|
|
|
Facilities and Resources
The Center for Genomic Sciences (CGS) is a research center within
Allegheny-Singer Research Institute. CGS has over 15,000 sq. ft (1500 sq.
meters) of laboratory space within the Allegheny-Singer Research Institute
(ASRI). These labs are located in recently renovated space on the 9th, 10th
and 11th floors of the South Tower of Allegheny General Hospital, the
teaching hospital associated with ASRI in Pittsburgh. The administrative and
executive offices of CGS, in which the PI and Co-PI have their offices, have
recently been relocated to a suite of newly renovated offices on the 11th
floor. This suite also contains a library/conference unit which can
configured as one large room or two smaller rooms depending on need for
meetings and research.
The CGS is a state-of-the-art facility for gene discovery, molecular
microbiology, functional genomics, PCR, comparative expressomics, molecular
diagnostics and confocal imaging. The facility has laboratories specifically
designed for PCR setup and amplification, automated sequencing and fragment
analysis, genetic linkage analysis, protein purification, biochemistry, differential
display, RNA and DNA array technology, cloning and gene expression, tissue
culture, planktonic bacterial growth, and bacterial biofilm growth. An
electronic data network digitally links all personnel, laboratories, and data
collection systems.
One of the most important renovation items involved re-engineering the
heating, ventilation, and air conditioning (HVAC) of wings on the 10th and
11th floors that permit controlled air flow in all of the laboratories. All
of the pre-PCR laboratories have positive pressure, and are physically
removed from the post-PCR labs which have negative pressure with respect to
the hallways. This HVAC system was designed to reduce to an absolute minimum
the incidence of false positive results caused by molecular carryover from
aerosolized PCR-amplified DNA.
Each laboratory in the CGS is used for a particular purpose with respect
to the overall goal of obtaining high-quality, accurate scientific results.
Special attention has been paid to the needs of PCR and other molecular
biological work requiring template amplification. Samples move through the
system in an orderly, unidirectional way, and individual investigators must
adhere to this policy. Certain procedures are done in each laboratory and the
sample is then transferred to the next.
Each laboratory is completely equipped and self-contained. There is no
trafficking of supplies, reagents, or equipment between labs. This is
particularly true for pipetting devices, which, if used indiscriminately, are
a likely source of contamination. All items are clearly marked as to which
laboratory they belong in and all workers are instructed to vigilantly check
and adhere to this policy.
Main laboratories
Imaging Facility. The new confocal imaging facility at the
Center for Genomics Sciences (CGS) is dedicated primarily to the study of
bacterial biofilms and human disease. The facility combines confocal scanning
laser microscopy and real-time time lapse imaging of bacterial biofilms
growing in situ without having to fix or dehydrate the sample
significantly reducing artifacts. The new CGS Imaging Facility will improve
our ability to investigate dynamic, living biofilms in situ in three
dimensions and in real time.
Specimen processing laboratory. This laboratory serves as the
sample collection, logging-in, processing and aliquotting facility. It
contains the tissue culture facilities which include three laminar flow
hoods, three sets of humidified CO2 incubators, a refrigerated clinical
centrifuge, a microfuge, and a kitchen area supplied with all necessary
equipment for reagent preparation. Aliquots from each specimen (except one
used for the initial assessment) are stored in this room at -80°C. This is a
"clean room"; no plasmid or amplified DNA of any kind is allowed in
it under any circumstances. No one who has handled amplified DNA without
having showered, shampooed, and changed clothing is allowed in this
laboratory at any time.
PCR setup laboratories. Two additional laboratories are used for
DNA/RNA extractions and PCR setups; one is for infections disease work and
the other for human genomics projects. In some cases individual aliquots
prepared in the tissue culture room may be brought into these labs,
particularly when lysis is being performed on one of the robotic
workstations. These are also considered "clean" laboratories and no
amplified DNA is ever allowed in them. In some cases the DNA preps are stored
here; in other instances they are used immediately to set up PCR reactions.
Any individual who has handled amplified DNA must shower and shampoo before
entering this lab. These labs contain: Hybaid thermal cyclers for RT
reactions, pipetmen, multichannel pipettors, electronic pipettors, electronic
multichannel pipettors, pipette aids, Sorvall refrigerated clinical
centrifuges; a Jouan refrigerated microfuge, 3 personal minimicrofuges, a
Savant speedvac; a Beckman UV-vis spectrophotometer; a Perkin Elmer LS 50
spectrofluorimeter, a two bay sliding glass door refrigerator which houses
the CGS working stock DNAs, three -20° C and a -80° C freezer.
PCR amplification laboratory. This lab houses six Perkin-Elmer 9600
thermal cyclers and a MJ research tetrad thermal cycler the latter outfitted
with both 96 and 384 well heads; it is considered a "dirty lab" and
individuals cannot go from this lab to the pre-PCR labs without showering and
shampooing. This lab is also approved for radioactive use and is equipped
with GM counters and a Bioscan instrument (radioactive labeling work is done
under the authority of the ASRI radiation safety committee, which inspects
the laboratory weekly). Most of the amplifications associated with the
infectious disease work and genotyping are performed in this laboratory. All
of the ID PCR analyses (by liquid hybridization) are also performed in this
laboratory on vertical electrophoresis gel rigs specifically designed by Dr.
Ehrlich for this purpose. In addition, this lab is used for storage of
amplified DNAs that are performed in this room. It contains: a two-bay
sliding glass door refrigerator, a -20° C freezer, eight power supplies, and
myriad electrophoresis rigs. This lab also has its own kitchen area for gels
and buffers as well as tabletop centrifuges and microcentrifuges, and water
baths. Nothing ever goes from this room into any clean lab. Any items for
further analysis in the darkroom or the gene mapping and sequencing
facilities are thoroughly wrapped prior to leaving the lab. Any items to be
disposed of are bagged and immediately removed, and are not allowed to sit in
the hallways outside other labs.
Cloning, gene expression and biochemistry facility. This facility
includes several labs and all of the equipment and reagents for bacterial,
yeast, plasmid and phage growth; library construction and subcloning; and
colony screening and hybridizations. This facility also contains the systems
for production and characterization of recombinantly expressed proteins from
bacteria and yeast and contains microbial culture equipment, centrifuges, a
refrigerator, and a kitchen area for the preparation of media used in this
laboratory and the cloning laboratory. Major equipment includes 2 two-bay
refrigerators; a refrigerated microcentrifuge; a tabletop centrifuge; -20°C
freezer; high speed centrifuge; New Brunswick shaking incubator; two 37°C
plate incubators; a CO2 incubator for pathogenic bacteria; electrophoresis
rigs and power supplies; as well as a selection of single and multi-well,
manual and electronic pipetting devices. This lab also houses the
Perkin-Elmer UV-Visible spectrophotometer and a new Perkin Elmer LS-50B
spectrofluorimeter, column chromatography equipment, a Waters HPLC system
with an auto-sampler, and refrigerated and heated water baths. All non-coldroom-based
biochemistry is performed in this laboratory, including protein purification,
fraction analyses, enzyme kinetics, and enzyme activation and degradation
studies.
|
|
Fluorescent fragment analysis laboratories. These three
laboratories (one each on the 9th, 10th, and 11th floors) collectively house
over $1 million worth of state-of-the-art automated genomic and expressomic
analytical equipment including: 1) two new Beckman capillary electrophoresis
automated DNA fragment analyzers; 2) three Applied Biosystems 377 PRISM
automated DNA sequencers with fragment analysis software and hardware
packages, each with its own Macintosh computer, upgrades for Genescan
genotype sequencing, and the running of variable-size gels; 3) three LiCor
automated fragment analysis instruments for DNA sequencing and mapping with
two computer/software systems; 4) the Genomyx (Beckman) automated fluorescent
differential display system which includes an automated electrophoresis/gel
drying system, multiple sequencing gel pouring system, a gel band excision
system and the automated cooled CCD camera-based fluorescent reader; 3
Sorvall refrigerated clinical centrifuge; a Jouan refrigerated microfuge, a
personal minimicrofuge, a Savant speedvac; 5 two-bay sliding glass door
refrigerators, a -20° C; and a full complement of pipettes and minor lab
equipment.
|
|
Gene Array Facility: This facility is composed of 4 labs and is
equipped with a Genetix Q bot (see figure) which is the only robot that can:
1) pick colonies (w/ color selection) and plaques, 2) grid DNA or bacteria
onto filters, 3) grid DNAs onto glass slides to make microarrays, 4) open and
close microtiter trays, and 5) re-grid cultures and automatically re-array
after normalization. This facility also houses: 1) a new state-of-the-art
Beckman FX 2-arm liquid handling robot with crossover capability for
massively parallel: plasmid preps for microarrays; PCR clean up; DNA
sequencing rxn set up and clean up; and genotyping set up. The FX is
currently being equipped with: 1) an integral 96-well Tecan fiber-optic
spectrofluorimeter for DNA quantitation which will be integrated with the CGS
laboratory relational database GENOMICS; and 2) a high speed microplate
centrifuge. This facility is also equipped with a Packard (GSI Lumonics) ScanArray
4000XL for multicolor (up to 5 lasers- currently configured with 3 lasers)
analysis of microarrays; and 3) a Cephied real time quantitative thermal
cycler - a Smart Cycler for verification of differential gene expression. The
Cephied thermal cycler has individual control for every PCR chamber.
Additional equipment includes ultra centrifuges, superspeed centrifuges,
vacuum centrifuges, microcentrifuges, controlled temperature rotary
hybridization ovens, roller bottle incubators, UV-Vis spectrophotometers,
bacterial growth chambers and a fume hood.
Protein Biochemistry laboratory. This lab houses 2 HPLC systems and
an FPLC unit; a Perkin Elmer LS-50 (research grade) spectrofluorimeter;
protein electrophoresis equipment, a UV-Vis spectrophotometer, centrifuges
and other assorted minor equipment.
Biofilm laboratory. This lab is equipped with a custom-designed
chemostat with peristaltic pumps built by Virtis for biofilm growth, and it
is also equipped to do fluorescence microscopy for biofilm analysis. It
contains an Olympus computer-controlled robotic micromanipulator which has
been mounted on an inverted fluorescence microscope equipped with a digital
imaging system. This setup, placed on a vibration isolation table, permits
the precise identification and manipulation of biofilms of any type for which
a marker has been defined. In addition this lab has a Jouan high-capacity
clinical centrifuge; a Dupont-Sorvall Microspin centrifuge; a Coulter counter
for determining cell number; a Fisher Isotemp water bath; an Olympus OK2
inverted phase contrast microscope; and all necessary pipetting devices
including pipette aids, pipetmen, repeater pipettors, etc.
Support laboratories and facilities
Specimen storage facility. This room contains four -80°C freezers;
a -135°C mechanical freezer; and two liquid nitrogen storage systems. All of
these units are alarmed and wired to a 24-hour-a-day monitoring service. One
of the -80°C freezers serves as a backup in the event of failure of one of
the others. All specimens are inventoried and their freezer, rack, and box
location are recorded in the CGS central database, GENOMICS, which was
designed by the PI and has been operational for almost a decade.
Core centrifuge room. This room houses a Sorvall superspeed and
ultra centrifuges and an automated LSC. These instruments are available to
all CGS staff on an as-needed basis. Appropriate tubes, fillers, and sealers
are located in this room as well.
Cold rooms. Each of the three laboratory floors have a 200-sq-ft 4°
C room with an integral -20° C room for large-scale frozen storage located
adjacent to the main labs. These facility house rotors, chromatography
columns, enzymes, media, etc.
Image analysis facility and darkroom. This laboratory houses a
Kodak digital image capture and analysis system which is networked for
transfer of all documentation types. The most recent additions to the imaging
capabilities of CGS are a Molecular Dynamics Phosphor Imaging system which is
connected via Ethernet to the laboratory database and the sequencing and
mapping laboratory. The photographic darkroom has been set up for developing
autoradiographic exposures of gel and filter hybridizations. A Kodak
automatic X-ray film developer and processor has been purchased and a
darkroom lighting system with outside warning light has been installed. This
system will greatly reduce exposure times, allowing more rapid reporting of
clinical results. A Polaroid MP4 camera system with UV trans-illuminator and
wall-mount white-light illuminators is also available. A portable Fotodyne
Trans-illuminator and Polaroid camera with various isolation hoods is also
available for bench-top photography of ethidium bromide-stained gels.
Data analysis facility. This office houses the data storage and
analysis center, which contains a high-end LINUX server with mirrored backup
of all critical systems throughout CGS. All users of the CGS can access and
input data through Windows NT/NOVEL client software. The server contains the
Progeny and BLAST commercial databases, as well as CGS's own Sybase and Rbase
databases, GENOMICS, designed by Dr. Ehrlich, which currently holds records
on over 30,000 specimens. Numerous software packages for data manipulation
are available locally and through Internet hookups. Planned expansions include
a Windows USER server which will serve as front end for the LINUX server and
provide an added level of security as we bring online a web-based version of
GENOMICS.
Animal facilities. The fully functional 10,000-sq-ft
Allegheny-Singer Research Institute animal facility (located in the same
building) houses both small and large animals and has facilities for animal
experiments and surgery. Around-the-clock husbandry and veterinary care is
available for all projects. The facility is fully approved under IACUC
protocols.
Other resources
Offices and personal computers. The CGS is also
equipped with office space, computers, storage and file areas (including
secure files for confidential records), a conference room, a photocopier, and
secretarial support. Each staff member in the CGS has a computer. Individual
computer workstations are mostly high-end Pentium PCs running Windows NT
4.0/2000 professional, plus a few Power Macs, with a full complement of
standard software (word processing, spreadsheets, database software,
pedigree-drawing programs, graphics packages, pedcheck programs, linkage
programs, e-mail, Netscape, etc.). All of the workstations are networked by
LAN and connected with the Internet. Several laser printers (black-and-white
and color) and several inkjet printers are placed throughout the center.
|