Project
Functional Microbiomics: Elucidation of the Functionality of the GI-tract Microbiota
The mammalian gastrointestinal tract (GI-tract) harbours a complex microbial ecosystem consisting of bacteria, fungi and yeasts; also called the microbiota. This intricate microbiota has been studied in great detail by anaerobic cultivation techniques. These studies have revealed that 300-500 different species of bacteria can be detected in an individual's GI-tract. Since the anaerobic bacteria outnumber the aerobic bacteria by a factor of 100-1000, cultivation on anaerobic selective media has long been a conventional method.
With the increase of available molecular techniques many methods to determine the microbiota composition have been developed that circumvent the traditional cultivation. Especially techniques based on 16S rRNA genes (rDNAs) amplified directly from the complex community provide a very efficient strategy to explore the biodiversity of the human gut. The information about the genetic capacity of the entire microbiota doesn't provide any information about the actual functionality of this microbiota. The functional efficacy of a group of organisms within this community is determined by factors like numerical abundance, competitiveness, metabolic activity and survival. Within this PhD project I'll try to elucidate the actual functionality of the GI-tract microbiota.
Aim
The objective of this project is to develop and evaluate high throughput approaches based on DNA or RNA that can be used as biomarkers for the microbial activity in the GI-tract.
Research Approach
The focus will be mainly on transcription studies based on metagenome analysis: the construction and analysis of a fosmid library of DNA isolated from adult faecal samples and DNA array of ileal perfusion effluent and ileostomy samples.
Fosmid library
Previous researchers of our group have started the construction of a metagenomic library from DNA extracted from GI-tract samples in E. coli. This part of the fosmid library will be tested for a number of approaches, like a multiplex PCR approach by the use of for instance ITS (Internal Transcribed Spacer) primers. This is the region between the 16S and 23S ribosomal genes. Biochemical analysis of the clones is another approach that can be tested. If a number of these approaches prove to work for the metagenomic library constructed so far, the construction of the library will be continued until the total metagenome is covered in the library (+ 16,000 clones). The availability of the complete metagenomic library may lead to the discovery of new genes and metabolic pathways and it will provide a more accurate identification of the microorganisms in the GI-tract, and hopefully a better understanding of microbial genetic diversity.
DNA array
Within the field of microbial ecology of the GI-tract, the colon is the most studied organ. The reason for this is that the availability of in vivo samples of the colon is much higher in comparison to the organs preceding the colon. Another reason is that investigation of easy to acquire faecal samples can be adopted to gain knowledge about the distal part of the GI-tract; the colon. Due to these facts, knowledge about the bacterial community of the ileum is very limited. In cooperation with a new member of WCFS, Nutrim, it will be possible to obtain ileal perfusion samples and ileostomy samples. The former samples can be taken from healthy volunteers while doing their normal activities, thus resulting in representative in vivo samples
of the ileal microbiota. The latter samples are from patients without a colon and an ileostomy in the distal part of their ileum. This ileostomy makes sampling rather easy although it should be taken into account that the microbiota of ileostomy patients will change over time towards a high resemblance to the colonic microbiota. The composition of the ileal community and its gene expression will be investigated with DNA microarray.
Researcher
Carien Booijink