Often a scientific protocol is not just a simple process of mixing liquids together in a single step. The addition of liquids into a mixture might be separated by incubation steps or prompts to do something manually. However, in the separate mixing stages, you may want to apply factors as a part of a DOE design, rather than all your factors being assigned to a single mixing stage.
With the Core Elements you have the flexibility to describe your scientific protocol so that it describes the experiment in the lab, while at the same time being able to apply DOE to that protocol throughout the different stages.
In this example experiment, there are three stages:
Media preparation
Inoculation with a bacterial starter culture
Incubation and induction of protein production
Here, a scientist may want to prepare bacterial culture media, consisting of various different components that they want to investigate with DOE. After preparing the media they want to be prompted to remove their bacterial starter culture from an incubator and inoculate the new culture, where the concentration of cells to be added is also investigated with DOE. Finally, protein production will be triggered by the addition of a small inducer molecule, but that concentration is also investigated by DOE.
This is a common process for a lot of biological scientists, however, it may not be an example in your wheel house. The fundamentals taught here still translate to other applications. For example, you may be investigating a cell-based assay in which you first need to grow your mammalian cells in a medium, and incubate for some time, before adding additional reagents in order to generate a signal from your assay.
In this tutorial you will learn:
How to build a DOE workflow that has multiple liquid handling stages, so that it is ready to apply a DOE across.
Get started
Building a flexible workflow for DOE
Media preparation
Add the Define Liquids and Plates and Make Mixture elements to the workflow builder.
Connect the output Liquids from Define Liquids and Plates to the Liquids To Make Mixtures input of Make Mixtures.
Optional. Add these elements to a group and annotate with the stage name.
Note: This is the most simple DOE workflow unit you can build and is best when you have defined concentrations for your stock liquids that can not vary. If you want Synthace to calculate the required stock concentrations and liquids, then you might want to build a DOE workflow that looks like this - to learn how see here:
Inoculation
Add another Define Liquids and Plates element and a Mix Onto element to the workflow builder.
Connect the output Liquids from Define Liquids and Plates 2 to the Source Liquids input of Mix Onto 1.
Connect the output Mixtures from Make Mixtures 1 to the Destination Liquids input of Mix Onto 1.
Optional. Add the new elements to an element group and annotate with the stage name.
Note: This is a combination of the most simple DOE workflow you can build and is best when you have defined concentrations for your stock liquids that can not vary. If you want Synthace to calculate the required stock concentrations and liquids, then you might want to build DOE workflows that look like this, or a combination of the different styles depending on your application:
Incubation and Induction
Add another Define Liquids and Plates and Mix Onto element and an Incubate element to the workflow builder canvas.
Connect the output Liquids from Define Liquids and Plates 3 to the Source Liquids input of Mix Onto 2.
Connect the output Mixed Liquids from Mix Onto 1 to the Liquids To Incubate input of Incubate 1.
Connect the output Incubated Liquids from Incubate 1 to the Destination Liquids input of Mix Onto 2.
Optional. Add the new elements to an element group and annotate with the stage name.
Note: This is a combination of the most simple DOE workflow units you can build and is best when you have defined concentrations for your stock liquids that can not vary. If you want Synthace calculate the required stock concentrations and liquids, then you might want to build a DOE workflow that looks like this, or a combination of the different styles, depending on your application:
That’s it, workflow building is complete. You will notice that each stage in the protocol has an element that supports applying DOE to, namely the Make Mixtures and Mix Onto elements. Let’s take a look at filling in the parameters to get to a simulation of this workflow.
Configure your element parameters
Media Preparation
Define the stock liquids that will make up your media and their concentrations. Here: glucose, yeast extract, NaCl, and a diluent, water.
Define two mixtures - one with high target concentrations of the stock liquids, and one with low concentrations.
Assign Water as the default diluent for all mixtures.
Select Let Synthace Optimise in the Replicate Grouping parameter, provide a plate name and select a plate type in the plate contents editor.
Inoculation
Define the cells and their concentration, and water. In this case, E.coli BL21 (DE3) at 1,000,000 cells/ul.
In the Mix Onto element select the Mix Mode “Mix By Composition” and the Addition Mode “Let Synthace Optimize”
Note: To learn more about how to use the Mix Onto element click here.
Set the Naming Mode option to “Do Not Rename”.
Note: When doing DOE in Synthace, the name of your mixtures is automatically assigned to the run number (for example, Run 1, Run 2, …). It is therefore important that you do not rename your mixtures throughout the workflow.
Define two mixtures, one with a high and one with a low concentration of cells, here 100,000 or 50,000 cells/ul, respectively.
Note: The mixture compositions parameter in Mix Onto allows you to define which mixture you want to mix liquids onto, which liquid you are going to mix, and the target concentration or volume you want to mix. So here, Mixture 1 will have cells mixed to 1,000,000 cells/ul and Mixture 2 to 50,000 cells/ul. This is why not renaming your mixtures is important, as Synthace will use the autogenerated mixture names via DOE to make sure the correct levels of liquids the right mixtures and overall DOE design.
Assign Water as the default diluent for all mixtures.
Set the Final Target Volume parameter to 100 ul for this example. This means that Synthace will calculate the required number of cells to reach the target concentrations in an overall total volume of 100 ul. However, the upstream mixture is already made up to 50 uL. Therefore, the available volume for cells is 50 ul, to to reach the Final Target Volume specified here.
Incubation and Induction
Define the incubation parameters.
Define the inducer and its concentration, here, IPTG at 500 mM and a diluent, Water.
Repeat Steps 2 and 3 from the previous section to set the Mix Onto and renaming modes.
Define two mixtures one with a high and one with a low concentration of IPTG, here 1 or 0.5 mM respectively.
Assign Water as the default diluent for all mixtures.
Set the Final Target Volume parameter to 110 ul for this example. This means that Synthace will calculate the required volume of IPTG to reach the target concentrations in an overall total volume of 110 ul. However, the upstream mixture is already made up to 100 uL. Therefore, the available volume for cells is 50 ul, to to reach the Final Target Volume specified here.
Simulate the workflow
Check that the device that you selected can follow the instructions that you prepared by simulating your workflow. To learn how, click here.
Preview the Simulation
After you simulate the workflow, click View Simulation to open the simulation details.
Open the Preview tab, then click through the steps to check that the instructions that Synthace has generated are as desired.
Note: This workflow starts with the preparation of the media mixtures before inoculating with cells. Following those steps you will notice a prompt message to indicate the incubation step before continuing with addition of Inducer.
We hope that this tutorial has been helpful!
To learn how to define factors in your workflow and calculate a DOE design, click here.