Core 1: Genetically-Defined Microbe Core - Gastroenterology & Hepatology

Director: John Kappes, Ph.D.

Co-Director: Charles O. Elson, M.D.

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Requests from Center investigators will be accepted on a first-come, first-served basis, subject to the requirement that the research be appropriate for the Center project and likely to contribute to its success.

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Microbiota Microarray List April, 2012

	Relation to Human Microbiome Project genome sequences.
Code	% identity	Name	function	Primary Species	Phylum	Class
rIB19	36	UvrD/REP helicase	transc/ transl	Propioni-bacterium acnes	Actinobacteria	Actino- bacteria
14-2	80	isolate 14-2 surface extract – flagellin	motility	Roseburia intestinalis	Firmicutes	Clostridia
3_1_57	100	Flagellin	motility	Lachnospiraceae 3_1_57	Firmicutes	Clostridia
BF30S		30S riboprotein	transc/ transl	Bacteroides fragilis	Bacteroidetes	Bacteroidia
Btheta	87	hypothetical protein	other/ unknown	Bacteroides thetaiotaomicron	Bacteroidetes
CBir1	83	Flagellin	motility	Butyrivibrio fibriosolvens	Firmicutes	Clostridia
CBir11	46	hypothetical protein/ flagellin	motility	Roseburia inulinivorans	Firmicutes	Clostridia
CBir14	90	GAPDH	metabolism	Lactobacillus salivarius	Firmicutes	Bacilli
CBir19	78	ABC transporter.ATP-binding protein	other/ unknown	Bacteroides vulgatus	Bacteroidetes	Bacteroidia
CBir23	85	elongation factor 1A	transc/ transl	Alistepes shahii	Bacteroidetes	Bacteroidia
Cbir45	59	hypothetical protein/ glycosyl hydrolase	metabolism	Bacteroides eggerthii	Bacteroidetes	Bacteroidia
CBir5	63	methyl-accepting chemotaxis protein	motility	Helicobacter cinaedi	Proteobacteria	Delta/ Epsilon
CBir56	60	methyl-accepting chemotaxis protein	motility	Helicobacter canadensis	Proteobacteria	Delta/ Epsilon
CBir63	47	Ig-like surface protein/ hypothetical protein	other/ unknown	Roseburia intestinalis	Firmicutes	Clostridia
CBir66	66	Flagellin/ hypothetical protein	motility	Roseburia intestinalis	Firmicutes	Clostridia
CBir8	90	elongation factor Tu	transc/ transl	Tannerella sp.	Bacteroidetes	Bacteroidia
CT-B	n/a	cholera toxin B subunit	other/ unknown	Vibrio cholerae	Proteobacteria	Gamma
EF20	100	Sal A	other/ unknown	Enterococcus faecalis	Firmicutes	Bacilli
Fla 2	72	flagellin 2 from A4 isolate	motility	Roseburia intestinalis	Firmicutes	Clostridia
Fla 3	81	flagellin 3 from A4 isolate	motility	Roseburia inulinivorans	Firmicutes	Clostridia
Fla X	56	flagellin	motility	Roseburia inulinivorans	Firmicutes	Clostridia
Flic2	0	flagellin	motility	Candidatus arthromitus (SFB)	Firmicutes	Clostridia
Keto			metabolism	Bacteroides fragilis	Bacteroidetes	Bacteroidia
MDR247	54	adenine deaminase	metabolism	Clostridium bolteae	Firmicutes	Clostridia
MDR254	73	flagellin protein	motility	Flavonifractor plautii	Firmicutes	Clostridia
MDR90	66	collagen adhesion protein	other/ unknown	Roseburia intestinalis	Firmicutes	Clostridia
OmpC	100	OmpC from UNC101 E. coli	other/ unknown	Escherichia coli	Proteobacteria	Gamma
rIB10	48	Nucleotidyltransferase/ hypothetical	transc/ transl	Roseburia intestinalis	Firmicutes	Clostridia
rIB12	60	homoserine dehydrogenase	metabolism	Flavonifractor plautii	Firmicutes	Clostridia
rIB13	98	protease IV /SppA	other/ unknown	Bacteroides vulgatus	Bacteroidetes	Bacteroidia
rIB14	84	helicase	transc/ transl	Ruminococcus sp	Firmicutes	Clostridia
rIB15	51	amidohydrolase	metabolism	Clostridium difficile	Firmicutes	Clostridia
rIB16	74	pyruvate synthase	metabolism	Flavonifractor plautii	Firmicutes	Clostridia
rIB17	39	NlpC/P60	other/ unknown	Flavonifractor plautii	Firmicutes	Clostridia
rIB18	30	surface array protein	other/ unknown	Campylobacter showae	Proteobacteria	Epsilon
rIB2	61	SAM domain protein	transc/ transl	Blautia hansenii	F&B
rIB20	0	ABC transporter	other/ unknown	Verminephrobacter eiseniae	Proteobacteria	Beta
rIB4	63	relaxase	transc/ transl	Clostridium asparagiforme	Firmicutes	Clostridia
rIB5	37	hypothetical protein – cytoplasmic	other/ unknown	Erysipelotrichaceae 3-1-53	F & P, chordata	Mollicutes
rIB6	47	hypothetical protein/ bIF-2	transc/ transl	Clostridium leptup	Firmicutes	Clostridia
rIB7	76	malonyl CoA carrier	metabolism	Bacteroides sp.	Bacteroidetes
rIB8	59	hypothetical protein/ glycosyltransferase	metabolism	Lachnospiraceae 3-1-57	Firmicutes	Clostridia
rIB9	45	methyl-accepting chemotaxis protein	motility	Roseburia intestinalis	Firmicutes	Clostridia
SFliC	n/a	Salomonella dublin Flagellin	motility	Salmonella dublin	Proteobacteria	Gamma
UFliC	100	FliC from UNC101 E. coli	motility	Escherichia coli	Proteobacteria	Gamma


Proteins are resuspended at 0.2 mg/ml in 1mM Tris, 0.1% SDS. The proteins are then be printed onto FAST 16pad slides (Whatman) using a MicroGrid II robot (Genomic Solutions). The printed slides are allowed to air-dry over night, then blocked with Protein Array Blocking Buffer (Whatman). Individual pads on the array are incubated with sera and secondarily with Alexa 647-anti-human IgG and Alexa 546- anti-human IgA. The resulting fluorescent slides are analyzed using a Genepix 4000B dual laser microarray reader (Molecular Devices) and Genepix Pro 6.0 software.

The software determines the net median pixel intensities for each individual feature (antigen spot) from a set of 10 measurements/feature. A median net digital fluorescence unit (DFU) for each feature represents the median values from 8 replicate antigen features on each array. A software program called “Significance Analysis of Microarrays” (SAM) (14, 23), which was developed at Stanford University will be applied to the data to identify antigens with statistically significant differences in antibody reactivity between groups. SAM computes a statistic for each feature, measuring the strength of the relationship between the feature intensity and the group. The software estimates a false discovery rate (FDR), i.e., a false positive rate, for each antigen by permuting the repeated measurements between groups. SAM results can be further analyzed using software called Cluster and Cluster results can be displayed using Tree View.

Retroviral Unit (Dr. Kappes)

Molecular construction of gene expression vectors (preparation of DNA; restriction analysis of DNA; insertion of gene; confirmation of gene expression; large-scale [Maxi prep] production of DNA.
Validation of vector trans-gene expression (DNA transfection and confirmation of gene expression.
Packaging of the vector and cryopreservation of the virus vector stock (preparation of DNAs for packaging and Env constructs; transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).
Concentration of the virus vector stock (large-scale transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).
Generation and titration of GFP-virions (preparation of DNA; large-scale transfection of mammalian cells; collection and preparation of culture supernatant [virus vector]; cryostorage).
Generation of HIV-1 stocks.
Infectivity titration of HIV-1 stocks.
Production of a stable gene-expressing cell line (packaging of vector DNA; transduction of mammalian cells; selection).
Production of a clonal gene-expressing cell line (packaging of vector DNA; transduction of mammalian cells; selection; limiting dilution cloning; expansion of [~25] clones).
Production of a shRNA-expressing cell line.
Confirmation of gene-expression cell line (analysis of cell line for trans gene expression by FACS or immunofluorescence, etc.).

Bacterial Unit (Dr. Elson)

Analysis of bacterial flora via 16S ribosomal DNA for validation of monocolonization or germ-free status (PCR and agarose gel; PCR and DGGE; Elution of bands, cloning, and sequencing).
Collection, 16s RNA characterization and cryopreservation of bacteria.
Cloning of genes for bacterial expression (PCR, cloning, screening, sequencing).
Expression and provision of bacterial protein relevant to inflammatory bowel disease, per protein.
Quantitation of microbial groups, for up to 25 samples, by quantitative PCR.
Printing antigen microarrays, per slide.

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