The Environmental Molecular Sciences Laboratory (EMSL) and the Joint Genome Institute (JGI) provide access to a variety of tools that leverage stable isotopes to measure carbon and nutrient flux, and identify microbes assimilating labeled compounds. During this webinar, EMSL’s Mary Lipton and the JGI’s Rex Malmstrom provided background on the various isotope-related technologies available at JGI and EMSL such as DNA-SIP, Protein SIP, Flux Balance analysis with metabolites, Isotope Ratio Mass Spectrometry, NanoSIMS, and other techniques.
Q&A from the Webinar:
1. Q(uestion): How much DNA is required for the JGI SIP metagenomics pipeline?
A(nswer): The more the better. We want to put at least a microgram of DNA in the tube so you’d need at least 2 micrograms in the sample to start with.
2. Q: What would a typical turnaround time be for data once samples are in hand at JGI?
A: It takes a few months to go from start to finish, i.e., QC to separation to library sequencing and assembly. Like others, our turnaround time has been impacted by the pandemic.
3. Q: What instrument / method does the JGI use to pre-screen DNA for enrichment?
A: The JGI uses liquid chromatography–mass spectrometry (LCMS). We do a hydrolyzation of the DNA and we look and quantify the isotopomers. We estimate the overall atom enrichment within the bulk community DNA, and that gives us a rough estimate of whether or not it’s worthwhile moving forward with density separation and fraction collection.
4. Q: Are there existing narratives, or plans to develop narratives in KBase for SIP studies? I’d be interested in how it could possibly be incorporated with some of KBase’s metabolic modeling narratives
A: KBase doesn’t currently have preconstructed SIP Narratives, but this is on the roadmap and will be piloted in the near future.
5. Q: How well-suited is the LA-IRMS for tracking N15? The example in the presentation was with C13.
A: Yes, EMSL can look at N15 as well as C13 as well as other isotopes using the IRMS. It’s just a matter of what you’re tuned to be looking for. we haven’t however worked out the ways to do that with the laser ablation IRMS so we don’t quite have the spatial resolution at tens of microns or 100 microns, whatever you want to call it. But NanoSIMS can do that at a really fine scale, and we can also easily measure the 15N at the bulk level, if that helps at all.
6. Q:How would field applications of the rhizoboxes mentioned in Jim Moran’s presentation work?
A: As long as we can harvest the sample, then we can put it into the laser system
7. Q: Can radioactive isotopes be used at EMSL?
A: No. We cannot take radioactive isotopes into EMSL and use them on the instruments because it has a long-term negative effect on utilization of those instruments.
8. Q: Related to Jim Moran’s talk, how long do plants need to be placed in a 13 CO2 chamber for sufficient labeling?
A: If you’re looking for the label in the plant, it could be as short as 24 hours. If you’re looking at the deposition into the rhizosphere, so the label has to go into the plant, out through the roots, into the soil, and then picked up by the rhizosphere, then that takes a little bit longer.
A lot of these questions are in some ways sort of experiment dependent. If you have a plant that’s in a very active growth phase, it would take potentially less time than a plant that’s beginning to go through senescence.
9. Q: With EMSL’s real-time trace gas monitoring, can this be conducted while you’re doing a 13CO2 plant labeling experiment, and if so–how do you resolve root respiration/soil respiration vs plant CO2 uptake?
A: We’ve accomplished this so far by physically separating the 13 CO2 that we’re using to label the leaves from the soil. So we have a separate air chamber around the leaves and then an air gap, regular atmosphere. It’s kind of hard around some stems of the live plant but we have an approach that we think that works. We currently don’t have a great way to resolve root respiration from microbial processes, but we’re not mixing the tracer with the CO2 produced at the end.
10. Q: Related to DNA-SIP, are there any special considerations for working with N15? Does the label need to be relatively stronger than C13?
A: Yes. Certainly, for DNA-SIP just because there are fewer nitrogen atoms than there are carbon atoms in DNA. So you need to get a little bit more labeling going on with nitrogen to see that separation. You can estimate from the relative amount of carbon and nitrogen in DNA and still try to target this density change equivalent to those found with >10% atom enrichment of 13C in your target organism in order to separate them by DNA-SIP.
For various isotope capabilities at EMSL, it depends on the capabilities. For speciation, protein-SIP, lipids, metabolites, it gets to the point where sometimes more label can be detrimental if you got up to too much, because it shifts your masses. And since we’re using a mass spectrometer to look at those masses, it makes it hard to identify the biomolecules in the mass spectrometer. If we’re using NMR, the opposite is true. The more the label we have, the better we can see it.
11. Q: Since Jim Moran was able to identify the major proteins that incorporated C13 and sort of the isotope of proteomics, what about doing this with metabolites at EMSL?
A: The answer to the question might also hinge on if you’re interested in a specific metabolite. If you could do some sort of targeted approach and you needed a lot of quantification, you could potentially link that with IRMS. A lot of people use MALDI (matrix-assisted laser desorption/ionization) imaging, but then you have to get the MALDI matrix into the soil and mix it up. There are likely some other imaging technologies that EMSL could use to target the metabolites.
12. Q: Can JGI work with labeled RNA?
A: Not right now but it’s on our radar.