this trick brings you good fortune and even better results; the day I discovered this forbidden work around I had to present protein purification data in less than 2 hours and the panic brain blast truly came through.
Few things bring the gentle molecular biologist to anger, but the experience of setting up your perfect ligation reaction only to find the DNA ligase tube as dry as the agarose gel that’s been on the bench for 3 weeks is one of them. You’ve put hours of work into the primer design you don’t trust your undergraduate to not mess up and several units of your highest fidelity polymerase (blessed be NEB’s Q5 hot-start) amplifying your gene inserts. You’ve even generously used half of your lab’s supply of uncommon restriction endonuclease in an effort to get your beautifully designed DNA construct, “faster”, for your PI. After all, when they were a grad student they would have had it done in less than 2 weeks! How then did you find yourself in this position: alone and without even a uL of molecular glue?
Perhaps it has to do with your lack of forsight, or maybe there’s a spare tube stashed away in your colleague’s -20C freezer box. Is it your fault? No- it must have to do with the simple fact that T4 DNA ligase, one of the most easily produced and common workhorse enzymes in molecular biology, costs an outrageous $64.00 for 20,000 units!
Figure 1. Highway robbery
Now I hear you say, “but certainly 20,000 units of T4 ligase is fair! How many units could one graduate student use?”, but I ask you to cast your eyes upon the blasphemy above once more. The concentration, 400,000 units/mL (400 U/uL), means that your $64.00 order will net you only 50 uL. God forbid you ordered the 2,000,000 units/mL (2,000 U/uL) tube, a measly 10 uL. Considering the manufacturer protocol calls for 1 uL of enzyme (at 400 U/uL) to be added per reaction and each ligation experiment typically consists of:
1:1 Vector:Insert Experimental
1:2 (or 1:3) Vector:Insert Experimental
Backbone only –
Insert only – (optional)
No ligase -,
your sweet T4 ligase will not be lasting long- perhaps 5 ligation attempts.
Now, to be completely transparent we should note that diluting and using lower concentrations of ligase with overnight incubation times is a viable alternative, however overnight incubations have been noted to decrease subsequent transformation efficiency by promoting long, linear DNA strand formation. Additionally, to use this strategy you will want to tackle the whole issue of unintuitive ligase unit definitions, per NEB – “One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer”. I won’t pretend this didn’t take me a few takes to understand conceptually and at least 30 minutes to see as any more practical than, “Just use one uL of stock.”
Anyway, to be honest my complaints about price and concentration are just a convenient excuse to procrastinate my dissertation work by manufacturing and validating a much cheaper (and sexier) alternative: recombinant p50-T4 ligase fusion. If you’re like me and want to be set on ligase for at least the next 4 PhD generations in your lab then join me on this adventure. In the end you’ll have so much ligase that you’ll be ethically bartering with other labs for reagents, undergrad labor, and perhaps even the ever-coveted RPMI 1640, 500 mL, cherry flavored.
What is p50-T4 Ligase
To put it simply, p50 is the somewhat promiscuous DNA binding domain of the ubiquitous transcription factor NF-κB. It also serves as the dimerization domain for the mature NF-κB protein, a heterodimer of p50 and p52. The natural palindromic sequence recognized by p50 binds to it with an impressive Kd ~8 pM, but nonspecific dsDNA also has a Kd ~5.7 nM. As noted by W.M. Patrick et al., the authors who conceived of this recombinant ligase originally, fusion of p50 to T4 ligase resulted in approximately 7-fold and 1.6-fold improvements in cohesive-end and blunt-end ligation, respectively [1].
If you’re familiar with recombinant fusion enzymes possessing enhanced activity- recently innovated high-fidelity polymerases such as Phusion come to mind- then you may find it initially puzzling that the holy grail of non-sequence specific DNA binding proteins sso7d was not utilized over p50. Saccharolobus solfataricus sso7d is remarkable in its activity and stability, but in that stability lies its weakness for this application. For efficient transformation of a ligation mix the ligase must be heat-inactivated and its grubby grip on the vector released. Sso7d happens to be so thermally tolerant that even incubation at 95C for 15 minutes is not sufficient to release its grasp fully, hence its utilization in PCR. p50, on the other hand, denatures at a (relatively) cool 65C in only 10 minutes which is conveniently the same conditions that denature T4 DNA ligase.
A number of other DNA-binding domains were fused to both N and C-termini of T4 ligase by the authors, but overall p50 showed robust and consistent activity and so this is the version I decided to manufacture for my lab. I highly encourage you to read the original paper, though [1].
The Goods (Protocol)
Note: this protocol is for overexpression and purification of p50-T4 ligase in E. coli from pET28-p50.T4ligase. The ligase has an N-terminal 6xHis tag. It’s pretty robust, yielding around 10-20 mg/L of culture in my hands but I haven’t tried it with other purification schemes so results may vary.
Transformation and Culturing (Days 1-3)
Transform BL21(DE3) E. coli or equivalent with pET28-p50-ligase vector.
(Optional) Transform DH5alpha E. coli for a glycerol stock for future generations.
The following day screen colonies and, on confirmation of plasmid identity, inoculate 6 mL LB supplemented with kanamycin. Grow overnight at 37C.
The next morning, inoculate 2x2L shaker flasks containing 500 mL LB + kan with 3mL of starter culture.
Grow at 37C with shaking to an OD600 of ~0.7 (about 2.5 hours).
Induce with IPTG to a final concentration of 0.4 mM (2 mL/flask of 100mM stock).
Incubate at 26C overnight (I had success sleeping in and going for 18 hours) for protein overexpression.
IMAC Purification (Day 4)
Collect your cultures in centrifuge bottles and centrifuge 5251xg/15 min/4C.
Resuspend each pellet in 25 mL of IMAC lysis buffer (see above) and split 1 tablet of Roche cOmplete Protease Inhibitor Cocktail tablet between the two fractions.
Sonicate each fraction as follows: 10s on, 30s off, 30% amplitude, for 15 cycles. Verify bacterial lysis on microscope if you find sonication to be arbitrary black magic as I do. Note: Remember to keep your culture in a large ice water bath to prevent overheating of the sample and denaturation of your ligase.
Pellet insoluble junk by spinning at 10,000xg/30 min/4C. You might have to aliquot your suspension into 1.5 mL Eppendorf tubes but trust me, if you try a lower speed for even an hour to avoid the tedious work you will reaponly cosmic punishment during the subsequent filtering step.
Syringe or SteriFlip filter your supernatant through a 0.2 um membrane. Alternatively, if you did not take my advice, wait an hour for your sample to pass through before finally giving up and returning to step 4 having wasted 2 hours.
Add a 1 mL bed volume of Ni-NTA resin to a 50 mL disposable column. As the resin is a 50:50 slurry of resin to ethanol, make sure you are adding 2 mL slurry.
Equilibrate the resin with 5x bed volumes (5 mL)of ddH2O, followed by 5x bed volumes of lysis buffer.
Incubate the filtered supernatant with the equilibrated resin on a rocker for 30-60 minutes.
Wash with 1x bed volume lysis buffer until the A280 of the flow-through levels off.
Wash again with 5x bed volumes of wash buffer until, again, the A280 of the flow-through levels off.
Elute 5+ fractions of 1x bed volumes elution buffer each, incubating the column for 10 minutes before each. Verify presence of p50-T4 ligase (~95kDa) in all fractions via SDS-PAGE. Pool eluate fractions containing bulk of yield.
Concentrate your ligase with an Ultracel 30kDa spin concentrator column and, once sufficiently reduced in volume, add glycerol to a final concentration of 40-50% to prevent freezing in the -20C.
Verify the concentration of your ligase with Nanodrop, a Bradford assay, or both. I concentrated my stock to around 5 mg/mL and found the ligase worked effectively at final concentrations between 0.3-3 uM, with minimal activity below 30 nM.
Activity of Purified Product
I verified the ability of p50-T4 ligase to join linear into circular dsDNA products with a very simple cohesive-end ligation test.
Using what I had on hand, the plasmid pSNAP-tag-m (5802bp) was linearized with XhoI overnight. The resulting linear plasmid was column purified and then ligated according to the standard protocol in NEB T4 ligase buffer with p50-ligase concentrations ranging from 3 uM to 3 nM, with both a no ligase negative and 400 units commercial NEB T4 ligase positive control. Reactions were in 20 uL total volume and were allowed to react for 10 minutes at room temperature before being inactivated at 65C for 10 minutes.
Note: When I tried to skimp on the deactivation by only incubating 5 minutes at 65C the resulting gel was disfigured and uninterpretable.
Figure 2. Lane 0: 1kb ladder. Lanes 1-5: dilution series of p50-T4 ligase. Lane 6: Commercial T4 ligase from NEB. Lane 7: No ligase, cut pSNAP-tag (m) only.
The ligated bands with our produced p50-T4 ligase look a bit blurry compared to the commercial ligase but I suspect this may have to do with insufficient heat-inactivation or more probably linear dimers and trimers of the plasmid (~12+ kb). I will be repeating this experiment in the near future with lower DNA concentrations as well as: 1. overnight incubation vs 10 minute incubation, 2. a heat-inactivation series at 65, 70, 75, 80, 85, 90, and 95C, and 3. a directional insert with 2 different sticky ends and subsequent transformation into E. coli.
Update on Activity After Storage & Transformation of Ligated Products – 2/5/2020
After working with this recombinant ligase for the last few months I am happy to report that our lab has switched over completely from commercial ligase (sorry NEB)!
Additionally, I successfully transformed numerous ligation products with no notable drop in efficiency.
Figure 3. Titration of p50-T4 DNA Ligase prior to a ligation attempt. I chose to use 98 nM concentration and had success after transforming with the above protocol (10min at RT in NEB T4 ligase buffer, 65C/15min heat inactivation, Transformed 1 uL into DH5alpha E. coli)
At this point I don’t have much more to add regarding this project. It was fun and if you want a copy of the plasmid for academic use send me a message and I’ll be happy to mail you over a copy, free of charge.
Peace.
References
Wilson, R. H., Morton, S. K., Deiderick, H. A., Gerth, M. L., Paul, H. A., Gerber, I., … Patrick, W. M. (2013). Engineered DNA ligases with improved activities in vitro. Protein Engineering, Design and Selection, 26(7), 471-478.
Chemically Incompetent 