Efficient Sampling of Genetically Encoded Biosensor Design Space Enabled with a Design of Experiments and Automation Workflow

The development and optimization of biosensors towards biotechnological applications is the scope of our research. Specifically, we aim to understand how to optimize and engineer biosensors through modulation of their genetic elements. Intuition-driven design choices, such as classical promoter engineering and fluorescence activate cell sorting are techniques routinely applied in the characterization and design of biosensors.

Design of experiment methodologies have not yet been widely adopted in genetic circuit design. Through this protocol, we seek to increase the uptake of these types of techniques in genetic circuit and biosensor design. To begin, determine the theoretical library size and calculate the number of individual variants needed to ensure greater than 95%library coverage.

Calculate the required volume of antibiotic supplemented lysogeny broth media according to the number of colonies that are to be screened. Open the liquid handler software and click on run next to the MTP liquid transfer program. Place the prepared media in the corresponding reservoir position.

Then fill the deck with empty microtiter plates according to the layout. Set the program to dispense 200 microliters of media. Click okay to confirm program start.

Now, transfer filled microtiter plate wells to a colony picker platform and unseal and transfer square auger plates of Pseudomonas putida transformed with plasmid variant library DNA into the colony picker platform. Use the colony picker to inoculate each prefilled well with a single colony from the transformant plates. Reseal the inoculated plates and transfer them to an offline shaking incubator.

Post-incubation, return the grown plates to the liquid handler platform and unseal. Click run next to the add glycerol to MTP protocol. Ensure that the plate layout on screen matches that of the liquid handler dock.

And sequentially, click okay for the protocol to run. Once finished, seal the plates and briefly mix them in an offline shaking incubator at 800 revolutions per minute for five minutes. Barcode the plates and store at 80 degrees Celsius until needed.

Calculate the required volume of antibiotic supplemented media for deep-well blocks. Click run next to the DWB liquid transfer program. Ensure that media is added to the correct reservoir.

The empty deep-well blocks are in correct layout positions and that an adequate supply of tips is available. Set the program to dispense 495 microliters of media. When ready, sequentially click okay to start the program.

Now, seal the filled deep-well blocks with breathable membrane and transfer to temporary storage at 4 degrees Celsius. Next, click run next to the inoculate from thawed MTP program. Ensure that MTP cryo stocks and fill deep-well blocks are transferred to the platform per layout and that sufficient tips are loaded.

Sequentially, click okay to initialize. When the program finishes, seal the inoculated overnight deep-well blocks with breathable membrane. Transfer them to an offline plate shaker incubator set for 16 hours at 30 degrees Celsius, 800 revolutions per minute, and 75%humidity.

Reseal, mix, and return cryostock microtiter plates to 80 degrees Celsius freezer. Now, calculate the required volume of media supplemented with various effector and antibiotic concentrations for the overnight deep-well blocks to be screened. Click run next to the DWB liquid transfer program.

Then ensure effector-supplemented media reservoirs are correctly placed. Add empty deep-well blocks into the liquid handler platform. When sufficient tips are available, sequentially click okay to start the protocol to generate assay deep-well blocks.

After filling, seal the assay deep-well blocks with breathable membrane. Refill the liquid handler with empty plates. Repeat inoculation until all assay blocks are filled.

Next, click run next to the transfer to assay DWB program. After unsealed assay deep-well blocks with effector-supplemented media are placed correctly, transfer and unseal overnight deep-well blocks containing grown P.putida per layout. Sequentially click okay to start the protocol.

When the program has finished, seal transfer assay plates to offline incubator. Discard the overnight deep-well blocks after inoculation. Next, transfer the deep-well blocks into a centrifuge.

Pellet cells at 4, 000 G in an ice-controlled rotor at 18 degrees Celsius for five minutes. After discarding the supernatant, place the centrifuge blocks onto the liquid handler platform. Calculate the volume of one times PBS required based on the number of deep-well blocks to be screened.

Click run next to the assay set up PBS resuspension DWB program, and set the dispense volume to 500 microliters. Then ensure PBS is added to the correct reservoir, and array the centrifuged plates according to layout. Click okay to start the program after confirming tip availability.

Reseal and remove resuspended deep-well blocks from the liquid handler. Check the underside of the plate to ensure pellets are completely resuspended. Click run next to the assay setup cells and PBS edition MTP program.

Then transfer resuspended deep-well blocks into the liquid handler according to layout. Load the empty microtiter plates according to layout and set dispense volume to 200 microliters before clicking okay. Transfer the filled microtiter plates to an offline multi-mode plate reader and measure the relative fluorescence and OD600.

Automated screening of 5, 000 promoter variants identified top candidates showing greater than 3.6-fold activation. The EC 50 values for 100 unique variants were plotted to visualize the sensitivity distribution and identify robust candidates. EC 50 values were computed for 226 enriched variants and ranked using Lin-log transformation to form a sensitivity-scaled library.

A definitive screening design was constructed using 1, 0, and 1 level variance from four modules. Transport, Regulator, P-out, and Output Ribosome-Binding Site model profiles revealed how changes in expression levels of the four modules non-linearly impacted EC 50 and Hill's coefficient. Identifying optimal expression combinations with RBS-Out, showing a strong positive effect on both sensitivity and slope.

The globally optimized biosensor variant incorporating ideal module strengths demonstrated enhanced sensitivity, and Hill coefficient compared to both the parental and DSD-optimized versions.