Bacterial cells require a variety of nutrients to grow, replicate, and produce proteins of interest. These nutrients include sources of carbon, nitrogen, and other essential components. Carbon is a critical element to bacterial growth as it serves as the backbone of all living organisms. Nitrogen is another essential nutrient required for bacterial growth, as it is a critical component in several biomolecules, such as amino acids, nucleotides, and proteins. Other essential nutrients include vitamins, minerals, and trace elements.
The specific components used in a given medium will depend on the nature of the cells being grown and the proteins being produced. For example, some bacteria require complex media that contain blood, serum, or other animal products, while others can grow in minimal media containing only a few essential nutrients. The composition of the medium and process conditions can also be adjusted to optimize bacterial growth and protein production, including the pH level, temperature, and oxygen availability.
Overall, optimizing the composition of bacterial cell media is critical to achieving high levels of protein production and efficient bacterial growth. By understanding the specific nutrient requirements of the bacterial cells used, researchers can design customized media that will support optimal growth and protein production.
The cell seeding density can also have an impact on how the cells grow and ultimately produce protein. This can be controlled by the number of cells added to the media, but also by varying the volume of the media to which a constant number of cells is added. Varying the volume also gives the opportunity to optimise for efficient cell suspension and oxygenation during shaking incubation.
In this example you will learn to
Define multiple continuous mixture component factors
Define a continuous volume factor
Calculate a 96-run space filling design
Simulate the DOE design against your workflow ready for execution in the lab
Build a simple DOE workflow
Build the following simple DOE Workflow
Note: To learn more about how to use the Core Elements click here. For more detail on how to build DOE workflows with the Core Elements click here.
Define the diluent, as well as the stock liquids and their concentrations for the different mixture components.
Define one or more mixtures in the Make Mixtures element along with all other required parameters.
Define both your input and output plates.
Simulate the workflow and confirm your mixtures are prepared as you would expect.
Continue editing the workflow to start creating your DOE design.
Define the DOE factors and calculate a design
Switch into DOE design mode.
Note: If the workflow has not already been added to a Synthace experiment you will be prompted to do so before being able to enter DOE mode.
Select the Make Mixtures element and toggle on both the Mixture Compositions and the Final Target Volume parameters as factors.
Launch the DOE Factor Table
Add the three continuous mixture component factors with the following settings:
Glucose (Carbon source) between 0.4 and 0.8 % w/v, sampled across a range, including 6 zeros.
Yeast Extract (Nitrogen source) between 1 and 5 g/l, sampled across a range, including 6 zeros.
NaCl (Salt source) between 50 and 200 mM, sampled discretely.
Note: For more detailed instructions on how to define a continuous mixture component factor click here.
Add and define the continuous volume factor with the following settings:
50 - 200 ul, sampled across a range
Note: For more detailed instructions on how to define a continuous volume factor, click here.
Click on Calculate Design at the top of the Factor Table panel.
Note: By default the space-filling design option is selected with 96 runs. To learn about other design types and how to calculate designs, click here.
Assess the quality of your DOE design with the design diagnostics.
Note: With 96 runs and an effect size set to 2, this space fill design is sufficient to capture all the main effects, some of the two factor interactions and quadratic effects if they exist. There are several correlated terms. However, for an initial screening iteration, this is considered a good start to identify where to focus subsequent designs. To learn more about design diagnostics, click here.
Simulate the DOE design
Click Simulate with Design and load the simulation once successful.
Click on the Design tab, in the simulation details page, to get an overview of the DOE design that was simulated against.
Note: When a DOE design spans multiple simulations you can filter this table so that it only shows the runs included in this simulation, or so that all runs are shown. The DOE design can also be downloaded from this tab, if you want to save this for reporting or to use in third party software.
Open the Preview tab, then click through the steps to check that the instructions that Synthace has generated are correct.
You can see that the media mixtures are prepared as per the DOE design.
Downloads
We hope that this tutorial has been helpful! If you got stuck anywhere, or you want to check that you did everything right, compare your workflow to the example below. The example is in the file at the bottom of this page, which you can download.
After you download the file, complete the following steps.
Create a workflow from the file. To learn how, click here.
By default the manual execution mode will be selected or select an alternative device on which you want to run the workflow. To learn how, click here.
Select input and output plate types compatible with the devices in your organisation if the default plate types are not supported in your environment. To learn how, click here.
To learn about other factor types, click here.