Storify on storing nucleic acids in unfriendly environments

Sometimes you want to collect samples in places that are hard to get to, or get out of. Sometimes those places are hot. Sometimes the people collecting your samples are not you, and they don’t have a lot of training. Sometimes there isn’t a freezer nearby. Or even electricity. Does this mean you can’t get nucleic acid samples from these places? Of course not. Here’s what Twitter had to say about this issue:

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Recent Collaborative Thrash Lab SAR11 research

There are some exciting developments in the world of SAR11 microbiology, and I’d like to take a moment to summarize recent research I’ve been involved in. All three papers have come out this year in The ISME Journal.

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The single-cell genomes from Thrash et al., 2014, aligned linearly according to scaffold size, showing (outside-in) GC content, scaffold boundaries, coding regions according to pan-genome status, and shared elements unique to the subclade Ic with orange lines

The final main project of my postdoc with Steve Giovannoni was a characterization of a deep-water subclade of the larger SAR11 group using single-cell genomics and metagenomics. This was a collaboration between our lab and that of Ramunas Stepanauskas and Ed DeLong, and although it began at Oregon State, the work carried over (like many projects do) into my appointment at LSU. The main findings of the paper were that this “subclade Ic” dominated deep-water metagenomic reads compared to extant surface-originated genomes, and that a few gene-content specific variations could be identified that may distinguish these organisms from the surface strains, but that, on the whole, the main observable differences were in global genomic footprints such as expected average genome size, average coding region size, intergenic spacer size, and preferential amino acid substitutions. The deep water ecotype, subclade Ic, has obviously been evolving separately from the surface subclades for some time based on these accumulated variations and phylogenomic branch lengths, however, it also appears these organisms share a similar metabolic niche with the surface strains: they are predicted to be obligate aerobic organisms with a particular appetite for common metabolic intermediates found in the cellular milieu of most organisms: simple carboxylic and amino acids and C1 and methylated compounds.

Read more here: Thrash, J. Cameron, Ben Temperton, Brandon K. Swan, Zachary C. Landry, Tanja Woyke, Edward F. DeLong, Ramunas Stepanauskas and Stephen J. Giovannoni. (2014) Single-cell enabled comparative genomics of a deep ocean SAR11 bathytype. The ISME Journal. doi:10.1038/ismej.2013.243.

 

Steve was also invited to write a Winogradski Review article in coordination with him recently being given the prestigious Jim Tiedge Award from ISME. Ben Temperton, who is also a former postdoc of Steve’s, and who worked closely with me on the subclade Ic study, above, was a co-author. Steve took the opportunity to review many of the observed variations between streamlining selection on free-living, small genomes like SAR11 and Prochlorococcus, and the small genomes of obligate intracellular symbionts like Rickettsia. Importantly, the review also pointed out that examples of streamlining selection are becoming more prevalent as data from single-cell genomics and assembled metagenomes continues to pour in. Indeed, the odd nutrient requirements that are sometimes a result of streamlining selection may be at the heart of the difficulty in cultivating many of these organisms in the lab.

Read more here: Giovannoni, Stephen J., J. Cameron Thrash, and Ben Temperton. (2014) Implications of streamlining theory for microbial ecology. The ISME Journal. doi:10.1038/ismej.2014.60

 

Dovetailing nicely with the theoretical concepts brought up in Steve’s review was a third piece from our lab, a detailed study on an unusual vitamin requirement in SAR11 that was discovered by Paul Carini, currently postdoc-ing with Alyson Santoro. This work identified that members of many SAR11 subclades are unable to make the vitamin thiamine by typical synthetic means, because they lack a single gene in an otherwise complete pathway, thiC. As a result, they are unable to synthesize 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), a direct precursor to thiamine. However, Paul also discovered that SAR11 has the capacity to use free HMP in media to relieve thiamine limitation, and identified a putative transporter for HMP in several SAR11 genomes. Additionally, HMP measurements from the Sargasso Sea and cultivation experiments pointed to a possible natural source of HMP from cyanobacteria, thus providing an explanation for how SAR11 can remain successful without the key thiC gene.

Read more here: Carini, Paul, Emily O. Campbell, Jeff Morré, Sergio Sañudo-Wilhelmy, J. Cameron Thrash, Samuel Bennett, Ben Temperton, Tadhg Begley and Stephen J. Giovannoni. (2014) Discovery of a SAR11 growth requirement for thiamin’s pyrimidine precursor and its distribution in the Sargasso Sea. The ISME Journal. doi:10.1038/ismej.2014.61

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LSUCC in full effect

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Jessica preparing medium components
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David and Brette making glycerol stocks
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Poised like an eagle ready to take flight: the Guava, at rest.

This semester, Jessica, Brette, and David have been working hard getting the high-throughput culturing lab (HTCL) up and running. Today Brette and David put samples of their first putative isolates in the freezer, thus christening the official LSU culture collection (LSUCC). We are naming this in the tradition of Stephen J. Giovannoni’s HTCC at Oregon State University, and that of the culture collections his former lab members Mike Rappé, Bob Morris, Uli Stingl and Jang-Cheon Cho. The HTCL relies on a couple principle features: dilution-to-extinction isolation techniques, and high-throughput/high accuracy counting of small cells at low cell densities. The former is assisted by use of a laminar flow hood, the latter with a Millipore Guava benchtop flow cytometer. The basic steps in the HTCL methodology are detailed in Connon & Giovannoni, 2002, and Stingl et al., 2007. Stay tuned as new microorganisms isolated by the HTCL at LSU are identified.

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Air sampling for BIOL4125

Today, with Professor Gary King’s help (who also teaches BIOL4125 but during the Fall), we did some sampling of the air in and around our classroom building, Lockett Hall. We have samples from multiple classrooms, bathrooms, hallways, and outdoors near the building. Some photos below. Students will be picking colonies next week, and later we’ll id them via 16S. Not terribly scientific, since we didn’t include appropriate controls, but it definitely scores high on nerdy coolness scales (I’ve checked them all).

The impactor sampler, into which we stuck plates with minimal medium:
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The basic supplies:
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One of the BIOL4125 students sampling the classroom- (glad Gary bought an impactor that was Tiger colors):
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Gary King processing some samples:
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The lab is coming together…

The lab is slowly coming together, and at this point most of the initial major equipment is present, if not unwrapped yet!

A panorama standing in front of the fume hood, the door to the culturing room is in the center.

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The culturing room without it’s crown jewel (Guava).

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But it’s here and ready for install…

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We’ve got a few other working items recently arrived:

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Mmm… water….

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My office

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