Evaluation of cell lysis procedures and use of a micro fluidic system for an automated DNA-based cell identification in interplanetary missions
Introduction
Investigating the possibility of extant or extinct life on planets or other celestial bodies outside of Earth has historically been performed by examining extra terrestrial meteoritic material that has landed on Earth or by limited investigations using direct probes deployed for planetary exploration (McKay et al., 1996). Opportunistic investigations of the former, such as Martian meteorites, while extremely useful, generally provide inconclusive and controversial (Steele et al., 2000; Barber and Scott, 2002; Treiman et al., 2004) information on the likelihood of biological material outside of Earth. More conclusive and direct evidence will likely be obtained using the latter approach of direct landing and sampling on the target of interest. Investigations to date such as the Mars landers Viking 1 and 2, and the Pathfinder, Spirit and Opportunity Rovers have focused mainly on geochemical analysis (e.g. Plumb et al., 1989; Whitfield, 2004). As technology develops for planetary probe exploration (e.g. Skelley et al., 2005) it is now possible to develop an analytical sample suite dedicated to remote and direct biological macromolecule detection providing more definitive evidence and data on the occurrence of biology outside of Earth.
A critical component of this next generation of instrumentation is the ability for sensitive and accurate detection of biological macromolecules such as nucleic acids, proteins and lipids and derivatives/precursors. The ability to incorporate non-culture dependent quantitative recovery of DNA, proteins and lipids in order to obtain quantitative and qualitative information about microbial communities is of critical importance given that of the 53 currently recognized prokaryotic phyla here on Earth, only 26 have cultured representatives available for investigation (Connon and Giovannoni, 2002). Whilst the benefits of developing this instrumentation for terrestrial microbiology and astrobiology investigations are clear there is the possibility that an extraterrestrial organism may not have DNA or cell walls similar to terrestrial organisms. Whilst this is correct in essence it does not preclude the use of DNA microarray technology and the extraction technology discussed in this paper for a variety of reasons. Namely, that the solar system must contain rocks released from earth during the time that life has been present on earth, these rocks are known to be able to harbor earth life (Schuerger et al., 2005). These may have impacted both Mars and Europa and be the basis of a surviving ecosystem there. Contaminated space craft have landed or crashed into the surface of Mars and organisms will be present on instrumentation sent to Mars and Europa to search for life, therefore DNA microarray technology incorporated into a MASSE like instrument (which will predominately use antibody/aptamer arrays for small molecule and biomarker detection), will allow any positive life detection result to be screened for similarity to earth microbes and/or contamination from the space craft. Finding a positive answer to complex life will also benefit for the understanding that DNA is not present within the sample. Finally this technique will allow screening of space craft and return samples from a planetary protection perspective, allowing rapid characterization of sources of contamination and implication of the metabolism of any contaminating organisms on the sample.
On the critical path of developing a remote biological analytical system is the successful ground truthing of instrumentation in environments on Earth that support life and are analogous to candidate locations favored for biological extraterrestrial investigation. In the results presented here, we have used Martian simulated regolith (JSC-1) where appropriate to maximize the applicability of the results to a relevant analytical scenario. Other valid experimental analogues that could provide development or ground truthing opportunities for biological suites of instruments include environments include satisfactory detection of microbial life reported in the frozen lakes of Antarctica (Priscu et al., 1999; Karl et al., 1999), deep-sea hydrothermal vents (Reysenbach et al., 2000) frozen volcanic vents from Svalbard (Steele et al., 2004) and deeply buried marine sediments (Parkes et al., 2000; Reed et al., 2002; D’Hondt et al., 2003). The detection of life in these environments provides ample evidence that microbes can exploit a remarkable range of harsh environments for survival.
Due to the limited potential sample characteristics of any environmental or extra-terrestrial sample, a design strategy under development at the Carnegie Institution of Washington is a Modular Assay System for Solar System Exploration (MASSE). This instrument combines macro-scale extraction processes based on kits commercially available coupled with micro-fluidic analytical systems for downstream investigation. In this paper we investigate some potential technologies under consideration for incorporation into front-end sampling handling and preparation systems in addition to proprietary microfluidic technology that has the potential to be applied to downstream detection of target analytes.
To date, limited data has been presented to test the efficacy of various cell lysis techniques such as bead beating, sonication, enzymatic lysis, and boiling (Kukse et al., 1998; Li and Mustapha, 2002; Miller et al., 1999). In addition, more recent efforts have evaluated the efficacy of commercially available kits for microbial DNA extraction (Orlandi and Lampel, 2000) with an aim to simplify and miniaturize the procedure (Ramesh et al., 2002). However, these studies lack analysis of the potential extraction bias between classes of environmentally relevant prokaryotes such as gram-negative bacteria, gram-positive bacteria, and Archaea. We evaluate the potential of five currently commercially available DNA extraction techniques, and a recently developed DNA extraction and storage technique on representative gram negative, gram positive and archaeal cells for upstream incorporation into the MASSE instrument.
When considering analytical systems for downstream investigation after initial sample pre-treatment, micro total analytical systems (μTAS) (Reyes et al., 2002) have been suggested as self-contained analytical systems that include sample pre-treatment, separation and the detection of target molecules (Auroux et al., 2004). For environmental microbiology applications, μTAS such as microfluidic “lab-on-a-chip” technology limits the consumption of potentially expensive reagents as well as enabling the near real time simultaneous detection of multiple target analytes, such as proteins (Li et al., 2004), amino acids (Skelley et al., 2005), nucleic acid (Wainwright et al., 2003) and cell membrane components (Yang et al., 2003).
In addition to addressing the current paucity of data on upstream macro scale sample handling and extraction, the work presented here also evaluates the potential for μTAS to be employed in the MASSE instrument by employing proprietary lab-on-a-chip development technology to investigate the potential for microfluidic cell lysis of different prokaryotic cells employing both chemical and biological lysis agents.
Section snippets
Cell culturing
Escherichia coli (ATCC 15669) and Haloarcula marismortui (ATCC 43049) were purchased from the American Type Culture Collection (Manassas, VA) and revived using recommended ATCC protocols. Bacillus megatarium was purchased from Wards Natural Science (Rochester, NY). E. coli and B. megatarium were incubated aerobically in 1.5% tryptic soy broth (TSB) (Becton, Dickinson and Company, Sparks, MD) on a shaking incubator (170 rpm) for 12 h at 37 °C. H. marismortui was cultured with ATCC medium 1218
FTA paper protocol development
FTA paper technology has been primarily used in medical applications (e.g. Belgrader et al., 1995; Del Rio et al., 1996) and to date a comprehensive study into protocols applicable to environmental microbiology applications is lacking. Fig. 1 illustrates the concentration of DNA produced after PCR using the ECf and ECr primer set from a 2 mm diameter sample of FTA paper with 100 μl of ∼1×108 E. coli added. Lanes 2–9 (Fig. 1) represent different processing protocols tested prior to PCR. The best
Discussion
The purpose of this work was to quantitatively and qualitatively evaluate commercially available DNA extraction kits to establish protocols and techniques that can be incorporated into an upstream soil handling system in addition to proving concept of prokaryotic manipulation and analysis using microfluidic chip technology as part of a life detection system under construction at the Carnegie Institute of Washington. After initial sample handling and pre-treatment, the life detection system then
Conclusion
This work illustrates the potential of FTA paper technology for environmental applications and is the first demonstration of quantitative multiple cell wall type lysis using microfluidic technology. We suggest that FTA paper technology displays promise for incorporation into a sample handling unit for MASSE and that microfluidic manipulation of either whole prokaryotic cells or target biological macromolecules offers great potential for incorporation into instrumentation designed and suitable
Acknowledgements
The authors wish to acknowledge Marilyn Fogel for helpful experimental comments, Rachel Schelble for manuscript editing in addition to the NSF Research Experience for Undergraduates program.
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