Learn how to use spectroscopy to measure and optimize the metabolic pathways in synthetic biology, and what are the challenges and solutions.

A specific isotope, often 13C, 15N, or 2 H, is introduced into a metabolic substrate. This isotope acts as a tracer, allowing scientists to track its movement and incorporation into various metabolites. In NMR spectroscopy, the magnetic properties of atomic nuclei (like those in the introduced isotope) are used to identify the structure of metabolites. When a metabolite incorporates the labeled substrate, its magnetic resonance signal changes, which can be detected by NMR. This technique is particularly useful for studying the dynamics of metabolic pathways in vivo. MS measures the mass-to-charge ratio of charged particles. When isotopically labeled substrates are metabolized, they alter the mass of the resulting metabolites.

How can you use spectroscopy to study metabolic flux in synthetic biology?

Synthetic biology is the engineering of biological systems to create novel functions and products. One of the key challenges in synthetic biology is to optimize the metabolic pathways that produce the desired molecules, such as biofuels, drugs, or materials. To do this, you need to measure how the flux of carbon and energy is distributed among different metabolic reactions and intermediates. This is where spectroscopy comes in handy.

Key takeaways from this article

Isotope tracing:

Use isotopes like carbon-13 as tracers in metabolic substrates to monitor their movement through different pathways via NMR or MS, revealing the dynamics of metabolic flux.
Consider homogeneity:

Be aware that uniform label distribution assumptions can oversimplify the system. Adjust for potential sample complexity and interference to ensure robust spectroscopic analysis results.

This summary is powered by AI and these experts

1 What is spectroscopy?

Spectroscopy is the study of how matter interacts with electromagnetic radiation, such as light, microwaves, or infrared. By exposing a sample to a specific wavelength or frequency of radiation, you can observe how it absorbs, reflects, or emits energy. This can reveal information about the structure, composition, and dynamics of the sample. For example, you can use spectroscopy to identify the chemical bonds, functional groups, or isotopes in a molecule.

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Dr Roy Awbery

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Spectroscopy, a powerful tool in organic chemistry and biological studies, faces challenges. In organic chemistry, complex spectra due to diverse functional groups require careful analysis. In biological studies, sensitivity issues limit detection in low-concentration biomolecules. Depth penetration constraints, sample interference, and the dynamic nature of biological systems add complexity. Advances in technology and interdisciplinary approaches help overcome these limitations, enabling researchers to push the boundaries and gain insights into intricate organic and biological landscapes.

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2 How does spectroscopy measure metabolic flux?

Metabolic flux is the rate at which metabolites are consumed or produced in a metabolic pathway. To measure metabolic flux, you need to track how a labeled substrate, such as glucose, is converted into different metabolites over time. The label can be an isotope, such as carbon-13, that has a different mass or nuclear spin than the natural abundance. By using spectroscopy, you can detect the presence and abundance of the label in different metabolites, and infer how the flux is distributed among different branches of the pathway.

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James Demmitt, MBA

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A specific isotope, often 13C, 15N, or 2 H, is introduced into a metabolic substrate. This isotope acts as a tracer, allowing scientists to track its movement and incorporation into various metabolites. In NMR spectroscopy, the magnetic properties of atomic nuclei (like those in the introduced isotope) are used to identify the structure of metabolites. When a metabolite incorporates the labeled substrate, its magnetic resonance signal changes, which can be detected by NMR. This technique is particularly useful for studying the dynamics of metabolic pathways in vivo. MS measures the mass-to-charge ratio of charged particles. When isotopically labeled substrates are metabolized, they alter the mass of the resulting metabolites.

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3 What are the types of spectroscopy for metabolic flux analysis?

Spectroscopy is an essential tool for metabolic flux analysis, with different types of spectroscopy being used depending on the type of label and the level of resolution required. Mass spectrometry (MS) is a popular choice, measuring the mass-to-charge ratio of ions in a sample to separate and quantify the isotopic composition of different metabolites and calculate fractional enrichment of the label. Nuclear magnetic resonance (NMR) is another technique, utilizing a magnetic field and radio waves to induce and detect spin transitions of different isotopes such as carbon-13 or hydrogen-2 to reveal the position and connectivity of the label in different metabolites, as well as providing structural information. Fourier transform infrared (FTIR) spectroscopy measures infrared absorption of molecules in a sample, using an interferometer and a detector to generate and record the infrared spectrum of different metabolites to identify functional groups and chemical bonds that contain the label.

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Dr Roy Awbery

Artist, children's author, illustrator, serial blogger, networker and innovator and occasionally a scientist who founded NuFor the nuclear forensics conference series.
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In addition to MS, NMR, and FTIR, other spectroscopic techniques could prove valuable in metabolic flux analysis. Raman spectroscopy, for instance, offers molecular vibrational information, complementing existing methods. Its ability to provide insights into molecular dynamics and structural changes enhances the understanding of metabolic pathways. Additionally, UV-Visible spectroscopy, sensitive to electronic transitions, can be employed to monitor specific chromophores, offering a different perspective on metabolite changes. Integrating these techniques expands the analytical toolkit, providing a more comprehensive view of metabolic processes.

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4 What are the advantages and limitations of spectroscopy for metabolic flux analysis?

Spectroscopy offers several advantages for metabolic flux analysis, such as high sensitivity and specificity, non-invasive and non-destructive measurements, and multiplexing and parallelization capabilities. However, spectroscopy also has some limitations, such as high cost and complexity, low spatial and temporal resolution, and interference and noise. Spectroscopy can detect and distinguish very low concentrations of labeled metabolites, and discriminate between different isotopes and molecular structures without requiring any chemical or physical modification of the sample. It can also measure multiple metabolites and pathways simultaneously to perform high-throughput analysis of many samples. On the flip side, spectroscopy requires expensive and sophisticated instruments and software, as well as skilled operators and analysts. Additionally, it cannot capture the spatial distribution and temporal dynamics of metabolic flux at the cellular or subcellular level, often requiring long sampling times and large sample volumes. Furthermore, it may be affected by the background signal, the matrix effect, or the overlapping spectra of different metabolites, necessitating calibration and normalization.

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Dr Roy Awbery

Artist, children's author, illustrator, serial blogger, networker and innovator and occasionally a scientist who founded NuFor the nuclear forensics conference series.
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This assumes uniform label distribution in metabolic networks, potentially oversimplifying dynamic biological systems. A limitation lies in the sensitivity of certain spectroscopic methods, impacting the detection of low-abundance metabolites. Advantages include the precision of mass spectrometry in quantifying isotopic compositions and the non-destructive nature of nuclear magnetic resonance for structural insights. However, assumptions about homogeneity and potential interference in complex samples should be considered for more robust analyses.

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5 How can you improve spectroscopy for metabolic flux analysis?

There are several ways to improve spectroscopy for metabolic flux analysis, such as designing and synthesizing new isotopic or fluorescent labels and tracers with higher specificity, stability, and compatibility. Additionally, you can adjust the parameters and settings of the spectroscopic instruments and methods to enhance the signal-to-noise ratio, resolution, and accuracy of the measurements. Furthermore, advanced data analysis and modeling tools can be used to process, interpret, and integrate the spectroscopic data, as well as construct and validate metabolic flux models and simulations.

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6 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?

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How can you use spectroscopy to study metabolic flux in synthetic biology?

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1 What is spectroscopy?

2 How does spectroscopy measure metabolic flux?

3 What are the types of spectroscopy for metabolic flux analysis?

4 What are the advantages and limitations of spectroscopy for metabolic flux analysis?

5 How can you improve spectroscopy for metabolic flux analysis?

6 Here’s what else to consider

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