Once you have selected a biological system you are confident with, you can start considering the factors that you can vary to study their effects on the system.

If you spend around 10 minutes brainstorming, you can easily come up with a list of more than 30 factors that could potentially influence your chosen system. However, for your first DOE and while you are still getting familiar with the methodology, it is advisable not to start with all of those factors at once. Taking on too much could overwhelm you. It's better for your first DOE to be feasible and simple enough for you to analyze and interpret the data, especially considering it's your initial experience.

For example, let's say you plan to optimize the composition of a microbial culture medium. Some obvious factor choices could include the contribution of Yeast Extract, the type and concentration of a buffer, the pH of the buffer, the type and concentration of salts, or specific substances required for the growth of your organism if it is auxotrophic. If you are interested in protein production, the concentration of amino acids becomes important. Now, you could independently vary the concentration of each individual amino acid, giving you 20 factors right there. However, for the sake of simplicity in your first DOE, you might choose to vary the concentration of an amino acid mix containing all 20 amino acids.

Take some time to think about the factors you might select and consider if there are any ways to simplify aspects of your experiment so that it doesn't become overwhelming too quickly. Choose between 5 and 10 of those factors to include in your first DOE design. It's important to pick factors that you believe will have a noticeable impact on your system, as well as a couple of factors you are unsure about and would like to investigate further. Perhaps you've come across a research paper mentioning the beneficial effects of a compound in an assay, but you haven't tested it yourself. This is a great opportunity to have some fun and try it out.

There are various DOE designs available in software like JMP or Design Expert, but at Synthace, we have aimed to simplify them to the essentials you need to get started.

If you've read the provided background material explaining DOE, you may already understand that it's an iterative process rather than a one-shot experiment. In most DOE campaigns, you begin with a screening design, which helps you identify whether your factors are affecting your system or not.

In Synthace, we recommend starting with an Optimal design where each of your selected factors has only two levels: one at a low set point and the other at a high set point. Optimal designs offer high power with minimal runs, making it an excellent starting point for your first DOE.

Utilize your scientific knowledge of the system to choose the high and low set points in a way that captures a range of activity from your system. Ultimately, you want to generate data that can be analyzed.

For instance, if you know that your system doesn't show any signal below 10 mM of Compound A, it wouldn't be wise to set your low and high levels at 2 and 8 mM, as you wouldn't observe any effect and might wrongly assume that the factor is unimportant.

Taking a simplified approach to DOE for your first attempt is a great way to familiarize yourself with the process. Once you've conducted your initial experiment, you'll realize that it's not as challenging as you may have imagined, and the biological insights you gain will showcase the strengths of DOE.