SIP Flashcards
What is SIP and what is it used for?
Stable isotope probing, a cultivation-independent technique that allows the identification of a group of microorganisms that are actively metabolising a substrate
When was SIP first used and what was it used to demonstrate?
1998, to demonstrate the importance of type I methanotrophs in methane mineralisation in lake sediments through the analysis of PLFAs that had incorporated 13C from 13CH4
(Methanotrophs important in controlling methane emission)
What has the original method been modified for analysis of?
DNA (2000)
RNA (2002)
Proteins (2008)
What is SIP typically performed with
i) Enriched cultures (natural or artificial communities) in the lab under conditions that try to mimic the natural environment
ii) in situ with natural communities
What is the general method of SIP?
Incubate culture with isotopically-lablled substrate, during which time the isotope is incorporated into the biomolecules of microorganisms actively degrading and utilising the subtrate
The labelled biomolecules are then extracted and analysed to identify which microorganisms had incorporated the isotope label
What is the method of DNA-SIP?
Apply a pulse of 13C, 15N, 18O to culture
Light and heavy DNA separated by CsCl density gradient ultra-centrifugation
Separated DNA is fingerprinted using various techniques, e.g. clone library construction; T-RFLP or DGGE with post-sequencing; pyrosequencing or MiSeq, to identify the microbial taxa that incorporated the label into newly synthesised DNA
What are the steps in CsCl density gradient ultra-centrifugation?
DNA placed in tube containing a solution of CsCl and ultra-centrifuged
CsCl will dissociate, heavy Cs+ ions will migrate to the bottom of the tube creating a gradient of Cs+ ions from low to high concentration own the tube
DNA of different densities will migrate to and position at a point that is the same density as the gradient i.e. neutral buoyancy
To visualise DNA, a DNA stain would have been added before ultra-centrifugation
DNA bands collected by a syringe or as fractions at the bottom of the tube
What are the issues of DNA-SIP?
To improve labelling efficiency, a highly labelled substrate and higher than in situ substrate concentration are used
This high concentration may inhibit some microbial groups in the community and not be realistic of natural environment conditions
Longer incubation times are required because label incorporation into DNA is dependent on cell replication
Longer incubation can lead to cross-feeding which is consumption of a metabolic by-product of the labelled substrate by a microbial group(s) that is not involved in degrading the original labelled “parent” substrate, resulting in incorporation of 13C label into other microbial groups
How can issue with DNA-SIP be resolved?
Performing time-resolved experiments:
1) Follow degradation over time
2) quantify the enrichment of microbial groups detected in the extracted heavy DNA e.g. by qPCR
3) by performing SIP analysis of DNA extracted at different time points throughout the experiment
What is the RNA-SIP methodology?
Similar to DNA-SIP.
Uses CsTFA gradient solution rather than CsCl because buoyant density of RNA is higher than DNA
Separated RNA is converted to cDNA by reverse transcription
The 16s rRNA can now be amplified and fingerprinted
What are the advantages of RNA-SIP?
High phylogenetic resolution
Higher sensitivity than DNA-SIP
Shorted incubation times than DNA-SIP because RNA turnover is a continuous process that is independent of cell replication
What are the limitations of RNA-SIP?
Tendency for RNA to self-associate and potentially precipitate thereby creating technical difficulties in resolving light and heavy communities
What is the methodology behind PLFA-SLIP?
Incubate sample with 13C-labelled substrate
Extract total PLFA
Analyse PLFAs by GC-C-IRMS
Data from detector is analysed by computer which identifies each PLFA detected and level of 13C labelling
Comparison to lipid profiles of characterised organisms on PLFA databases
What are the advantages of PLFA-SLIP?
High sensitivity
Can use in situ substrate concentration
Amenable to field studies
No separation of labelled and unlabelled molecules required
What are the disadvantages of PLFA-SLIP?
Low phylogenetic resolution
Poor quality and availability of databases of phylogenetic PLFA profiles
Inability to identify novel organisms
Limited to 13C substances
