SUPPLEMENTARY INFORMATION FOR ONLINE PUBLISHING
Archaeal Diversity Analysis of Spacecraft Assembly Clean Rooms
Christine Moissl†, James C. Bruckner, and Kasthuri Venkateswaran*
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Biotechnology and Planetary Protection Group, Jet Propulsion Laboratory, California
Institute of Technology, Pasadena, CA 91109
*Corresponding author:
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Kasthuri Venkateswaran
Biotechnology and Planetary Protection Group
M/S 89, Jet Propulsion Laboratory; California Institute of Technology
4800 Oak Grove Dr., Pasadena, CA 91109
818-393-1481 Phone; 818-393-4176 Fax
kjvenkat@jpl.nasa.gov
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Sampling locations and facilities
Samples were collected from selected surface areas within clean rooms of two distinct NASA
facilities; namely, the Jet Propulsion Laboratory spacecraft assembly facility (JPL-SAF) and
Johnson Space Center Genesis Curation Laboratory (JSC-GCL). Clean room certification
was based on the maximum number of particles that are greater than 0.5 m per cubic foot of
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air (ISO 14644-1 Part 1: Classification of air cleanliness; www.iest.org/iso/iso1.htm). The air
within class 10 clean rooms was maintained at fewer than 10 particulates per cubic foot, class
10K clean rooms were allowed to harbor a density of 10,000 particles per cubic foot, and so
on. In addition to air quality, several environmental parameters of clean rooms were also
controlled, such as humidity, temperature, and air circulation. NASA quality assurance
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engineers performed periodic audits to ensure that certified-facility cleanliness levels
conformed to the requirements delineated (La Duc et al., 2007). The air of all facilities was
filtered through High Efficiency Particle Air (HEPA) filters with the exception of JSC clean
rooms which utilized Ultra Low Particle Air (ULPA) filters to achieve higher cleanliness. In
addition, the floors of JSC <5K certified clean rooms were raised to prevent the accumulation
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of organic-laden debris (La Duc et al., 2007). Details of the sampling areas and other
characteristics are given in Table 1.
Sample collection
The sampling at JPL-SAF was conducted during six different times (once a month from
October 2005 to April 2006 except November 2005) at three fixed locations within a class
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100K clean room whereas JSC-GCL was sampled from 8 different locations of various clean
rooms (four from class 10, three from class 1K, and one from uncertified room). To sample
the JPL-SAF, Biological Sampling Kits (BiSKit, Quicksilver Analytics, Abingdon, MD)
were used according to the manufacturer’s instructions. As the BisKiT was not certified for
use in class <1K clean rooms, the JSC-GCL samples were obtained using sterile polyester
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wipes pre-moistened with phosphate buffered saline (PBS, pH 7.2). After sampling, wipes
were placed in 50 mL tubes containing 35 mL PBS and vortexed. Samples were immediately
divided into various portions and frozen (-80°C) or processed without interruption. Air
sampling was done as described elsewhere (Moissl et al., 2007b).
DNA extraction, amplification of archaeal 16S rRNA genes and cloning
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The JSC samples (collected using sterile wipes) were subjected to standard phenolchloroform DNA extraction procedures (Johnson, 1981; Moissl et al., 2007). However, a
modified procedure was used to extract nucleic acids from the samples collected from JPLSAF. Briefly, sample solutions obtained from BiSKit were incubated at 65°C (30 min) after
the addition of 1% sodium desoxyl sulphate (SDS). The reaction mixtures were subsequently
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subjected to three freeze- thaw cycles (-80°C, 65°C). After the addition of Proteinase K
(Sigma-Aldrich, final concentration 500µg/ml), the samples were incubated at 37°C for 30
min. This step was followed by a phenol-chloroform extraction (Johnson, 1981) and the
DNA was precipitated overnight at -20°C in 2 volumes of ice-cold ethanol. After
centrifugation the ethanol was removed and the nucleic acid pellet resuspended in 50 µl
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sterile pure water.
In each step of the DNA extraction and PCR, suitable negative controls were used. In
addition to water controls, an unopened BisKit buffer was included as a negative control in
DNA extraction and PCR. All PCR reactions were performed with the following controls: a
positive control (DNA of Acidianus brierleyi) and two negative controls (buffer extract and
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water). In order to check whether any inhibitory molecules were present in the extract all
samples that exhibited negative PCR amplification for archaea were spiked with known DNA
of A. brierleyi. To find the optimal amplification conditions for archaeal sequences from our
samples, several preliminary tests were done with the first samples obtained (JPL October
2005). All possible primer combinations (forward: 8aF (Burggraf et al., 1992) and 345aF
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(Burggraf et al., 1997), reverse: 1119aR (Burggraf et al., 1997) and 1406uR (Lane, 1991))
were used for preliminary PCR reaction, as well as the nested PCR- primer combination 8aF
- 1406uR, and 344aF - 1119aR. The brightest PCR signals were obtained when PCR was
performed with 8aF and 1119aR, followed by a reamplification with the same primer
combination. This primer combination was used for all other subsequent studies. The optimal
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annealing temperature for the PCR was found to be 55°C or 60°C. A 50°C annealing
temperature resulted in weaker PCR signals.
The PCR was performed under the following conditions: 95°C for 4 min; 33 cycles of 95°C
for 50 s, 55°C for 50 s, and 72°C for 1 min 30 s; and final incubation at 72°C for 10min.
Based on the preliminary testing, reactions were performed using 2 μl of the extracted nucleic
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acid as a template for the amplification of archaeal 16S rDNA using the forward and reverse
primers 8aF and 1119aR, respectively. The amplicons of samples and controls of the initial
PCR were used as templates (2µl) for an immediate second amplification with the same
primer combination. The length and quantity of the amplicons were visualized on a 1%
agarose gel. Amplified PCR fragments were purified immediately (QIAquick PCR
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purification kit, Qiagen, Valencia, CA) and subjected to cloning using a TOPO® TA cloning
kit (Invitrogen, Carlsbad, CA) according the manufacturer’s instructions. The presence of
inserts of the expected size was verified by direct PCR screening of up to 120 transformants
without plasmid extraction. For restriction fragment length polymorphism (RFLP) analyses,
the PCR products were digested with a mixture of four different restriction endonucleases
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(AluI, HhaI, HinfI and RsaI; Fermentas Inc, Hanover, MD). The resulting restriction patterns
were compared, and representative transformants were selected and their plasmid DNA
extracted (Qiaprep kit, Qiagen, Valencia, CA) as recommended by the manufacturer.
Sequencing of the 16S rRNA gene sequences was performed by Agencourt Bioscience
Corporation (Beverly, MA).
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Rarefaction analyses
The coverage of clone libraries was calculated according to Good (Good, 1953) using the
equation: C= [1-(n1/N)]*100, where C is the homologous coverage, n1 is the number of
OTU’s appearing only once in the library, and N is the total number of clones examined.
Phylogenetic analyses
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All sequences obtained were submitted to the CHECK_CHIMERA program of the
Ribosomal Database Project to detect possible chimeric artifacts (Cole et al., 2003). For
phylogenetic analyses, an alignment of approximately 40,000 homologous full and partial
sequences available in public databases was used (ARB project, (Ludwig et al., 2004)). The
new 16S rRNA gene sequences were added to the 16S rRNA alignment using the
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corresponding automated tools of the ARB software package (Ludwig et al., 2004). The
resulting alignment was checked manually and corrected if necessary. For tree
reconstruction, methods were applied as implemented in the ARB software package.
Nucleotide sequence accession numbers
The 16S rRNA gene sequences of the clones were deposited in the NCBI nucleotide
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sequence database. The accession numbers are given in Fig. 1.
References (Supplementary Information)
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Aquifex pyrophilus. Syst Appl Microbiol 15, 352-356.
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Burggraf, S., Huber, H. & Stetter, K. O. (1997). Reclassification of the crenarchael orders
and families in accordance with 16S rRNA sequence data. Int J Syst Bacteriol 47, 657-660.
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