Inside Silantes Lab

Silantes uses a cascade of biotechnology fermentations without any chemical synthesis steps to produce stable isotope labeled biomolecules for NMR and mass spectrometry applications. Utilizing a variety of fermenters, Silantes produces cell growth media, ready-to-use NMR standards, labeled nucleotides, phosphoramidites, amino acids and many other biomolecules. Our state-of-the-art instrumentation ensures excellent quality control.

StabIe isotope labeled biomolecules are used for applications in NMR and mass spectrometry. SI labeled amino acids are in particularly high demand for quantifying and analysing proteins by mass spectrometry.

Autotrophic Fermentation

In the fermentation shown in Fig. 1, Ralstonia eutropha (Cupriavidus necator) is the host strain of choice due to its facultative chemolithoautotrophic metabolism, which indicates that the organism is able to change its metabolic pathway according to the substrate available.

While both autotrophic and heterotrophic fermentations are possible, the latter is considerably more expensive due to the required organic carbon sources.

The metabolic end product of this fermentation is cyanophycin, which forms the base for the production of the stable isotope labeled amino acids arginine and aspartate.

SI labeled amino acids are of particular interest for quantitative proteomics applications with arginine used for the SILAC method in cell culture. A major advantage of this method is the fact that cells growing in the presence of labeled amino acids incorporate the label into their proteins without any perturbing effects on their metabolism

Silantes ¹³C labeling workflow benefits

Ralstonia eutropha is fermented in the presence of ¹³CO₂ and incorporates the label in all components of its metabolic pathways. One metabolic product is cyanophicin, which is an intermediate for the production of the amino acids arginine and aspartic acid.

With regard to the production of ¹³C-labeled substances, autotrophic organisms that utilize ¹³CO₂ as their sole carbon source, such as algea or Ralstonia eutropha, are very cost efficient. In addition to the fact that ¹³CO₂ is cheaper than ¹³C-labeled sugars, heterotrophs in comparison to autotrophs incorporate only a fraction of the label, with the rest lost due to respiration.

Article: https://journals.asm.org/doi/full/10.1128/AEM.01972-12

Zero waste principle

Once the Ralstonia eutropha fermentation is completed, the SI labeled biomass is introduced as an organic carbon source in a second cascade step of another heterotrophic fermentation to harvest additional complex biomolecules.

Green Compact

Silantes also uses green algae as another autotrophic production organism that utilizes photosynthesis to generate organic material with water, CO₂ and light. Plate or tube reactors are most commonly used for these applications. Silantes has designed the “Green compact” with incorporated glass rods that support the fermentation with illumination from the inside (Fig. 2-5). Compared to other fermenters the “Green compact” has an optimal space time yield, improved ergonomics,and increased robustness and stability.

Microalgae, in this case Chlorella vulgaris, have the ability to convert inorganic into organic compounds. When they are cultured in the presence of ¹³CO₂,¹⁵NO₃– or ²H₂O their biomass is labeled with the respective stable isotope, and a variety of stable isotope-labeled compounds can be extracted and purified.

Article: https://www.sciencedirect.com/science/article/abs/pii/0304416570900929

Silantes laboratories are equipped with state-of-the-art instrumentation to ensure proper quality control of our products. Here are some of the newly acquired instruments, their applications and functionality. 

Alpha II Fourier Transform IR Spectrometer

What is Infrared Spectroscopy?

Most molecules absorb light in the infrared range of the electromagnetic spectrum and then transform the absorbed energy into molecular oscillation. The absorbance is characteristic for type and strength of the chemical bond of the material. The resulting infrared spectrum represents a molecular fingerprint allowing the identification of unknown organic and inorganic samples.

What is FTIR Spectroscopy?

Instead of irradiating samples in sequence with single wavelengths, a slow and time-consuming process, all infrared wavelengths can be analyzed with only one measurement.

This interferogram depicts the light intensity not as function of the wavelength, but as a function of the position of a mirror located in the interferometer. Therefore, the signal first has to be fourier-transformed, to create the usual infrared depiction of light intensity as a function of the wavenumber. Advantages are a very good signal to noise ratio and a high wavelength accuracy.[1]

What does Silantes use the FT IR Spectrometer for? 

Silantes uses the Alpha II FT IR spectrometer to measure the purity of deuterium oxide (D₂O). The desired atomic purity is 99.9%.

D₂O, also known as heavy water, is applied in many biological and chemical processes. In biological processes, D₂O is used for metabolic labeling of biomolecules, which can then be analysed by NMR spectroscopy or mass spectrometry. Especially for NMR spectroscopy, D₂O is important for the production of deuterated solvents.

Another area of great potential is the deuteration of drugs. The pharmaceutical industry uses the selective replacement of hydrogen with deuterium to enhance the metabolic profile (e. g. increased bond strength) of drugs. Other areas of application are optical fiber technology and OLED.

Pfeiffer Mass spectrometer

For the process control of gas in fermentations Silantes uses a mass spectrometer (Figure 2). By measuring the mass/charge ratio of the gas components in a fermentation, one receives a fingerprint, which characterizes the gas of interest.

Such an analysis of residual gases provides valuable data on the state of the vacuum system. Calibration with a testing gas provides information about partial pressure and concentration of gas components. [2].

Two features of the instrument are of particular relevance for Silantes’ applications:

• The entire system has to be explosion proof, due to a hydrogen content of 80%.
• Different isotopes must be distinguished: ¹³CO₂ from ¹²CO₂ and D₂O from H₂O.

Labwit cooled shaker/ incubator

Preculture for cells

For cells to survive a long time outside of their natural environment, they have to be cultured in a customised medium. However, not all media are used with the same intent. Some are applied to ensure cells survival, others aim for unlimited cell growth. The latter has to contain the necessary nutrients for growth, such as amino acids.

The essential amino acid glutamine is of special importance for energy supply. The biosynthesis of glutamine requires a shaking incubator (Fig. 3), because the probecause the process is time and temperature dependent.

Such an analysis of residual gases provides valuable data on the state of the vacuum system. Calibration with a testing gas provides information about partial pressure and concentration of gas components. [2].In addition, Silantes uses incubators for the cultivation of algae. The Labwit incubator has microprocessor detection settings and PID control of both temperature and shaking speed, which guarantees maximum accuracy, uniformity and reliability [4].

In addition, Silantes uses incubators for the cultivation of algae. The Labwit incubator has microprocessor detection settings and PID control of both temperature and shaking speed, which guarantees maximum accuracy, uniformity and reliability [4].

Sources

[1] https://www.bruker.com/de/products-and-solutions/infrared-and-raman/ft-ir-routine-spectrometer/alpha-ii-compact-ft-ir-spectrometer.html
[2] https://www.pfeiffer-vacuum.com/de/produkte/messung-analyse/analysegeraete/gasanalyse/
[3] https://www.chemie.de/lexikon/Glutamin-Synthetase.html
[4] http://www.labwit.com.au/shaking_incubators.html

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