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Loren Launen

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ASM Microbe Day 3

Hard to even describe how exciting today’s Microbe was. Going backwards through the day – it ended with Kate Rubins, NASA astronaut (yeah – as in, spent months on the space station) and virologist, giving a keynote that was organized as a conversation with science writer extraordinaire Ed Yong. She was/is inspiring. Listening to Kate talk about her work on the space station, which was focused on figuring out how to do science in space, meant hearing phrases like “When we go to Mars we ….”. Not IF, when. I haven’t been so hopeful, and so inspired, for many years. We can solve big problems and do great things. 

And there was a lot of other great stuff! I really enjoyed listening to the talk given by Harmit Malik who was the recipient of the Eli Lily Elanco award and focuses on the molecular arms race between host and viral pathogen. From this I learned that focusing on conserved regions of proteins is not necessarily helpful in understanding evolutionary change in systems where competition is driving the bus. Better to focus on regions that change quickly because that’s where genetic innovation shows up. Time to read up on the Red Queen hypothesis! I also learned that a gene essential to making the placenta in placental mammals got into mammalian genomes from a retrovirus. Bizarre and cool (the env gene, in case you are interested). 

There was a great session on invasive group A strep that helped me understand the global importance of these infections and also reminded me of the famous tale of Ignaz Semmelweis. I would like to writ a book on this topic from an historical perspective for lay folk. Yup this is maybe a commitment on that. Maybe. Andrew Steer spoke in this session and also kindly answered many of my questions. 

And last but most important I learned more about Vibrio vulnificus, the creature I now research. Specifically we need to look (in our isolate genomes) for the rtxA gene which encodes the MARTX protein, an important virulence factor and a means of biotyping. I had a great conversation with Douglas Teague, doctoral student at the U of S Alabama, who is working on this protein and was really helpful. I also learned a lot yesterday and today from posters out of Charles Lovell’s group (U of S Carolina) on vibrio. 

Tomorrow is the day I present my own poster (which makes me nervous), and there is lots of great science to see and learn! More on day four tomorrow! 

ASM Microbe 2017 day 2 comments

Another great day at Microbe! This morning I attended a session on microbes and sex (lateral gene transfer). Big takeaway – how do we make phylogenetic trees with organisms that have so much of their genome derived from lateral gene transfer? For example, Camilla Nesbo discussed the Thermotoga (specifically, members that are mesophilic from oil wells) – a group that has at least 10 percent of it’s genome derived from lateral gene transfer in from archaea (Theotoga is a bacterial taxon). Tal Dagan spoke on transduction. This was fascinating. While I teach about transduction, transformation and conjugation as the three LGT mechanisms, there are more (i.e. Cytoplasmic bridges, namotubules and outer membrane vesicles).  This was news to me. I also learned that ALL protein families have been transferred laterally at least once in evolution! And of course, this is especially relevant in the prokaryotes. I learned we could use the tool PHAST to map prophages in genomes (prokaryotic ones). And about the fact that transduction is an important way in which gene duplication events occur (coined autologs). And… genome similarity strongly predicts the likelihood that two donor cell types / species / strains / whatever can share the same phage. As a last point – LGT by transduction has allowed some bacteria to acquire whole new metabolic abilities like acquisition of Photosystems! Wow! In the same session, James McInerney spoke on many ideas including evolution of eukaryotic cells and how to analyze data without making phylogenetic trees (which oversimplify). This was all great and amongst other tidbits I learned that in yeast the archaeally derived genes (which are less of the genome than the bacterially derived genes) – are more important to the cell by 100x fold. Even when not of clearly

 important function – whaaaat??? So what is a yeast cell? Eukaryotic? Bacterial? Or archaeal? 

At the poster session I got a lot of great V. vulnificus info. More later. 

MEGA

MEGA stands for Molecular Evolutionary Genetics Analysis, a **fairly easy program to use that creates phylogenetic trees from bacterial isolate sequence fasta files. I say fairly easy because it took me a few tries to get back into my basic proficiency using the software. It’s free for both Mac and PC.

I think the biggest issue with MEGA that I’ve run across is not the alignments, pairwise distance analysis or phylogenetic tree software suites, nor all the other options one can do to spruce up the data or make it more visually appealing – it’s figuring out what format the sequence files need to be in from step to step. So, I’ve written out a flow chart of sorts that has helped me…

Read more on my lab e-notebook

Short read sequence typing (srst2) for Vv isolates

The next step in analyzing our Vibrio vulnificus isolates is DNA analysis, specifically by short read sequence typing (srst2). Srst2 allows the facile analysis of specific loci within whole genome data for molecular typing and evolutionary analysis by phlogenetic trees. It is sensitive enough to detect genes and alleles at >5x coverage (Molecular Typing by srst2 Analysis Poster, Sanders 2016).

Basically how this works is that you have your sample sequence (ex: Sample_7274) in a fastq.gz format in a folder. These are large files containing the sample’s whole genome sequence. Then, I added a 37 gene custom database, a mix of housekeeping and virulence genes that the program will seek to find, match and extract from the whole genome sequence…

Read more on my lab e-notebook

 

 

Vibrio vulnificus under Fluorescence

Despite a rather bumpy beginning with finding viable broth cultures to plate (that was yesterday’s adventure), plates grew pretty well overnight. Interestingly enough, the plate with the ideal CFU count was 1:1,000,000 at 1 mL plated instead of the previous dilution (1:10,000 at 0.1 mL plated). I was able to count 83 distinct colony forming units which fits between 30-300 ideal CFU.

Amount Plated Dilution Factor CFU CFU/mL
0.1 mL 1:1,000,000 13 1.30 x 108
1.0 mL 1:1,000,000 83 8.3 x 107
0.1 mL 1:10,000 4 large clumps together
1.0 mL 1:10,000 too many to count

With that in hand, I smeared a tiny amount of cells onto a slide, put a drop of DAPI on top, then covered with a coverslip. DAPI binds strongly to A-T regions of DNA and can stain both live and heat fixed cells (Wikipedia). We don’t heat fix Vv because of potential aerosols…

Read more on my lab e-notebook