I got asked how long it takes to publish a paper, from when you start the experiments to when it’s finally accepted, and it gave me an idea to write this post!
The short answer is that it’s like asking ‘how long is a piece of string?’ (the correct answer to that, kids, is ‘double half its length’). It varies, from paper to paper, but the main difference comes from the types of experiments you need to do. Please feel free to stop reading if you wanted the short answer! Beyond this is me writing my to do list for the upcoming few months.
Some experiments just take longer to do. Sometimes it’s the set up, sometimes its the experiment itself, and other times it might be the data analysis after the experiment (worst case it’s all three). Most experiments require some trial and error before you can actually do them, because- this is research. Chances are nobody else in the whole world has done it before. I’ve had to learn brand new techniques from external departments and institutions because I didn’t have a local expert in my department to get help from. Lots of groundwork is required before you can even get started.
So, just as an example, here’s an experimental outline of what I’m currently working on. It’s for one experiment, technically speaking, but in order to do said experiment, this is the long list of the things I need to achieve beforehand (and a rough time frame for how long it might take me):
Be warned- long read with lots of technical jargon ahead!
1. Streak out two strains of Escherichia coli (E. coli), each with specific plasmid DNA (circular piece of DNA found in bacteria) that I want loads of (we use E. coli to make tonnes of DNA for us, which we can harvest by killing them and purifying the plasmid DNA- mobid, I know).
Streak dilution onto Luria-Bertani (LB) agar plates, incubate overnight at 37 degrees to let the bacteria grow up to the point we can actually see them under the naked eye.
2. Pick one single colony and grow in LB broth
Inoculate (i.e. touch colony on to) LB broth and incubate overnight (~16-18 hours) while shaking at 37 degrees celsius.
3. Extract plasmid DNA
Use a kit to break open the bacterial cell and purify just the plasmid DNA – depends how many samples you have but the whole process (including all the centrifugations and incubations) can mean processing one sample takes minimum 30 minutes or so (usually much longer).
4. Measure DNA yield (how much DNA do I have?), then digest with enzymes that cut specifically designed areas of my plasmid DNA.
By ‘digestion’ I don’t mean the enzyme eats and poops out whatever is left of the DNA (that would be hilarious), but it just means allow the enzyme to cut the DNA. There’s a library of enzymes that cut certain sequences of DNA, and you can genetically engineer plasmid DNA so that they contain those sequences. Means you can literally cut and paste DNA into different plasmids at your leisure/toil.
Setting up a digest (I set up multiple that I pool and concentrate into one tube at the end) can take anything from 5 to 15 minutes, depending on how many you set up. Digestion can take a minimum 1 hour at 37 degres, but to give my enzymes the best chance at cutting all the available DNA sequences, I leave them overnight. Times can vary depending on your enzyme and your plasmid.
5. Isolate cut product, clean up digest (inactivate enzymes so they stop doing the choppy choppy), and set up ligations.
In this instance of ‘cloning’ (this process I’m outlining here is what most scientists are talking about when we say ‘cloning’), I’m essentially cutting out an inserted piece of DNA from one plasmid, and pasting it in another. That means for one plasmid, I have an ‘insert’ I need to isolate. I can do this by running a DNA gel to separate out my cut DNA bands using an electrical current (very similar to the gels used to prepare westerns). If things have worked in this particular instance, I should see one band for a big piece of DNA (my plasmid backbone), and another, smaller band for my ‘insert’. I can then use a scalpel blade to cut out my ‘insert’ DNA from the gel, then proceed to purify the DNA using a kit. This entire process of running a gel and cutting the band out can take about an hour.
The time required to do the clean up can also vary depending on how many samples you have, too. I’d probably allocate at least 30 minutes if it’s just a couple samples.
Ligation is when you stitch your insert DNA into your new plasmid backbone using special enzymes. Setting it up can take about 10-15 minutes (again, depends how many samples and conditions you have), and the incubation can be 1 hour on the bench, or overnight at 4 degrees. I always choose the 4 degrees overnight for a gentler, longer chance at successful ligation.
6. Transform ligated DNA into E. coli
Transformation is the technical term for ‘putting foreign DNA into bacteria’. This whole process of forcing the E. coli strain to take up your hopefully stitched together plasmid DNA takes a minimum 2 hours before you can even plate your bacteria (i.e. spread them on to LB agar to grow) at which point, you need to incubate overnight once again to grow the bacteria to the point you can actually physically see them.
7. (Provided the science gods granted me with bacterial colonies) Perform a colony PCR and check whether whatever bacteria I’ve got growing on my plate has the DNA I want.
Sometimes (often times?), you do the transformation, you come in the next day, and see absolutely no colonies. It happens a lot. If you get nothing, you can put the plates back in the 37 degree warm room and just hope something crops up, but… usually you just go back and set up another ligation (you can play around with how much insert:plasmid you put into your ligation reaction, maybe incubate differently, and generally alter some parameters), but… yeah, unfortunately it’s a case of rinse and repeat.
However! If the gods grant you some bacterial colonies (you only need one, right?), you can use a technique called polymerase chain reaction (PCR) to screen each colony for the presence of your inserted DNA. Basically PCRs amplify specific DNA sequences of interest, so you can use it to look for your insert. PCRs (including set up) can take about 2-5 hours depending on the reagents used, the size of your insert (the larger your inserted DNA, the longer the reaction), and you also need to run a DNA gel to look for your amplified DNA (see running DNA gel in step 5 for times), so… I’d give it a good 3-6 hours of your day just to do this.
8. (Provided I have positive clones) Inoculate overnight LB broth cultures with my positive colony of bacteria (and repeat the process of harvesting plasmid DNA again).
Now at this point, if you got no positive clones, you’d start back at the ligations and repeat everything from there on.
But, if you had a particular colony carrying plasmid with your inserted piece of DNA, then you can grow them up overnight (to make lots of DNA) to repeat the plasmid isolation step again.
9. Purify plasmid, send off to get it genetically sequenced.
Once you repeat the plasmid isolation step (see step 3), you can once again measure the DNA yield and send the plasmid off to get it sequenced. We have a company across the road that does all this for us, so we simply drop samples off by walking across the road. Others may not be so lucky and have to rely on couriers or postal services. The company policy is that provided samples get dropped off before 3pm, the results should be sent back to you in 24-48 hours time (sometimes longer). We can then download the results and check (using a special software program) that all the DNA sequences are correct, inserted in the right orientation (depending on what you’re doing, it can sometimes go in backwards), and is ready to use.
10. (Provided my sequencing results showed correct insert) Repeat this whole digest, ligate, transform process with my newly made plasmid, to insert additional, different pieces of DNA.
In my case, my first ‘insert’ was a special piece of DNA that lets me put even more pieces of DNA into plasmids using more kinds of digestion enzymes (it’s called a multiple cloning site). So now that I’ve got that site in there, I can then proceed to cut and paste all sorts of DNA from already made plasmids that my lab has stocked up.
Phew! So that’s the end of the cloning step. The next step for me is generating the bacterial strains I want to do the big experiments with! Essentially I want to put the plasmids I made into my bacteria of interest (which is Coxiella burnetii).
1. Grow up my strain of Coxiella
Different bacteria have different growth rates (as well as nutrient requirements). Unfortunately for us, Coxiella takes a minimum 6-7 days before we have enough of them to use for transformations. They’re also extremely fussy, so not only is the liquid that they grow in more complicated, but they have a strict, 37 degrees, 5% CO2, 2.5% O2 (microaerophilic) atmospheric condition requirement. No 16-18 hour incubation in basic media under normal atmospheric conditions for us (people who work on E. coli and the like should count themselves somewhat fortunate).
2. Transform my plasmid into Coxiella
Sounds simple on paper, but the whole process takes ages. Day 1, you prepare your grown up Coxiella, make them turn into a balloon (in shape) and zap them with electricity to force them to take up foreign DNA (takes roughly 2 hours if you include going to different floors to use the zappy machine). Then you need to give them some recovery time (that’s the technical term, the lay term would be some ‘chill out’ time) so that they can get over the fact you zapped them with 1.8 kV of electricity, so that’s another 24 hours. They’re in shock… hahahaha.
At this point, you can then plate them on their own, specialised agarose plates, so that they can grow another 6-7 days before you can even think about picking bacterial colonies.
3. Check colonies to see if they express the protein encoded on the plasmid
DNA is essentially an instruction manual to tell a cell to make a specific kind of protein. So, my end goal when I make my bugs take up DNA is that they make the protein I instructed them to produce. That leads to a western blot. I get some of these transformed bacteria, put them in a special buffer and boil them to kill the bacteria (sorry!), then look for my protein of interest. For more on how westerns work, please see my previous post outlining the process. Depending on the primary antibody used, this whole process could take a whole working day, or possibly span two days.
If, at that point, I find colonies which are successfully making my protein, I can then grow them up in a larger volume of liquid, both to stock down (so that we have frozen backups), but to also finally use them for experiments.
If, like most instances, I don’t see any protein, they’re the wrong size to what’s expected, or there’s not a lot of protein being made… You literally have to repeat this whole transformation step again. From growing up the strain to transforming and confirming presence of protein, the whole process can take 2-3 weeks.
So that’s the overall process of finally generating my bacterial strains. NOW, we can do the experiments. If you’re still with me, you can see how long this whole process can take, and I haven’t even done the actual experiments I want to do yet! Experiments can take a lot of time and effort just to prepare.
The actual experiment itself I won’t highlight in detail like above, because, well, if you think the above was complicated- the actual experiment is an order of magnitude higher in complication, but my variation means it’s even more complex than the standard experiment. My head hurts just thinking about it. Basically it’ll take me a few weeks to do at least three biological replicates (i.e. repeat the same experiment a minimum of three times on different days/weeks so that you can demonstrate your results are reproducible and consistent).
And that’s just one experiment. Most papers have multitudes of experiments showcased (the higher the prestige of the journal, the more figures and experiments required to publish a manuscript in it), so it means that at any given moment, we have to be running different but concurrent experiments, writing, going to meetings, and also going about our daily business of eating and sleeping. You’ll definitely struggle if you’re not good at organising yourself! But- it’s a skill that you develop over time, so you get more efficient as you learn.
So the experiments, again, are where the biggest variations come up. The writing up can be quick or slow depending on how much time you can commit to it, and how fast and efficient you are as a writer. Even if you write super quick, it still has to be read and edited by everyone whose name appears on your paper (and anyone who has emailed multiple people to ask them to do something will know how long it can take to receive feedback), so that’ll take some time… and we haven’t even sent the paper off to a journal for peer-review yet!
The peer-review process can take anything from a few weeks (my current paper turnaround was three weeks), to over 6 months, before you receive any feedback. It’s soul destroying if you’re relying on it to apply for grants, especially if, after the long wait, it gets rejected. For more info on the writing process, see my previous post here.
Even once your paper is accepted for publication, you still need to go through the editorial process of getting it proofed (i.e. the process of making it pretty and publication ready). What you send to the journal is a Word document, but what you get at the end is an edited, formatted, prettied up PDF. While it’s the journal’s job to make it pretty, the authors also need to check the finalised product before publication to make sure everything is in order. All the fonts are correct, there aren’t any spelling errors that have somehow not been noticed until now, images still look high res. and pretty… Once you give the all clear (or comments on required changes), the journal can then prepare the manuscript for full publication. That whole process can take a month or two.
If you learn anything from this, it’s that patience and resilience is a must in this world. You just won’t cope, otherwise. On the plus side, the first time you see your hard-earned paper all prettied up on the journal’s webpage, or hold a printed PDF copy in your hands, it’s quite exciting (also weird, because it’s something you’ve been staring at on your laptop for months or years in a less professional looking format). Makes the entire process (somewhat) worth it. I guess the alternative could be something like the comic below…