Name: Author Spotlight: Understanding Microbe Adaptation Using Innovative Techniques for Exploring Thermophilic Evolution
Uploaded: 2024-06-14T13:10:00
Description: The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures ...

The authors thank Prof SV Albers (University of Freiburg), Prof Eveline Peeters (Vrije Universiteit Brussel), and Dr Rani Baes (Vrije Universiteit Brussel) for advice and the S.&#160;acidocaldarius DSM639 strain. This work was funded by a Royal Society Research Grant (awarded to DRG: RGS\R1\231308), a UKRI-NERC &#34;Exploring the Frontiers&#34; Research Grant (awarded to DRG and CGK: NE/X012662/1), and a Kuwait University PhD scholarship (awarded to ZA).

1. Preparation of S. acidocaldarius growth medium (BBM+)
NOTE: To cultivate S. acidocaldarius, this protocol uses Basal Brock Medium (BBM+)23. This is prepared by first combining the inorganic stock solutions outlined below to create BBM&#8722;, which may be prepared in advance. BBM+ is then prepared as needed by adding the organic stock solutions to BBM&#8722;. Stock solution recip...

Experimental Evolution Protocol for Thermophile Adaptation Using Low-Cost Thermomixers

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+dangerous+missense:+Thermophiles+display+especially+low+mutation+rates.">Avoiding dangerous missense: Thermophiles display especially low mutation rates.</a> PLoS Genet. 5 (6), e1000520 (2009).</li><li>Grogan, D. W., Carver, G. T., Drake, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+fidelity+under+harsh+conditions:+Analysis+of+spontaneous+mutation+in+the+thermoacidophilic+archaeon+Sulfolobus+acidocaldarius.">Genetic fidelity under harsh conditions: Analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius.</a> Proc Natl Acad Sci U S A. 98 (14), 7928-7933 (2001).</li><li>Wagner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+genetic+tool+box+for+the+Crenarchaeote+Sulfolobus+acidocaldarius.">Versatile genetic tool box for the Crenarchaeote Sulfolobus acidocaldarius.</a> Front Microbiol. 3, 214 (2012).</li><li>Suzuki, S., Kurosawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+gene+encoding+restriction+endonuclease+SuaI+and+development+of+a+host-vector+system+for+the+thermoacidophilic+archaeon+Sulfolobus+acidocaldarius.">Disruption of the gene encoding restriction endonuclease SuaI and development of a host-vector system for the thermoacidophilic archaeon Sulfolobus acidocaldarius.</a> Extremophiles. 20 (2), 139-148 (2016).</li><li>Baes, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+and+translational+dynamics+underlying+heat+shock+response+in+the+thermophilic+crenarchaeon+Sulfolobus+acidocaldarius.">Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius.</a> mBio. 14 (5), e0359322 (2023).</li><li>Gonz&#225;lez, A. 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This work has developed an experimental evolution protocol for thermophiles, here tailored for the archaeon S. acidocaldarius, but adaptable to other microbes with high-temperature growth requirements. This protocol builds on techniques initially designed for mesophilic bacteria but is specifically modified to overcome the technical challenges associated with high-temperature aerobic growth2,4,5,<sup class="x...

Early life on Earth may have originated in extreme environments, such as hydrothermal vents, which are characterized by extremely high temperatures and acidity1. Microbes continue to inhabit extreme environments, including hot springs and volcanic solfatara. Characterizing the evolutionary dynamics that occur under these extreme conditions may shed light on the specialized physiological processes that enable survival under these conditions. This may have wide-ranging implications, from our understanding of the origins of biological diversity to the development of novel high-temperature enzymes with biotechnological applications.
The understanding of microbial evolutionary dynamics in extreme environments remains limited despite its critical importance. In contrast, a significant body of knowledge about evolution in mesophilic environments has been acquired through the application of a technique known as experimental evolution. Experimental evolution involves observing evolutionary change under laboratory conditions2,3,4,5. Often, this involves a defined change environment (e.g., temperature, salinity, introduction of a toxin or a competitor organism)7,8,9. When combined with whole-genome sequencing, experimental evolution has enabled us to test key aspects of evolutionary processes, including parallelism, repeatability, and the genomic basis for adaptation. However, to date, the bulk of experimental evolution has been performed with mesophilic microbes (including bacteria, fungi, and viruses2,3,4,5, but largely excluding archaea). A method for experimental evolution applicable to thermophilic microbes would enable us to better understand how they evolve and contribute to a more comprehensive understanding of evolution. This has potentially wide-ranging implications, from deciphering the origins of thermophilic life on Earth to biotechnological applications involving &#39;extremozymes&#39; used in high-temperature bioprocesses10 and astrobiological research11.
The archaeon Sulfolobus acidocaldarius is an ideal candidate as a model organism for developing experimental evolution techniques for thermophiles. S. acidocaldarius reproduces aerobically, with an optimal growth temperature at 75 &#176;C (range 55 &#176;C to 85 &#176;C) and high acidity (pH 2-3)4,6,12,13,14. Remarkably, despite its extreme growth conditions, S. acidocaldarius maintains population densities and mutation rates comparable to mesophiles7,15,16,17,18. In addition, it possesses a relatively small, well-annotated genome (strain DSM639: 2.2 Mb, 36.7% GC, 2,347 genes)12; S. acidocaldarius also benefits from robust genome engineering tools, allowing for a direct assessment of the evolutionary process through targeted gene knockouts19. A notable example of this is the availability of genetically modified strains of S. acidocaldarius, such as the uracil auxotrophic strains of MW00119 and SK-120, which can serve as selectable markers.
There are significant challenges with conducting experimental evolution with thermophiles like S. acidocaldarius. Extended incubation at high temperatures required for these studies imposes considerable evaporation for both liquid and solid culturing techniques. Extended operation at high temperatures can also damage the traditional shaking incubators that are commonly used in experimental evolution in liquid media. Exploring multiple temperatures necessitates a substantial financial investment for acquiring and maintaining several incubators. Furthermore, the high energy consumption required raises significant environmental and financial concerns.
This work introduces a method to address the challenges encountered in performing experimental evolution with thermophiles like S. acidocaldarius. Building upon a technique developed by Baes et al. for investigating heat shock response14,21, the method developed here utilizes bench-top thermomixers for consistent and reliable high-temperature incubation. Its scalability allows for the simultaneous assessment of multiple temperature treatments, with reduced costs in acquiring additional incubation equipment. This enhances experimental efficiency, enabling robust statistical analysis and extensive investigation of factors influencing evolutionary dynamics in thermophiles22. Moreover, this approach significantly reduces the financial initial investment and energy consumption compared to traditional incubators, offering a more sustainable and environmentally friendly alternative.
Our method lays the groundwork for experimentally investigating evolutionary dynamics in environments characterized by extreme temperatures, which may have played a key role during the early stages of the diversification of life on Earth. Thermophilic organisms have unique properties, but their extreme growth conditions and specialized requirements have often limited their accessibility as a model system. Overcoming these barriers not only expands research opportunities for investigating evolutionary dynamics but also enhances the broader utility of thermophiles as model systems in scientific research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m syringe-driven membrane filters</td>
			<td>StarLab</td>
			<td>E4780-1226</td>
			<td>For filter sterilising media components that cannot be autoclaved.</td>
		</tr>
		<tr>
			<td>1 &#956;L inoculation loops</td>
			<td>Greiner</td>
			<td>731161, 731165, or 731101</td>
			<td>For inoculating cultures. Other loops can be used.</td>
		</tr>
		<tr>
			<td>1000 &#956;L pipette tips</td>
			<td>StarLab</td>
			<td>S1111-6811</td>
			<td>Other pipette tips can be used.</td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tubes</td>
			<td>StarLab</td>
			<td>S1620-2700</td>
			<td>For culturing S. acidocaldarius in thermomixers.</td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>StarLab</td>
			<td>S1111-0816</td>
			<td>Other pipette tips can be used.</td>
		</tr>
		<tr>
			<td>50 mL polystyrene tubes with conical bottom</td>
			<td>Corning</td>
			<td>430828 or 430829</td>
			<td>Other tubes may be used. Check performance at 75 &#176;C. Tubes with plug seal caps may not allow sufficient aeration; check before using.&#160;</td>
		</tr>
		<tr>
			<td>50 mL syringe</td>
			<td>BD plastipak</td>
			<td>300865</td>
			<td>For use with syringe-driven filters.</td>
		</tr>
		<tr>
			<td>96 well microtitre plates (non-treated, flat bottom)</td>
			<td>Nunc</td>
			<td>260860</td>
			<td>For measuring OD at 600 nm in spectrophotometer.</td>
		</tr>
		<tr>
			<td>Adjustable width multichannel pipette</td>
			<td>Pipet-Lite</td>
			<td>LA8-300XLS</td>
			<td>Optional, but saves time when transferring between microcentrifuge and 96 well plates.</td>
		</tr>
		<tr>
			<td>Ammonium sulfate ((NH4)2SO4)</td>
			<td>Millipore</td>
			<td>168355</td>
			<td>For Brock stock solution I.</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Priorclave</td>
			<td>B60-SMART or SV100-BASE</td>
			<td>Other autoclaves can also be used.</td>
		</tr>
		<tr>
			<td>Breathe-EASY gas permeable sealing membrane</td>
			<td>Sigma-Aldrich</td>
			<td>Z763624-100EA</td>
			<td>Cut to size to use on pierced microcentrifuge tubes. If substituting other gas permeable memrbanes, ensure performance is adequate at 75 &#176;C</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td>For Brock stock solution I.</td>
		</tr>
		<tr>
			<td>CELLSTAR Six well plates (suspension/non-treated)</td>
			<td>Greiner</td>
			<td>M9062</td>
			<td>Other manufacturers&#39; six well plates can likely be substituted. Check performance at high temperatures.</td>
		</tr>
		<tr>
			<td>Cobalt(II) sulfate heptahydrate (CoSO4&#183;7H2O)</td>
			<td>Supelco</td>
			<td>1025560100</td>
			<td>For Trace element stock solution.</td>
		</tr>
		<tr>
			<td>Copper(II) chloride dihydrate (CuCl2&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>307483</td>
			<td>For Trace element stock solution.</td>
		</tr>
		<tr>
			<td>D-(+)-glucose anhydrous (C6H12O6)</td>
			<td>Thermo Scientific Chemicals</td>
			<td>11462858</td>
			<td>Other pentose and hexose sugars may also be used (e.g. D-xylose, D-arabinose). Glucose is not a preferred carbon source for S. acidocaldarius (SV Albers, personal communication)</td>
		</tr>
		<tr>
			<td>Double-distilled water (ddH2O)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gelrite</td>
			<td>Duchefa Biochemie</td>
			<td>G1101.1000</td>
			<td>Gelrite (gellan gum) is used in place of agar to make solid media due to its higher melting point.</td>
		</tr>
		<tr>
			<td>Glass 100 mm Petri dishes</td>
			<td>Brand</td>
			<td>BR455742</td>
			<td>Glass Petri dishes are used because most standard polystyrene 90 mm Petri dishes deform at 75 &#176;C (brand-dependent). Alternatively, six well plates can be used as these do not deform at high temperatures.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>New Brunswick</td>
			<td>Innnova 42R</td>
			<td>Other incubators can also be used. Check the operating temperature for equipment prior to purchase/use, as many incubators are not capable of temperatures higher than 65&#176;C.</td>
		</tr>
		<tr>
			<td>Iron(III) chloride hexahydrate (FeCl3&#183;6H2O)</td>
			<td>Supelco</td>
			<td>103943</td>
			<td>For Fe Stock Solution</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (Epsom salt) (MgSO4&#183;7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td>For Brock stock solution I.</td>
		</tr>
		<tr>
			<td>Manganese(II) chloride tetrahydrate (MnCl2&#183;4H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>SIALM5005-100G</td>
			<td>For Trace element stock solution.</td>
		</tr>
		<tr>
			<td>Mini Smart Wi-Fi Socket, Energy Monitoring</td>
			<td>Tapo</td>
			<td>Tapo P110</td>
			<td>To monitor energy consumtion&#160;</td>
		</tr>
		<tr>
			<td>N-Z-Amine A - Casein enzymatic hydrolysate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C0626-500G</td>
			<td>N-Z-Amine-A is used as a source of amino acids.</td>
		</tr>
		<tr>
			<td>Paper clip (or other sturdy wire)</td>
			<td>none</td>
			<td>none</td>
			<td>For piercing 2 mL microcentrifuge tubes.</td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate (Monopotassium phosphate) (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td>For Brock stock solution I.</td>
		</tr>
		<tr>
			<td>Promega Wizard Genomic DNA Purification Kit</td>
			<td>Promega</td>
			<td>A1120</td>
			<td>Optional, to extract genomic DNA in the lab</td>
		</tr>
		<tr>
			<td>Sodium molybdate dihydrate (Na2MoO4&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>M1651-100G</td>
			<td>For Trace element stock solution.</td>
		</tr>
		<tr>
			<td>Sodium tetraborate decahydrate (Borax) (Na2B4O7&#183;10H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>S9640</td>
			<td>For Trace element stock solution.</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>BMG</td>
			<td>SPECTROstar OMEGA</td>
			<td>For measuring OD at 600 nm. Other spectrophotometers that can read OD at 600 nm can be used.</td>
		</tr>
		<tr>
			<td>Sulfuric acid (Diluted in a 1:1 ratio with water) (H2SO4)</td>
			<td>Thermo Scientific Chemicals</td>
			<td>11337588</td>
			<td>Used to adjust pH of Brock stock solution II/III to a final pH of 2&#8211;3.</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>DLab</td>
			<td>HM100-Pro</td>
			<td>Other thermomixers can also be used; key consideration is the ability to maintain 65&#8211;75 &#176;C temperatures and 400 RPM</td>
		</tr>
		<tr>
			<td>Uracil (C4H4N2O2)</td>
			<td>Sigma-Aldrich</td>
			<td>U0750</td>
			<td>Deletion of pyrE is a common genetic marker used in S. acidocaldarius. Deletion strains must be supplemented with uracil for growth. Supplementation is not strictly required for the DSM639 wild-type strain, but is included here as future experiments may involve deletion strains.</td>
		</tr>
		<tr>
			<td>Vanadyl sulfate dihydrate (VOSO4&#183;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>204862</td>
			<td>For Trace element stock solution.</td>
		</tr>
		<tr>
			<td>Zinc sulfate heptahydrate (ZnSO4&#183;7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>221376</td>
			<td>For Trace element stock solution.</td>
		</tr>
	</tbody>
</table>

adaptation at the extremes of life: experimental evolution with the extremophile archaeon sulfolobus acidocaldarius

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures is a key requirement, not only for understanding fundamental evolutionary processes but also for developing S. acidocaldarius as a chassis for bioengineering. One major obstacle to conducting experimental evolution with thermophiles is the expense of equipment maintenance and energy usage of traditional incubators for high-temperature growth. To address this challenge, a comprehensive experimental protocol for conducting experimental evolution in S. acidocaldarius is presented, utilizing low-cost and energy-efficient bench-top thermomixers. The protocol involves a batch culture technique with relatively small volumes (1.5 mL), enabling tracking of adaptation in multiple independent lineages. This method is easily scalable through the use of additional thermomixers. Such an approach increases the accessibility of S. acidocaldarius as a model system by reducing both initial investment and ongoing costs associated with experimental investigations. Moreover, the technique is transferable to other microbial systems for exploring adaptation to diverse environmental conditions.

Author Spotlight: Understanding Microbe Adaptation Using Innovative Techniques for Exploring Thermophilic Evolution

Adaptation at the Extremes of Life: Experimental Evolution with the Extremophile Archaeon Sulfolobus acidocaldarius

Research in our group, focuses on understanding the rules of evolution. With this project, we've developed a new protocol to study how thermophilic microbes evolve, using controlled laboratory experiments. This will let us answer questions like how they respond to environmental change through adaptive evolution.A major challenge is controlling cultivation conditions. Thermophiles require high temperature environments for growth, leading to high evaporation rates, and the risk of dried cultures, and growth plates over the incubation period. Another challenge is the slow growth rates of some thermophiles which can make rapid iteration testing challenging.Our protocol addresses the major challenges associated with cultivating thermophilic microbes at multiple temperatures. We now have a better ability to control environmental conditions, ensuring the results we observe are consistent, and reflect the experimental conditions we impose. This will enable us to study thermophile adaptation in real-time.Our protocol offers a high throughput method, not just for sulfolobus research, but adaptable to a variety of microorganisms. Using thermo mixers, we can carry out simultaneous studies at different temperatures, without requiring multiple large shaking incubators. It's also reduces energy costs, offering a greener pathway for this type of research.Our findings pave the way for evolution experiments in sulfolobus and other thermophiles. Much of what we think we know about evolution comes from studying mesophilic organisms, and there's a risk that we're missing, the key rules from thermophiles that will help us explain how the diversity of life on earth has evolved.

Growth curve measurements 
Growth curves for S. acidocaldarius DSM639 are shown in Figure 3A. Growth was found to be similar when comparing incubation using thermomixers with that in conventional incubators. Average growth rate parameters were estimated by fitting a logistic curve to each replicated growth curve and calculating the mean and standard error. Times to mid-exponential phase on the thermomixer and incubator were 27.2 h &#177; 1.1 h and 31.1 h &#177;...

Here, we present an experimental evolution protocol for adaptation in thermophiles utilizing low-cost, energy-efficient bench-top thermomixers as incubators. The technique is demonstrated through the characterization of temperature adaptation in Sulfolobus acidocaldarius, an archaeon with an optimal growth temperature of 75 &#176;C.

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures ...

adaptation-at-extremes-life-experimental-evolution-with-extremophile

Research

JoVE Journal

Biology

Reviving Sulfolobus acidocaldarius Cultures for Temperature Evolution Experimentation

To begin, use a bunsen burner to carefully heat a blunt steel wire. Pierce it into the lid of a two-milliliter microcentrifuge tube to create a one-millimeter-wide hole. Place the pierced tubes in an autoclavable vessel for sterilization.Once the tubes have been autoclaved and cooled, fill them with 1.5 milliliters of prewarmed BBM+Now use a pipette tip to scrape about 50 to 60 milligrams of the Sulfolobus acidocaldarius culture from a glycerol stock. Transfer the inoculum into the BBM+filled tubes. Keep an uninoculated tube with BBM+as the negative control.To prevent any aerosols from escaping from the pierced tube lid, place a heat-tolerant gas permeable membrane on the top of the lid. Place the inoculated tubes in a thermomixer for inoculation. After the cultures have reached the required optical density, centrifuge the tubes at 5, 000 G for two minutes at room temperature.Then, pipette out the supernatant to discard it. Add 1.5 milliliters of warm BBM+to the pellet and resuspend the cell pellet in the medium. The growth was found to be similar when comparing incubation using thermomixers with that in conventional incubators.

Initiation of Independent Lineages of Sulfolobus acidocaldarius Cultures for Temperature Evolution Experimentation

To begin, use a spectrophotometer to measure the optical density of a sulfolobus acidocaldarius starting culture. Next, create serial dilutions of the starting culture from 10 to the 1 to 10 to the 6. Pipette 100 microliters each from the 10 to the 5 and 10 to the 6 dilutions, then transfer them into the wells of a six well plate containing solid BBM plus.Place the plate in a box with damp cloth to a static incubator at 75 degrees Celsius for five to seven days until single colonies emerge. To establish the desired number of evolving lineages from single colonies, with an inoculation loop, pick a colony at random. Resuspend the colony in 1.5 milliliters of prewarmed BBM plus contained in a pierced two milliliter tube.Label the tube appropriately. After repeating the resuspension for several colonies, fill a tube with 1.5 milliliters of the medium as negative control. Seal the tops of the tubes with a breathable membrane, then incubate them in a thermomixer until the cultures become turbid.Centrifuge the clonal cultures at 5, 000G for one minute at room temperature. After discarding the supernatant, pipette 200 microliters of BBM plus into the tube and resuspend the pellet. Add 200 microliters of 50%glycerol to each tube to create glycerol stalks.Then, store the stalks at 80 degrees Celsius until further experimentation.

Temperature Evolution Experimentation Using Sulfolobus acidocldarius

To begin, use the revived ancestral Sulfolobus acidocaldarius populations. Add BBM plus to dilute the cultures to an OD600 of 0.01. Pipette appropriate volume from each of the seven cultures into three separate pierced two milliliter tubes.Seal the tubes with a breathable membrane, then place each set of seven ancestral populations into separate thermomixers. Set each thermomixer to the required temperature and 400 RPM to begin the evolution experiment. After 48 hours, transfer 15 microliters of each of the transfer zero cultures into 1.5 milliliters of warmed BBM plus medium contained in a pierced two milliliter tube labeled transfer one.Seal the tubes with a permeable membrane before placing them back in randomized positions into the respective thermomixers. Measure the OD600 of transfer zero in a plate-based spectrophotometer to complete the first transfer. Repeat the culture transfer and measurement of optical density every two days to perform transfers until the desired number of transfers is reached.Prepare glycerol stocks of populations after every 10th transfer and on the final transfer, and label the tube with the ancestral population identifier and the transfer number. Thermomixers should be set to constant temperatures of 75 degrees Celsius and 65 degrees Celsius. A third thermomixer will be gradually decreased to observe the response to gradual change.

Post-Evolution Growth Assays of Sulfolobus acidocaldarius Ancestral and Evolved Lineages

To begin incubate the revived ancestral and temperature evolved cultures of Sulfolobus acidocaldarius at the final temperatures. Once incubation is complete, measure their optical densities at 600 nanometers. Next, add 1.5 milliliters of BBM plus into six sets of seven tubes for each culture.Then add 15 microliters of each culture into the six sets of tubes. Set three thermomixers to 75 degrees Celsius and three thermomixers to 65 degrees Celsius. Place the tubes in the thermomixer corresponding to the temperature and replicate ID.After a 48-hour incubation transfer, 200 microliters from each culture into a 96-well plate.Measure the optical density with a spectrophotometer. Finally, plot and analyze the data with statistical software. Lineages from the constant 75 degrees Celsius condition increased in OD600 from an initial range by the end of the experiment.In contrast, lineages from the constant 65 degree Celsius treatment displayed a drop. Populations in the temperature drop treatment increased from the initial optical density to transfer six, then showed a steady decline.