Arking:JCAOligoTutorial19
The Critical Path and time management
One thing you'll quickly realize about synthetic biology is that the implementation is really slow. There are many efforts underway to speed this up, but the fact that it is so slow means you have to think about managing your experiments. If you don't, you're never going to get anywhere. The concept of the "Critical Path" is something you should have in mind. In many engineering fields, the critical path is a very quantifiable and manageable concept. In synthetic biology, you can't really implement it rigourously, but it's an important concept. Since you are probably just starting out with research, you aren’t going to be very efficient, and you don’t know yet how long things take. So, just do your thing and don’t worry about this too much yet. I want to plant the seed for this concept in your mind, though, so that you will start to think about it. In the end, if you want to be an effective researcher, you’ll need to do analysis of your experimental plans and make decisions about when to start various things.
Basically, the idea is this--there are many small tasks that you need to do in order to complete your project. Many of these tasks have to be done in a specific order successfully in order to get to another task. You have to successfully get through the entirely list of tasks in order to get to your goal. The critical path is the length of time it's going to take to reach that goal that is the sum of all the dependent tasks. That isn't a perfectly rigorous definition, but we're not going to be using this concept rigorously anyway. Let's take an example to understand it better: executing the basic construction file. We’ll examine all the things that affect how long it will take and describe how to examine the situation and decide what you should be doing and when.
Hopefully, your lab is already set up for you and you don’t have to do ‘’everything’’ needed to execute the construction file. You might have noticed, though, that there are many instruments (thermocyler, pipettman, incubators, centrifuges) and consumables (microcentrifuge tubes, miniprep kits, polymerase, restriction enzymes, competent cells, petri dishes, media) that are needed for ‘’any’’ construction file. Additionally, there are two things you’ll need specifically to execute ‘’your’’ construction file: the oligonucleotides, the template, and the vector into which you are going to put your part. To simplify things, let’s assume your lab is perfectly managed and you have access to the materials you need at any time. So, the question is this, what should you do first? You might be thinking design your oligos. That may or may not be correct—it depends a little on how long it takes you to design your oligos, how long it takes them to arrive, and the availability of the vector digest and template. Let’s consider several scenarios: Case 1: I made the vector digest last week, and the miniprepped template is already in my freezer box. OK, if this is the case things are simple. Design your oligos. You have all the materials you need to set up your pcr, and you can’t do the digestion/ligation/transformation business until you’ve done the pcr. So, get on ApE and do your thing. Case 2: I made the vector digest last week, I don’t have a template for my PCR. Things are pretty simple here too. Acquiring a template for doing PCR is probably going to take quite a while. Even if you know someone who is willing to mail the DNA to you, it’s still going to take them some time to answer your email request, put the sample in an evenlope, and fedEX it to you. So, let’s say minimum 3 days. So, the question is this: what should I be doing ‘’right now’’? Depending on the supplier and their schedule with your institution, obtaining oligonucleotides can be as fast as 24 hr turnaround. We have this situation, so it really doesn’t help me to order the oligos right now. They will just sit on my desk for 2 days while I wait for the template. What I need to do right now is get on my email and request the template since that is the longer path within my critical path to the basic part’s construction. In practice, acquiring a template could be an incredible slow process…like months if you have to sign an MTA agreement. If you are going to use gene synthesis to get the template, that’s slow too—maybe 2 weeks or so. Get going with acquiring the template by the shortest route possible—the oligos can wait.
Case 3: I made the vector digest last week, I acquired the template as an agar stab. OK, great. I got the template…sortov. I can’t do the PCR on the agar stab as these are cells containing the template plasmid not the pure plasmid. So, I’m going to have to grow it up and then miniprep it. I might also want to first restreak the cells from the stab onto a plate, pick a single colony, grow it to saturation, and then miniprep before starting my PCR. If I am going to do the restreaking, it will take about 16 hrs to get the colonies. I sleep at night, so in reality this means I restreak today and get colonies tomorrow. I then pick a colony and grow it in culture to saturation. Again, I sleep, so this means it will take another day to get culture. I then miniprep it, and that will take maybe 20 min. So, total, it’s going to take me about 48 hrs to get through it. So, most likely, restreaking the template should be a higher priority than ordering the oligos, but I probably should go ahead and do both.
Case 4: I have my template, but I need to put it in a new vector. If I have the template, I could order oligos and be doing PCR tomorrow afternoon. That isn’t going to help me much if I have nothing to put the PCR product in. Let’s say my construction file for making the new vector requires that I do an in vitro cut and paste, transform, pick a colony, miniprep, and sequence. That’s going to take a few hours to do the cut and paste, a next day turnaround for getting colonies, a day to pick and grow the colonies, a quick miniprep, and then submission and receipt of sequencing which takes 1 to 2 days. At best, this will take 3 days just to get the vector. In practice, it usually takes longer. You then have to do the digest of the vector, and gel purify it. That should take a few hours, and really you could run that in parallel with the PCR. So, what you should be doing ‘’right now’’ is start making the vector.
So, basically this all boils down to getting a feeling for how long various tasks take and keeping in mind that there is usually a thread of tasks that is needed to get you to the point where you can even begin doing something else. We assumed that all the common materials for doing the project were readily available. This is often not the case. The thing you maybe ‘’should’’ be doing ‘’right now’’ to avoid increasing the length of your critical path is let the lab manager know that you are out of PCR tubes.
Again, don’t worry about this too much just yet if you’re starting out. But do reflect on it—what made your experiment take so much longer than it should have? I think you will find that seemingly trivial things like waiting an extra day to send out a materials request are very often the primary culprits in extending the critical path. As you become more aware of these things, you’ll begin to prioritize your activities and recognizing when you need to rush the completion of a task and when you can put it off until tomorrow.
