Automatisierte Modular High Throughput Exopolysaccharid Screening-Plattform mit hochsensiblen Carbohydrate Fingerabdruck-Analyse Gekoppelt

The overall goal of this approach is the fast and reliable screening for microbial exopolysaccharide producers and the identification of their carbohydrate fingerprint. This is an essential step in order to utilize the unexplored diversity of exopolysaccharides. Our method can help to speed up the polymer research in field of microbial exopolysaccharides.

It enables the fast identification of novel polysaccharide variants with different properties for specific applications. The main advantage of this technique is the combination of different exopolysaccharide detection systems in a modular and completely automized screening concept. This makes it extremely fast and reliable.

This technique can also be performed manually in laboratories where there is no automatization platform available, thereby its use is very flexible, and can be adjusted to different screening needs. This exopolysaccharide, or EPS, screening platform has a modular setup, which allows a separation of the protocol into two major parts, Automated Screening and Carbohydrate Fingerprinting. The first task is to cultivate the strains for screening and remove the cells by centrifugation.

The strains are cultured on glucose as carbon source in 96-well plates covered with breathable sealing film on micro titer plate shaker at 30 degrees Celsius and 1, 000 RPM. On the day of the screen, prepare the robot worktable and storage carousel as described in the protocol text. Remove the breathable sealing film from the plates and allocate the main cultures in carousel positions 1-1 to 1-4.

Start the automated robotic program. To remove cells after cultivation, transfer the main culture deep well plates from the carousel into the centrifuge, and centrifuge at 4, 300 x g and 20 degrees Celsius for 30 minutes. For the precipitation before filtration, move the micro titer plates, as well as the pH-indicator micro titer plates, from the carousel to the worktable.

Also transfer the filtration plates, which ensure the complete removal of all bacterial cells, together with the collector plates to their worktable positions. After centrifugation, transfer 180 microliters of the main culture supernatant to the filtration plate. Aspirate 150 microliters from the main-culture supernatant and dispense 50 microliters into the micro titer plate for precipitation before filtration, and 100 microliters to the pH-indicator plate.

Precipitation of the supernatant with 2-propanol is performed to assess EPS production. Use a 12.5-milliliter multi-step pipette to manually add 150 microliters of 2-propanol to each well. After shaking the micro titer plates at room temperature for 10 minutes at 900 RPM, visually observe fiber or flake formations that indicate EPS production.

After the main culture plates are stored back to the carousel, examine the fermentation broths, which should show decreased sedimentation after centrifugation. The increase in viscosity is another indication of EPS production. The second task is to ensure complete cell removal from the viscous fermentation broth via a 96-well filtration.

Centrifuge the filtration plates at 3, 000 x g and 20 degrees Celsius for 10 minutes. Then return the plates to their carousel home position and perform equilibration of the gel-filtration as described in the protocol text. Next, pipette 35 microliters of filtrate to the center of the equilibrated gel-filtration plate, and 50 microliters to the micro titer plate that is used for precipitation.

Move the gel-filtration plates to the centrifuge, and move all other plates from the worktable back to the home positions in the carousel. After the storage of all plates back in the carousel, take note of the highly viscous supernatants as indicated by residuals in the filter plate. A lack of filtrate can lead to false negative results in the following analytical modules.

The third task in automated screening is the removal of the remaining monomeric sugars from the growth media via a 96-well gel filtration. Centrifuge the gel filtration plates at 1, 000 x g and 20 degrees Celsius for two minutes. Prepare the worktable via the robotic manipulator for processing the gel filtrates.

To detect the remaining glucose after gel filtration, us 50-microliter tips to transfer 25 microliters of doubly distilled water to a fresh micro titer plate. Aspirate 20 microliters of doubly distilled water, five microliters of air, and 5 microliters of gel-filtrate, and dispense into the same plate. Take 200-microliter tips and aspirate 50 microliters of glucose-assay reagent-mix from worktable position 1-2 to start the first assay.

Move the micro titer plates into the incubator. After 30 minutes of incubation, move the plates from the incubator to the micro titer reader, and record absorbance at 418 and 480 nanometers. To determine total carbohydrate content by the phenol-sulfuric-acid method, pipette 20 microliters of gel-filtrate to the micro titer plates.

Manually remove the plates from the carousel and place them into the liquid handling station. Include the calibration plate with 20 microliters of different glucose concentrations in triplicates. Place a waste container at position 1, a 300-microliter tip box at position 2, and a 250-milliliter trough with 110 milliliters of freshly prepared phenol-sulfuric acid at position 3.

Use an 8-channel pipette with 300-microliter tips to transfer 180 microliters of phenol-sulfuric acid into each row of all the plates. Cover all micro titer plates with lids and mix by shaking for five minutes at 900 RPM at room temperature. Incubate for 35 minutes at 80 degrees Celsius in an oven for the color reaction.

After cooling down the plates under a fume hood, measure the extinction at 480 nanometers. Strains from the automated screening are designated as EPS-positive if they fulfill at least two of the following three criteria. Viscosity control, detection of polymer, and calculated glucose equivalent.

The glucose equivalent is calculated using this equation. The Carbohydrate Fingerprint of the screening platform contains the last three tasks which are manually performed. The remaining filtrate of the positive hits from task two provide the basis for the gel-filtrate in task five.

Task six is the 96-well micro hydolysis of the gel-filtrate. To accomplish this, first use a 12-channel 50-microliter pipette to transfer 20 microliters of gel-filtrate to a new PCR-plate. Then use a 1.25-milliliter multi-step pipette to add 20 microliters of four molar trifluoroacetic acid to each well.

Cover the PCR-plate with a thermoplastic elastomer cap mat, and place the PCR-plate in a special clamping device. Mix the solutions via inverting the clamping device 10 times. After centrifuging the PCR-plate, and securing it in the clamping device, placed the secured clamping device in a preheated sand bath and incubate at 121 degrees Celsius for 90 minutes.

Using a 12-channel 200-microliter pipette, add a 3.2%ammonia solution to adjust the pH to approximately eight. Cover the PCR-plate with a thermoplastic elastomer cap mat, and shake it manually using the clamping device. The final task is to analyze the Carbohydrate Fingerprint via the high-throughput-1-phenyl-3-methyl-5-pyrazolone, or HTPMP, method.

To begin this procedure, use a 12-channel 50-microliter pipette to transfer 25 microliters of the neutralized hydrolysate to a fresh PCR-plate. Add 75 microliters of derivatization reagent-mix and cover the plate with a thermoplastic elastomer cap mat. Place the PCR-plate in a PCR-cycler at 70 degrees Celsius for 100 minutes, followed by cooling down to 20 degrees Celsius.

Transfer a 20-microliter aliquot into a new micro titer plate, then use a 12-channel 200-microliter pipette to add 130 microliters of 19.23 millimolar acetic acid to each line. Mix directly via aspirating and dispensing at least six times, and transfer all the liquid to a 0.2-micrometer filter plate with a micro titer collector plate. Centrifuge the plate at 1, 000 x g for five minutes, remove the filter plate, and seal the micro titer collector plate with a silicone cap mat.

Place the micro titer plate into the UHPLC-UV-ESI-MS for the determination of the Carbohydrate Fingerprint. This table shows the results of three exemplary novel strains as successfully identified with this screening platform. The left part of the table displays the results of the automated screening modules concerning viscosity formation, polymer production, and the glucose equivalent from the total hydrolysis, which were used as evaluation parameters for detailed Carbohydrate Fingerprint analysis.

The Carbohydrate Fingerprint based on calibrated sugars as well as unknown sugars, dimers, and substituents are given in the right part of the table. By use of this information, the monomeric composition can be calculated and compared with already known polymer structures. Furthermore, a targeted screening for interesting monomeric compositions and rare carbohydrates can be performed.

Once mastered, this technique can be done for 386 strains within only five hours. Following this procedure, additional assays like the already existent pyruvate or further assays for acetates, or phosphate can be included in order to answer additional question of exopolysaccharide declaration. After its development, this technique, by its high sensitivity, paved the way for our research in the field of genetic engineered, microbial exopolysaccharide producers to explore the effect of altered exopolysaccharide biosynthetic pathways on the residing chemical structure.

After watching this video, you should have a good understanding of how to identify novel exopolysaccharide producers in a very fast and reliable way.