Stanford University DNA Extraction in Atacama Soils Lauren Fletcher – NASA Ames Research Center/Stanford University Christopher McKay – NASA Ames Research Center Spaceward Bound: Atacama Season 1

Uranium P-36 Explosive Space Modulator Goal: Venus Viewing Improvements Plan: Development of an Earth Shattering Explosive Device Allied Tactical Attack Command Armed Martian Association

Stanford University Looking For Martians In the Atacama Lauren Fletcher – NASA Ames Research Center/Stanford University Christopher McKay – NASA Ames Research Center Spaceward Bound: Atacama Season 1

Stanford University A Little Bit about Mission Design Two General Laws Govern Mission Design: Objectives Exploration Political Planetary Defense Science Limitations Monetary Political Physics Technology Science Assume our Objective is the discovery of Life on Mars and that principle limitations are Scientific & Technology

Stanford University Stuff We Know About Mars We Know: Conditions on early Mars are theorized to have been similar to that of Earth at the time of the genesis of life on Earth. *LONG* Periods when there were *LARGE* bodies of free flowing water on the Mars surface. Continue to be periods when the conditions are right for to produce water vapor if not liquid water. Large deposits of Subsurface and Polar Ice. Thin Atmosphere. We Conclude: Life was at least *possible* on Mars It is *still* possible that there is Life on Mars Therefore, We Assume: Life Does exist on Mars….we just havent found the right place or have had the right tool to find it.

Stanford University Same Song, Second Verse (The Origin of Life on Earth/Mars) Life is the Same Interchange of Planetary Materials Extra-solar system meteorites hitting both planets. Separate Genesis Life is Different Conditions on *both* planets acceptable for the initiation of Life, but a completely different genesis Leads to Further Refinement of our Objective: Is there Life? Does it look like us? Did we come from the same source or a separate Genesis?

Stanford University The Complexity of Life on Mars The Answer is.....Not Very! Current Scientific data shows a very stark environment devoid of all (VISIBLE) life. Provides a Key Scientific Limitation: Life on Mars is likely to be mono-cellular or not very complex multi-cellular organisms. Therefore, We Conclude: Looking for Microbes is the best way to find our Martians.

Stanford University Direct and Indirect Detection of Martians Indirect – Looking for the *Signs* of Biology: Respiration Detection of Organic Material Direct – We see it, therefore it is: Cultivation Microscopy Direct Visual Florescent Stain Sublimation of Nucleic Acids Phospholipid Fatty Acid Analysis DNA Detection and Analysis Comparison of the Methods used for the Direct Detection of Martians in the Atacama (following)

MethodReported Results What does it really detect? How related to # bact/gm? SensitivityForms of Error Cultiva- tion 10^4 CFU/gm 10^6 – S25˚ CFU/gmDirect measurement of cultivatable heliotrophic bacteria/gram >10^4 CFU/gm Only cultivatable bacteria QPCR166 bact/gm peaks at 10^4 Copies/gmDirect measurement of all amplified copies of this 16sDNA sequence. Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. >9 bact/gm DNA Shearing Not a pan-generic primer Assumed #copies/bact % efficiency in removing DNA from soil Interference of humic acid in amplification DAPI10^6 – 10^7 bact/gm Total Biomass by staining DAPI DNA Bacteria determined during examination through microscope Stains viable & nonviable only 4 Stains DAPI chain of DNA 1, and stains total biomass. The microscope operator is responsible for differentiating between bacterial and non-bacterial points. Not Known. No reference to sensitivity in publications read. % efficiency in removing microbes from soil 4 Results decrease significantly after 1-2 weeks of storage time 1 Operator error in determining what constitutes a countable bacteria 4 Methods of extrapolation of total bacteria from counts 4 Masking of bacteria by soil particles Sublima- tion 5x10^6 bact/gm 1 sample only Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells Cant be directly related. No way to isolate bacteria from eukaryotes, and viruses 5x10^6 #gms per species is variable 70% of result could be dead 2 Interference of mineral surfaces, salts and larger amounts of organic matter in sublimation 3 %efficiency of sublimation of Adenine from natural soils unknown 3 Over-estimation due to eukaryotes and viruses. Non-biologically produced Adenine present in sample PLFATo be Determined from last set of samples. Total Biomas by #pmoles PLFA/gm of soil subdivided by composition ~10^6 cells or 50pmole of PLFA required for analysis. # of pmoles of PLFA/bact is variable % efficiency in removing PLFA from soil- bound microbes.

MethodReported Results What does it really detect? How related to # bact/gm? SensitivityForms of Error Cultiva- tion 10^4 CFU/gm 10^6 – S25˚ CFU/gm Direct measurement of cultivatable heliotrophic bacteria/gram >10^4 CFU/gm Only cultivatable bacteria QPCR166 bact/gm peaks at 10^4 Copies/gm Direct measurement of all amplified copies of this 16sDNA sequence. Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. >9 bact/gm DNA Shearing Not a pan-generic primer Assumed #copies/bact % efficiency in removing DNA from soil Interference of humic acid in amplification DAPI10^6 – 10^7 bact/gm Total Biomass by staining DAPI DNA Bacteria determined during examination through microscope Stains viable & nonviable only 4 Stains DAPI chain of DNA 1, and stains total biomass. The microscope operator is responsible for differentiating between bacterial and non-bacterial points. Not Known. No reference to sensitivity in publications read. % efficiency in removing microbes from soil 4 Results decrease significantly after 1-2 weeks of storage time 1 Operator error in determining what constitutes a countable bacteria 4 Methods of extrapolation of total bacteria from counts 4 Masking of bacteria by soil particles Sublima- tion 5x10^6 bact/gm 1 sample only Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells Cant be directly related. No way to isolate bacteria from eukaryotes, and viruses 5x10^6 #gms per species is variable 70% of result could be dead 2 Interference of mineral surfaces, salts and larger amounts of organic matter in sublimation 3 %efficiency of sublimation of Adenine from natural soils unknown 3 Over-estimation due to eukaryotes and viruses. Non-biologically produced Adenine present in sample PLFATo be Determined from last set of samples. Total Biomas by #pmoles PLFA/gm ~10^6 cells or 50pmole of PLFA required for analysis. # of pmoles of PLFA/bact is variable % efficiency in removing PLFA from soil- bound microbes.

MethodReported Results What does it really detect? How related to # bact/gm? SensitivityForms of Error Cultiva- tion 10^4 CFU/gm 10^6 – S25˚ CFU/gmDirect measurement of cultivatable heliotrophic bacteria/gram >10^4 CFU/gm Only cultivatable bacteria QPCR166 bact/gm peaks at 10^4 Copies/gmDirect measurement of all amplified copies of this 16sDNA sequence. Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. >9 bact/gm DNA Shearing Not a pan-generic primer Assumed #copies/bact % efficiency in removing DNA from soil Interference of humic acid in amplification DAPI10^6 – 10^7 bact/gm Total Biomass by staining DAPI DNA Bacteria determined during examination through microscope Stains viable & nonviable only 4 Stains DAPI chain of DNA 1, and stains total biomass. The microscope operator is responsible for differentiating between bacteria l and non- bacterial points. Not Known. No reference to sensitivity in publications read. % efficiency in removing microbes from soil 4 Results decrease significantly after 1-2 weeks of storage time 1 Operator error in determining what constitutes a countable bacteria 4 Methods of extrapolation of total bacteria from counts 4 Masking of bacteria by soil particles Sublima- tion 5x10^6 bact/gm 1 sample only Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells Cant be directly related. No way to isolate bacteria from eukaryotes, and viruses 5x10^6 #gms per species is variable 70% of result could be dead 2 Interference of mineral surfaces, salts and larger amounts of organic matter in sublimation 3 %efficiency of sublimation of Adenine from natural soils unknown 3 Over-estimation due to eukaryotes and viruses. Non-biologically produced Adenine present in sample PLFATo be Determined from last set of samples. Total Biomas by #pmoles PLFA/gm of soil subdivided by composition ~10^6 cells or 50pmole of PLFA required for analysis. # of pmoles of PLFA/bact is variable % efficiency in removing PLFA from soil- bound microbes.

MethodReported Results What does it really detect? How related to # bact/gm? SensitivityForms of Error Cultiva- tion 10^4 CFU/gm 10^6 – S25˚ CFU/gmDirect measurement of cultivatable heliotrophic bacteria/gram >10^4 CFU/gm Only cultivatable bacteria QPCR166 bact/gm peaks at 10^4 Copies/gmDirect measurement of all amplified copies of this 16sDNA sequence. Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. >9 bact/gm DNA Shearing Not a pan-generic primer Assumed #copies/bact % efficiency in removing DNA from soil Interference of humic acid in amplification DAPI10^6 – 10^7 bact/gm Total Biomass by staining DAPI DNA Bacteria determined during examination through microscope Stains viable & nonviable only 4 Stains DAPI chain of DNA 1, and stains total biomass. The microscope operator is responsible for differentiating between bacterial and non-bacterial points. Not Known. No reference to sensitivity in publications read. % efficiency in removing microbes from soil 4 Results decrease significantly after 1-2 weeks of storage time 1 Operator error in determining what constitutes a countable bacteria 4 Methods of extrapolation of total bacteria from counts 4 Masking of bacteria by soil particles Sublima- tion 5x10^6 bact/gm 1 sample only Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells Cant be directly related. No way to isolate bacteria from eukaryotes, and viruses 5x10^6 #gms per species is variable 70% of result could be dead 2 Interference of mineral surfaces, salts and larger amounts of organic matter in sublimation 3 %efficiency of sublimation of Adenine from natural soils unknown 3 Over-estimation due to eukaryotes and viruses. Non-biologically produced Adenine present in sample PLFATo be Determined from last set of samples. Total Biomas by #pmoles PLFA/gm of soil subdivided by composition ~10^6 cells or 50pmole of PLFA required for analysis. # of pmoles of PLFA/bact is variable % efficiency in removing PLFA from soil- bound microbes.

MethodReported Results What does it really detect? How related to # bact/gm? SensitivityForms of Error Cultiva- tion 10^4 CFU/gm 10^6 – S25˚ CFU/gmDirect measurement of cultivatable heliotrophic bacteria/gram >10^4 CFU/gm Only cultivatable bacteria (1-10% of Resident Population) QPCR166 bact/gm peaks at 10^4 Copies/gmDirect measurement of all amplified copies of this 16sDNA sequence. Divide # copies detected by 5.5 copies/bact. in order to convert to # bacteria/gm. >9 bact/gm DNA Shearing Not a pan-generic primer Assumed #copies/bact % efficiency in removing DNA from soil Interference of humic acid in amplification DAPI10^6 – 10^7 bact/gm Total Biomass by staining DAPI DNA Bacteria determined during examination through microscope Stains viable & nonviable only 4 Stains DAPI chain of DNA 1, and stains total biomass. The microscope operator is responsible for differentiating between bacterial and non-bacterial points. Not Known. No reference to sensitivity in publications read. % efficiency in removing microbes from soil 4 Results decrease significantly after 1-2 weeks of storage time 1 Operator error in determining what constitutes a countable bacteria 4 Methods of extrapolation of total bacteria from counts 4 Masking of bacteria by soil particles Sublima- tion 5x10^6 bact/gm 1 sample only Total Biomas, by #gms Adenine/gm of soil to include viable, non-viable and dead (intact and non-intact) cells Cant be directly related. No way to isolate bacteria from eukaryotes, and viruses 5x10^6 #gms per species is variable 70% of result could be dead 2 Interference of mineral surfaces, salts and larger amounts of organic matter in sublimation 3 %efficiency of sublimation of Adenine from natural soils unknown 3 Over-estimation due to eukaryotes and viruses. Non-biologically produced Adenine present in sample PLFATo be Determined from last set of samples. Total Biomas by #pmoles PLFA/gm of soil subdivided by composition ~10^6 cells or 50pmole of PLFA required for analysis. # of pmoles of PLFA/bact is variable % efficiency in removing PLFA from soil-bound microbes.

Stanford University Summary There are also Advantages to Each Method: Sensitivity Ease of Sample Preparation Have to Balance Advantages and Limitations in the selection of a specific method in order to meet your Objectives.

Stanford University Proof of Martians on the Mars Surface The two recent Mars rovers have provided the proof of Martians on the Mars Surface!!!!

Stanford University Martian on Mars Surface Photos

Stanford University The Secret Plot!!! The two designers of the Optical Sensors Secretly Etched Two Martians on the Silicon Chip There are four Martians on the Mars surface.

Stanford University A Burning Man Duck-Mobile!