Stable isotope probing (SIP)

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If you feed isotopically-enriched substrates to an organism, the biomass of that organism will in turn be enriched in those same isotopes. Some of the labeled molecule are phylogenetically informative, e.g. the rRNAs, and so by separating and sequencing these molecules you should be able to determine who, in a mixed culture, is eating the labeled substrate. Because this process usually uses non-radioactive isotopes, it is called stable-isotope probing, or SIP.

For example, if you feed 13C-labeled phenol to an environmental sample in which you know phenol is being degraded (for example in a wastewater aerobic digestor), the organisms that take up and use this phenol will end up with 13C-labeled rRNAs. These can then be separated from the unlabeled rRNAs because of their higher density in cesium density gradients, PCR amplified, cloned (or separated by DGGE), and sequenced. This process has been used a great deal to identify organisms resonsible for degrading pollutants (using the 13C-labeled pollutant compound), to identify primary producers (using 13-CO2), to identify organisms in carbon cycle bottlenecks (e.g. propionate-turnover in rice patty sediment), and organisms in key steps of the nitrogen cycle (using 15N-labeled nitrate, nitrite, or ammonium).

Grow population with labeled substrate added,...

isolate RNA and separate heavy and light RNAs in Cs-TFA gradients,...

do reverse transcriptase PCR to amplify ssu-rDNA from both types of RNA, ...

compare heavy and light rDNAs by DGGE (or tRFLP),...

and finally sequence the rRNAs of interest & study phylogenetically.

How cesium tetrafluoroacetate density gradients work

The key to this process is to get the heavy isotope label into the organsisms, and then separate labeled & unlabeled molecules - molecules with phylogenetic information. How does this separation by density work? By using cesium density gradient centrifugation, and in specific, usually Cs-TFA ultracentrifugation.

TLA 100.2 rotor
A TLA 100.2 rotor, capable of carrying as many as 10 4ml samples at 435,000 x g at 100,000 RPM. (JWBrown)

Solutions of cesium salts will form stable density gradients when subjected to very high gravitational fields in a centrifuge for many hours - more concentrated (and therefore more dense) at the bottom, less concentrated (less dense) at the top. In other words, although still in solution, cesium ions "settle" somewhat in very high g forces. If the solution also contains macromolecules, these will float up or sink down to form bands where the densty of the macromolecule matches the density of the surrounding cesium solution. RNA binds lots of cesium, and is therefore fairly dense in cesium solutions - about 1.7 g/cm3. DNA binds less cesium and so has a lower density of about 1.5, whereas protein doesn't bind much cesium at all and so has a density of 1.1 or 1.2. Within these regions, the molecules will separate slighty based on small differences in their density, because of differences in their base composition or because of differences in their isotopic composition. In other words, molecules (in this case RNAs) with a higher fraction of heavy-isotope atoms will settle into bands that are just a little bit lower in the gradient that those with lighter isotopes.

The tetrafluoroacetate salt is used rather than chloride or sulfate (often used for other kinds of cesium density gradients) because it is more soluble. In order to band RNAs (with a density of about 1.7) in about the middle of a gradient, the density of the solution at the bottom might be 1.8, well above the solubility of CsCl, or even (depending on the details) that of Cs2SO4. If the salt begins to precipitate at the bottom of the tube, it will initiate a chain reaction in which the solid salt forms a large, heavy knob at the bottom of the tube, unbalancing the rotor. These rotors typically spin at 50-100KRPM, in a vacuum because the outer parts of the rotor are traveling supersonically. An off-balance rotor will will tear the centrifuge apart, fracture the rotor, come off the motor spindle, or fail in any of several other equally catastrophic ways. Ultracentrifuges have armour plates on all sides for this very reason, but a rotor failure is nevertheless dangerous, expensive, and messy. A great description of a typical rotor failure can be read at: http://www.ehs.cornell.edu/chem_lab_safety/centrifuge_safe.cfm