Methods

The Orphan Lab has pioneered the application of nanoscale secondary ion mass spectrometry (nanoSIMS) to problems in microbial ecology. Using stable isotopes as probes, and using nanoSIMS to quantitatively measure isotopic abundances within intact microbial samples with nanometer resolution, we answer questions such as, "How active are microbes within their native environments? How does the spatial organization of microbes affect their activity? Which nutrients do microbes metabolize and cycle through the ecosystem?" There are currently only about 50 nanoSIMS instruments around the world, one of which is housed here at the Caltech Microanalysis Facility.

Bio-orthogonal non-canonical amino acid tagging (BONCAT) was collaboratively developed in the laboratories of Erin Schuman (Max Planck Institute for Brain Research) and David Tirrell (Caltech); (Dietrich et al. 2006, PNAS) and adapted by our lab to enable rapid and inexpensive fluorescence-based identification of translationally active uncultured microbes (Hatzenpichler et al 2014, Environ. Microbiol and Hatzenpichler et al 2016 PNAS) and newly produced viruses (Pasulka et al 2018 Environ. Microbiol; and Martinez-Hernandez et al. in prep) directly in diverse environmental samples. BONCAT is now used routinely in microbial ecology and geobiology labs globally. Here, a non-canonical methionine surrogate is introduced into environmental samples and incorporated into newly synthesized proteins using the native translational machinery of the cell. After incubation, translationally active cells and viruses can be fluorescently labeled through click chemistry and viewed by microscopy or alternatively recovered for downstream sequencing using fluorescence activated cell sorting. This approach is also compatible with environmental proteomics.

The intense light generated by synchrotrons enables the examination of materials and organisms at resolutions and in modes that are otherwise unattainable. We use X-Ray Fluorescence (XRF) and X-ray Absorption Near Edge Spectroscopy (XANES) to analyze the elements and species within microbial cells and their habitats in order to answer fundamental questions about how cells store, share, and cycle nutrients.

The Orphan Lab employs a proteomic approach to better understand the relationship between genes and function in microbial communities. Using techniques such as stable-isotope labeling and BONCAT, we identify actively synthesized proteins in energy-constrained systems. Through these approaches, we can determine proteins and pathways that are critical for community homeostasis and response to environmental stimuli.

Incubating microbes at high pressure requires specialized equipment. In the Orphan Lab, portable 1.4 L high-pressure vessels are used. These aluminum-alloy cylindric containers are capable of creating hydrostatic pressure up to 6000 psi (about 410 atm or 41 MPa) using an oil reservoir and hydraulic pump to compress the sample within the vessel. Temperature is controlled by placing the vessel within a cold room to mimic in situ temperature conditions.

The Orphan Lab also uses a variety of other resources on campus for analysis and imaging of microbial and mineral samples. These methods include infrared and Raman microscopy, bulk X-ray fluorescence spectroscopy, SEM, benchtop XRF imaging, and X-ray powder diffraction. More information about these instruments and resources can be found below.