It’s been a hot minute since last update on this lab note page. Things since last write up had been quite hectic, both in good and bad sense.

Binomica Labs

Setting up GNU Guix package manager

I had an idea to look into the history of academic posters while working on the Nanopore Community Meeting 2022 material – almost a whole year ago now.

Rough translation – the history of academic poster

It’s six in the evening where I am – sitting aside a window looking out into a field of almost complete darkness.

It’s six in the evening where I am – sitting aside a window looking out into a field of almost complete darkness. Winter evenings set in faster and darker than ever, and I’m constantly reminded of a small part of my brain still stuck in late summer and its long, drawn out fading of the day into the night. I’m pretty sure that part of my brain still thinks it’s 2021 too.

Wrapping up 2023 – review as an amateur biologist

Here’s a quick test running Dorado basecaller on ONT long read files in a Google Colab GPU instance, based on previous notes written by Miles Benton.

Dorado basecalling on Google Colab

I’ve been away from this lab notebook for a little while – for good reasons. Pace of projects I’m involved in moved at breakneck speeds, and some of them being publication facing projects ended up enforcing a bit of self-imposed embargo for the stuff I’d normally write about.

I’ve been away from this lab notebook for a little while – for good reasons. Pace of projects I’m involved in moved at breakneck speeds, and some of them being publication facing projects ended up enforcing a bit of self-imposed embargo for the stuff I’d normally write about. The first of the preprints should be coming out soon, so hopefully I’ll be able to share some interesting stuff here in a couple of week’s time.

Polishing long reads

Quick note on what might be a second-to-last test of the HMW Deinococcus genome extraction. I set up a 24 hour ethanol precipitation reaction following the usual lysis step.

Quick note on what might be a second-to-last test of the HMW Deinococcus genome extraction. I set up a 24 hour ethanol precipitation reaction following the usual lysis step. Deinococcus pellets after the 2×15 minute 37C incubation with lysozyme+TE+RNase was eluted with 140ul of the lysis buffer and 10ul of proteinase K, incubated at 60C for one hour.

Deinococcus note November 2nd

Continuing on with optimizing the Deinococcus genome extraction for long-read sequencing.

Continuing on with optimizing the Deinococcus genome extraction for long-read sequencing. While swapping out the Thermo kit lysis buffer for Sambrook bacterial lysis buffer improved the secondary lysis step greatly (Deinococcus absolutely requires double lysis, using lysozyme to dissolve the outer envelope and then using another lysis step to disrupt the bacterial membrane), it wasn’t quite enough to get improved genome yield.

Deinococcus note November 1st

Lots of little maintenance activities today: None of the later inoculations of the recent Deinococcus acquisition show the weird clumping behavior seen in the first flask, documented on previous post. At this time I’m almost certain that the clumping behavior in liquid culture was a contamination from the sponge topper for the flask.

Lots of little maintenance activities today:  None of the later inoculations of the recent Deinococcus acquisition show the weird clumping behavior seen in the first flask, documented on previous post. At this time I’m almost certain that the clumping behavior in liquid culture was a contamination from the sponge topper for the flask.

Lab activities Oct 18th

EDIT: The author of the CoLoRd paper responded with clarification and some parts of the note were corrected with corresponding EDIT tag. His response is copied at the bottom of the post (also in the comments section way below). Thank you!

EDIT

: The author of the CoLoRd paper responded with clarification and some parts of the note were corrected with corresponding EDIT tag. His response is copied at the bottom of the post (also in the comments section way below). Thank you! I recently found out about a new raw sequencing data compression and archiving tool called CoLoRd (reference Kokot M, Gudys A, Li H, Deorowicz S. CoLoRd: Compressing long reads. bioRxiv.

Raw sequencing data archiving test II- with CoLoRd

A particular midpoint rooted Deinoccoccus-Thermus phylogenetic tree I made during the last SCG hmm set curation have been on my mind for the last few days.

Rethinking Truepera radiovictrix among Deinos – an amateur note

I recently wrote about the 16S barcoding results for our mysterious East River bacteria – it’s quite likely that the bacteria is a novel Methylobacterium from class alphaproteobacteria, and we’re going to try a full genome sequencing soon.

I recently wrote about the 16S barcoding results for our mysterious East River bacteria – it’s quite likely that the bacteria is a novel Methylobacterium from class alphaproteobacteria, and we’re going to try a full genome sequencing soon. Here’s a casual record of observation and study so far, in preparation for an eventual manuscript, starting with the initial course that started all this eight months ago.

East River screening so far: Oct 2020-June 2021

One thing I used to do with some of the environmental phage sampling runs in the past (both soil and liquid) was to simply crash them with cracking buffer and see what sort of bands I would get on a gel – of course, most of the time whole environmental samples would result in genomic […]

One thing I used to do with some of the environmental phage sampling runs in the past (both soil and liquid) was to simply crash them with cracking buffer and see what sort of bands I would get on a gel – of course, most of the time whole environmental samples would result in genomic smears, but occasionally I would see some defined bands indicative of some dominant culture (with a genomic compartment small enough to be resolved on your typical

Cracking buffer for East Water sample

In a past episode of our slow misadventures into nature-poking, we PCR’d out 16s barcoding sites from a East River bacterial sample and a peculiar branching bacteria sampled from an iridescent biofilm formed around a mangrove culture.

Barcoding strange bacteria part II

I decided to do a quick pigmentation extraction and spectral sweep test for the East River bacterial sample B and D today, with older Halobacteria NRC-1 cultures acting as controls. The extraction protocol is the same one I described in a previous post.

Spectrum checking East River bacteria

Another delayed post, with a bit of a hectic schedule.

East River screening re-plating, and note on Deinococcus radiophilus raw reads

The day is finally here! Back one year ago, when we were just beginning to hear about the COVID-19 being an issue (good grief that feels like a distant past) – we discovered a strain of Halobacterium NRC-1 that seems to thrive in acidic media with pH of about 5.5~ from an older, neglected culture vessel.

One year anniversary of mutant Halobacterium NRC-1, and finishing the research paper.

The main media used in our lab for Halobacteria NRC-1 propagation is ATCC 213, consisting of below components for a 500ml batch NaCl 125g MgSO4 5g KCl 2.5g CaCl2 0.1g Yeast Extract 5g Tryptone 1.25g The media straight after mixing usually has a pH of 5.9~ or so. I keep two batches at a time, […]

The main media used in our lab for Halobacteria NRC-1 propagation is ATCC 213, consisting of below components for a 500ml batch NaCl 125gMgSO4 5gKCl 2.5gCaCl2 0.1gYeast Extract 5gTryptone 1.25g  The media straight after mixing usually has a pH of 5.9~ or so. I keep two batches at a time, one at the acidic pH for the Halobacteria mutant and another one adjusted to be + pH 7 with 1M KOH solution.

Hypersaline media and its discontents

Earlier than expected results on the East River water screening for bacteriochlorophyll a producing alphaproteobacteria. Both of the homemade media plates from the 19th show healthy colony growth as of the morning of 23rd, mere three days after inoculation.

Earlier than expected results on the East River water screening for bacteriochlorophyll a producing alphaproteobacteria. Both of the homemade media plates from the 19th show healthy colony growth as of the morning of 23rd, mere three days after inoculation. What surprised me is the number of carotenoid producing colonies on the plates – the 100ul undiluted inoculation plate shows five colonies.

East River screening II

Or rather, there was an attempt. Things had been a little hectic with our final dash to finish the final draft of the Deinococcus research project that dragged on for close to two years – and make no mistake, those were some tumultuous two years of research.

Or rather, there was an attempt. Things had been a little hectic with our final dash to finish the final draft of the Deinococcus research project that dragged on for close to two years – and make no mistake, those were some tumultuous two years of research. Keeping true to the spirit of amateur biology, my full time job isn’t anything even remotely biology related.

Quick and dirty TLC analysis of carotenoid

I ran a quick test of a makeshift kitchen media for East River screening, based of the PPES-II used in some older Deinococcus and Alphaproteobacteria research papers.

I ran a quick test of a makeshift kitchen media for East River screening, based of the PPES-II used in some older Deinococcus and Alphaproteobacteria research papers. I’ve had some good results with milk powder and marmite based media for Deinococcus radiophilus before, and now might be as good time as ever to formalize some kitchen media for routine lab processes.

Kitchen media test – East River screening

It’s been a rather eventful week, something I’ll need to catch up on the lab notes later. For now, please enjoy the pictures of our new Flongle adapter (and our flowcells, but I really don’t want to take them out of the packaging yet)! Chances are we’ll be using these for organelles and phage sequencing.

It’s been a rather eventful week, something I’ll need to catch up on the lab notes later. For now, please enjoy the pictures of our new Flongle adapter (and our flowcells, but I really don’t want to take them out of the packaging yet)! Chances are we’ll be using these for organelles and phage sequencing.   The adapter is literally an adapter that would allow us to use Flongle flowcells on our Minion sequencer. Watch out for the contact ports!

Flongles arrived!

The screening study for aerobic anoxygenic photoheterotrophic alphaproteobacteria in the East River continues, even as we are on the verge of the seventh draft for our Deinococcus radiophilus paper.

The screening study for aerobic anoxygenic photoheterotrophic alphaproteobacteria in the East River continues, even as we are on the verge of the seventh draft for our Deinococcus radiophilus paper. While the skeleton of the physical study is being planned out I’m focusing on the initial survey of the known bacterial genomes to get a better idea of what I could expect in the field (“

 Seventy percent of all archeology is done in the library.

Bacteria-alphaproteobacteria bchl screening

As I have mentioned in other places before, one of my primary goals as an amateur researcher is to sample and find phages specific to the Deinococcus genus, mostly through testing samples from the East River.

Deinococcus soft agar experiment continues

The weekend continues with the repeat run of the previous Deinococcus radiophilus soft agar run. In our last run, the plates still showed anomalous growth behavior even in the absence of CaCl2, and without U.V. activation to a certain degree.

The weekend continues with the repeat run of the previous Deinococcus radiophilus soft agar run. In our last run, the plates still showed anomalous growth behavior even in the absence of CaCl2, and without U.V. activation to a certain degree. I should eliminate the most straightforward source of the error first, focusing on an even mixture of soft agar and culture.

Deinococcus soft agar – Oct 3rd

It’s a common practice for amateur researchers, community labs, and some of the more cash-strapped research labs (there’s more than you might imagine!) to use common grocery store variety distilled water for non-essential media and reagents needs.

It’s a common practice for amateur researchers, community labs, and some of the more cash-strapped research labs (there’s more than you might imagine!) to use common grocery store variety distilled water for non-essential media and reagents needs. Well, if you’re one of the many in such situation, you might want to keep your eyes on the pH level of dH2O.

pH for frugal water sources

I maintain two different Halobacteria lines as a kind of off-time activity to cool my head with.

I maintain two different Halobacteria lines as a kind of off-time activity to cool my head with. They both started out as typical Halobacterium sp. NRC-1, but after a year or so of culturing them I was able to isolate a rather oddly behaving subculture of the original cells and are currently in process of analyzing them.

Quick Halobacteria genome extraction

As people say, time flies. Unbearable heat of the summer at the warehouse lab, compounded by emanations from overheating computers are still vivid in memory as if it was yesterday. Now, I’m looking at the last of autumn leaves as the flora sheds their colors and turn gaunt one by one.

As people say, time flies. Unbearable heat of the summer at the warehouse lab, compounded by emanations from overheating computers are still vivid in memory as if it was yesterday. Now, I’m looking at the last of autumn leaves as the flora sheds their colors and turn gaunt one by one. We’ll be welcoming winter in the next few weeks at most. I recently took a small sojourn to a friend’s place, hoping to recover from a recent health issue.

Treebuilding common E. coli strains

While on the usual internet lam traveling down the endless piles of papers reporting on fascinating new mysteries from the microbial world, an MRA stood out and caught my attention.

Looking into Pangean relics

Noting an interesting observation on culturing the new Deinococcus species in the lab. I’ve been running some tests regarding growth conditions of the new Deinococcus species, and am pretty confident the particular species grows far better under room temperature conditions, as far as liquid cultures are concerned.

Noting an interesting observation on culturing the new Deinococcus species in the lab. I’ve been running some tests regarding growth conditions of the new Deinococcus species, and am pretty confident the particular species grows far better under room temperature conditions, as far as liquid cultures are concerned. Attached above is a picture of two cultures inoculated six days apart from the same colony on a plate.

Deinococcus note October 20th

It’s been a while since the last lab note update – August 26th post on Halobacterium genome extraction was the last post, more than a month ago.

It’s been a while since the last lab note update – August 26th post on Halobacterium genome extraction was the last post, more than a month ago. The Halobacterium sequencing process had run through the traditional Binomica labs path in the meantime, which usually involves starting out as a quick weekend project and then running into unexpected difficulties, requiring far more time and resources than was originally anticipated.

East River screening continued

Last Halobacteria genome extraction test (for ONT Flongle sequencing) happened without using any real reagents like proteinase K and RNase – both to see how the skeleton protocol fares with the culture (which is a pretty standard way we do things around here) and because we’re completely out of the said reagents at the time.

Last Halobacteria genome extraction test (for ONT Flongle sequencing) happened without using any real reagents like proteinase K and RNase – both to see how the skeleton protocol fares with the culture (which is a pretty standard way we do things around here) and because we’re completely out of the said reagents at the time. The results from the initial test was very promising.

Halobacterium high molecular weight genome extraction test 2

It’s finally that time of the year – our annual Halobacteria mutant strain genome sequencing and assembly. Quick recap; our lab has an acidophilic/tolerant (robust growth at pH 4.0~5.0) Halobacteria mutant strain from around 2019.

4th annual Halobacteria sequencing: genome extraction

It’s a special week over at Binomica Labs.

Halobacteria mutant sequencing; 3 years in

With the dusts of a hot and tumultuous September finally in tow, I decided to spent the month’s last few days at my friend Sebastian’s new place. His new lab is situated atop a hill overlooking a small pebble strewn Long Island beach side, surrounded by fresh air and lush greenery.

Salt marsh sampling and Opentrons

This is the first post under the ‘history’ heading on this online lab note, so I’ll start with a bit of a tangent on why there will be more science history related notes and posts on this site going forward.

Searching for Phi X 174

As I’ve written about a couple of times in this lab note, I have ongoing still-cultures of Halobacterium NRC-1 control and its tentative acidotolerant mutant strain going on in our studio-lab.

Halobacteria note; sedimentations from stoppered still culture flasks of NRC-1 mutant and control

Tonight we’re continuing on with the weekly study of the Tree Thinking textbook, proceeding to chapter 2. “Tree thinking and its importance in the development of evolutionary thought.” The chapter covers the transition from ladder thinking around the concept of evolution to tree thinking, beginning with Jean-Baptiste Larmarck’s concept of Scala Naturae- the ladder of […]

Tonight we’re continuing on with the weekly study of the Tree Thinking textbook, proceeding to chapter 2. “Tree thinking and its importance in the development of evolutionary thought.”   The chapter covers the transition from ladder thinking around the concept of evolution to tree thinking, beginning with Jean-Baptiste Larmarck’s concept of Scala Naturae- the ladder of life- (described in his 1809 work Philosophie Zoologique) as an example of

Tree Thinking Chapter II – Tree thinking and its importance in the development of evolutionary thought.

I’ve mostly stumbled my way into the amateur pursuit of biology, lacking a realistic path to studying the topic in advanced settings.

I’ve mostly stumbled my way into the amateur pursuit of biology, lacking a realistic path to studying the topic in advanced settings. While the unconventional choice led me down a more curious path than experienced by many others, I’ve always been dogged by a constant suspicion that the lack of structured learning in my background left me wanting in deeper understanding of the fundamental concepts in biology.

Thinking about amateur learning – and diving into the Tree Thinking Chapter 1

Our last attempt at 16s barcode sequencing the East River bacteria wrapped up with out a solid conclusion, owing to the less than spectacular sequence fidelity, and the question of Methylobacterium hit from the salvaged sequence remaining suspicious on the account of it being a contaminant of sequencing reagents.

Barcoding strange bacteria part III

Sometimes availability of a good tool marks the beginnings of great research questions, just like the case with the wonderful GToTree– a phylogenetics tool I view as the gold standard for utility, ease of use and documentation for researchers and students alike.

Writing the tree – Single Copy Gene set generation with pfam

Last year’s experience working with MacLea lab brought with it quite the varied ensemble of microbial sequencing side-projects, involving both long and short read platforms.

Nanopore: archiving signals

Continuing on with the Halobacterium long-term culture observations. So out of the ‘unknown status’ cultures recorded on January 28th, 2020/Mar17 and 2020/Mar30 cultures remain questionable. I think Mar30 culture will end up picking up soon, but it could be safe at this stage to assume Mar17 culture is simply dead.

Continuing on with the Halobacterium long-term culture observations. Confirmed living as of February 11 2022  1) 2021/Jan14 Halo R control : doing well, I don't see a difference between this culture and other, far fresher inoculations. Same goes for all other cultures (including the mutant culture in acidic media) inoculated on the same day.

Lab note Feb 11

Alphabet representation for protein amino acid residues can fun to play around with – let’s see what a season’s greeting looks like with a quick blast search: Dumping above into NCBI blast shows: Interesting! I’m surprised we got any hits at all – let’s look at the alignment quality: Ah – this is no good.

Alphabet representation for protein amino acid residues can fun to play around with – let’s see what a season’s greeting looks like with a quick blast search: &gt;greetings MERRYCHRISTMASANDHAPPYNEWYEAR  Dumping above into NCBI blast shows:  Interesting! I’m surprised we got any hits at all – let’s look at the alignment quality:  Ah – this is no good.

MERRYCHRISTMASANDHAPPYNEWYEAR

The surprisingly eventful high molecular weight genome extraction tests for the new Deinococcus species continues. All this is a bit of a pain point inherent in self-funded amateur research, since purchasing specialty kits for different lab processes needs to be avoided whenever possible, both for sake of knowledge and for financial restraints.

Deinococcus note October 30th

Prior to our deep dive into sequencing and assembling the Deinococcus radiophilus genome, we shared the common wisdom among the laypeople, and perhaps more people in the research community than most would like to admit;

Prior to our deep dive into sequencing and assembling the Deinococcus radiophilus genome, we shared the common wisdom among the laypeople, and perhaps more people in the research community than most would like to admit; that any bioinformatics processes on the prokaryotic and phage genome scale are such trivial processes on any modern hardware, one would simply need a laptop (perhaps not even a good one) to crunch through all research questions.

Frugal bioinformatics – genome archiving bench test

A quick note for the day – primary media used in the lab for anything related to Deinococcus, phages or otherwise, is TGYM recipe as mentioned in the paper by Lewis, N.F. Studies on a radio-resistant coccus isolated from Bombay duck (Harpodon nehereus) describing the original isolation and characterization of the Deinococcus radiophilus.

A quick note for the day – primary media used in the lab for anything related to Deinococcus, phages or otherwise, is TGYM recipe as mentioned in the paper by Lewis, N.F.

 Studies on a radio-resistant coccus isolated from Bombay duck (Harpodon nehereus)

describing the original isolation and characterization of the Deinococcus radiophilus.

Deinococcus growth media note

2022 finally draws to a close with coming of an unusually warm and humid first week of December in the city.

2022 finally draws to a close with coming of an unusually warm and humid first week of December in the city. This year was a tumultuous one, but life goes on. Completion (of a sort) of the Oxalis stricta chloroplast genome this summer prompted me to submit an abstract for this year’s Nanopore Community Meeting in NYC, coincidentally the first in person/hybrid meeting since the pandemic.

Nanopore Community Meeting 2022

My initial attitude during the planning stages of Deinococcus radiophilus sequencing project was that the organism would provide a good first step for delving deeper into nitty gritty details of modern microbiology, and that I’ll eventually grow out of it. However, the more I study the genome of Deinococcus radiophilus, the more interesting the entirety […]

My initial attitude during the planning stages of Deinococcus radiophilus sequencing project was that the organism would provide a good first step for delving deeper into nitty gritty details of modern microbiology, and that I’ll eventually grow out of it. However, the more I study the genome of Deinococcus radiophilus, the more interesting the entirety of the Deinococcus genus becomes.

Weekend crispr tree trip

A rather busy week, I’m trying to finish up the final draft of our Deinococcus research paper before the year is done, so we can get with formatting and other final touches necessary to publish it officially early next year.

A rather busy week, I’m trying to finish up the final draft of our Deinococcus research paper before the year is done, so we can get with formatting and other final touches necessary to publish it officially early next year. I’ve been on an edge lately when it comes to handling the Halobacteria culture in the lab, now that a contamination in hypersaline media is a known possibility.

Quick updates – Halobacteria mutant behavior and new lab equipment

Our preprint addressing a fascinating Haloarchaeal genome discovery is right around the corner! There’s always a chance what we’re looking at is erroneous, but due diligence so far suggests otherwise. I’m really excited about the initial feedback- it looks like my first official publication could start off with a bang.

Schoop, G. Halococcus litoralis, an obligate halophilic colorant. German veterinary weekly. 1935;43:817-20.

Quick lab note update for today’s new arrival. Our small amateur biology lab’s microscopic zoo continues to grow in size- some plates and agar stabs of a new Deinococcus species (to us) just arrived from our collaborator’s lab, along with a fresh rapid sequencing kit for round two of rapid long-read sequencing experiment.

Shipment from our collaborator – a new addition to our library of microbes

The initial test recorded in the blue shift DNA mutagenesis test II entry failed, most likely due to the state of the competent E.coli. We ran the same experiment described in the previous post again, with freshly prepared NEB turbo competent E.coli.

The initial test recorded in the blue shift DNA mutagenesis test II entry failed, most likely due to the state of the competent E.coli. We ran the same experiment described in the previous post again, with freshly prepared NEB turbo competent E.coli. 24 hours of incubation with 660nm red LED gave us the same result as before, with the methylene blue containing sample significantly degraded through photo reaction.

Blue shift DNA mutagenesis test III

Before going further – Jan 27th was something of a momentous day. Our Deinococcus radiophilus genome submission finally became the representative genome on Genbank! Now I just need to get the preprint out there.

Before going further – Jan 27th was something of a momentous day. Our Deinococcus radiophilus genome submission finally became the representative genome on Genbank! Now I just need to get the preprint out there. I’m in middle of a big re-arrangement of the warehouse lab, finally dusting our bench and wiping down all the grime accrued over the pandemic period.

Lab note: Jan 17-Jan 28

Note: This particular lab note is from an ongoing project in collaboration with a different lab. Please pardon some of the necessary vagueness.

Note:

This particular lab note is from an ongoing project in collaboration with a different lab. Please pardon some of the necessary vagueness. All this is pre-publication data, so I have to blur out some details until we can at least get a preprint out (which should happen soon, fingers crossed!). I’ve been working on a particularly befuddling genome of a previously unsequenced Archaeal microbe for the last couple of months.

ONT long-read assembly note: on unusually difficult archaeal genome

Our warehouse lab has a new addition – a Thelio desktop computer from System76, fully decked out with 32 thread AMD Ryzen 9 5950X and 64GB of ECC RAM.

Our warehouse lab has a new addition – a Thelio desktop computer from System76, fully decked out with 32 thread AMD Ryzen 9 5950X and 64GB of ECC RAM. Of course, I couldn’t afford a machine like this with our amateur biologist resources – we were fortunate enough to have been chosen for the “Unleash your potential” program ran by System76 earlier this year aimed at the maker/hacker crowd.

Genomes in the machine

A quick note on minor housekeeping stuff for the unidentified Methylobacterium species we recovered from the East River (December 25th, 2021). Some of the culture plates started exhibiting anomalous growth patterns on the PPES-K plates.

A quick note on minor housekeeping stuff for the unidentified Methylobacterium species we recovered from the East River (December  25th, 2021).  Some of the culture plates started exhibiting anomalous growth patterns on the PPES-K plates.

Methylobacterium update

The Halobacterium genome extraction optimization for Nanopore Flongle sequencing continues. After a couple of weeks of some strange hectic schedules, I was finally able to sit down for the last couple of days to really think about the protocol at hand.

The Halobacterium genome extraction optimization for Nanopore Flongle sequencing continues. After a couple of weeks of some strange hectic schedules, I was finally able to sit down for the last couple of days to really think about the protocol at hand. 82nd culture of Halobacterium was used a little earlier than I normally process them – these cultures were two days old by the time of extraction.

Halobacterium high molecular weight genome extraction test 4

Following is an early work-in-progress draft for an upcoming zine “Genome assembly for artists.” The content here is released under CC BY-NC-SA license. Normally I work with publicly accessible databases full of assembled genomes and other biological data, like the almost eponymous NCBI Genbank.

“Genome assembly for artists” draft part 1

Tonight marks the completion of our third Deinococcus genome extraction test. First two processes were adopted from my Halobacterium protocol – alas, they were both subpar compared to the Deinococcus radiophilus genome extract we made two years ago.

Deinococcus note October 26th

A quick update on the third high molecular weight extraction test on our Halobacterium – the culture used for this test is our ~80th subculture. With this we’ve been running continuous acidic media subculture of Halobacterium mutant strain for 551 days, or a little over a year and a half.

A quick update on the third high molecular weight extraction test on our Halobacterium – the culture used for this test is our ~80th subculture. With this we’ve been running continuous acidic media subculture of Halobacterium mutant strain for 551 days, or a little over a year and a half. I can’t wait to wrap up the genome extraction tests and finally sequence this mystery microbe.

Halobacterium high molecular weight genome extraction test 3

Just a quick post for record’s sake. Initial liquid culture inoculation made on the 9th seems oddly slow, and displays clumping in the flask.

Liquid media subculture of the new Deinococcus species

The cultures of red-tinted bacteria (hopefully a bacteriochlorophyll utilizing species of some sort) from previous East River sampling run were getting a little long in the tooth, so I subcultured them into the new, smaller autoclavable plates with better seal using the last of the PPES-K media.

East River sample housekeeping, and barcoding strange bacteria

The new Deinococcus species we received for a collaboration grew on our TGYM media without any issues. New plates were struck out on October 6th, and colonies formed by early October 8th, somewhat less than two day turnaround in line with our experience with Deinococcus radiophilus.

The new Deinococcus species we received for a collaboration grew on our TGYM media without any issues. New plates were struck out on October 6th, and colonies formed by early October 8th, somewhat less than two day turnaround in line with our experience with Deinococcus radiophilus. The colonies look as ‘wet’ as we’ve seen with D. radiophilus on the TGYM plate, though it displayed a minor crumbling behavior when inoculated to liquid TGYM.

First subculture of the new Deinococcus strain

The Halobacterium flongle sequencing data is approaching rapidly – I decided to take the week to clean up our in house high-molecular weight DNA extraction protocol, adapt them for the S-layer membrane of Halobacterium (for example, the extensive freeze-thaw process we used for Deinococci and gram-negative microbes would not be necessary here) and test out […]

The Halobacterium flongle sequencing data is approaching rapidly – I decided to take the week to clean up our in house high-molecular weight DNA extraction protocol, adapt them for the S-layer membrane of Halobacterium (for example, the extensive freeze-thaw process we used for Deinococci and gram-negative microbes would not be necessary here) and test out the process against our Halobacterium strains.

Halobacterium high molecular weight genome extraction test 1

The Flongle sequencing of our potentially mutant Halobacteria NRC-1 is right around the corner (as soon as we get that interminable computer hardware issue out of the way), and I’ve been busy catching up on literature and compiling tools and data we might need for post-sequencing analysis.

Many eyes of Halobacteria – screening Archaea for retinalphototrophy

In a previous lab note entry I outlined a workflow for curating a subscreened single copy gene HMM set from a selection of genomes and pfam-HMM, courtesy of the GToTree pipeline. The process works quite robustly, and I was able to make more than a couple of very interesting datasets through it since then.

Making use of single copy gene HMM set – in mostly bash way

We’re almost there – final Sanger sequencing confirmation of Par A type protein and an integrase in our Deinococcus radiophilus genome before updating our draft for final publication. At last! Tonight’s run was a straightforward Taq PCR to check our primer design against our genome extraction stock.

Last PCR before the publication!

The blue shifted plasmid samples from yesterday’s test was taken out of the red light exposure chamber and transformed with NEB turbo E.coli strain. Hopefully the full 24 hour exposure was enough to induce purine mutation in the naked DNA, we’ll find out soon.

The blue shifted plasmid samples from yesterday’s test was taken out of the red light exposure chamber and transformed with NEB turbo E.coli strain. Hopefully the full 24 hour exposure was enough to induce purine mutation in the naked DNA, we’ll find out soon. The methylene blue certainly reacted with the red LED at 660nm during the incubation.

Blue shift DNA mutagenesis test II

Another round of observation from the initial East River sampling, on the road to discover bacteriochlorophyll a producing aerobic alphaproteobacteria strains from our own local environment (who can afford ATCC strains out of pocket these days?

Another round of observation from the initial East River sampling, on the road to discover bacteriochlorophyll a producing aerobic alphaproteobacteria strains from our own local environment (who can afford ATCC strains out of pocket these days? Har har).   The 1/1 East River inoculation plate is looking quite overgrown by this point, with signs of some obvious fungal growth.

East River screening IV

We’re running a quick and dirty test of methylene blue based DNA mutagensis method (based off their photosensitive quality) today, something to occupy our mind while waiting for the new system 76 rig to arrive (for our upcoming mutant Halobacterium NRC-1 genome sequencing project). The test is a simple one involving methylene blue diluent and […]

We’re running a quick and dirty test of methylene blue based DNA mutagensis method (based off their photosensitive quality) today, something to occupy our mind while waiting for the new system 76 rig to arrive (for our upcoming mutant Halobacterium NRC-1 genome sequencing project).   The test is a simple one involving methylene blue diluent and 660nm LED exposure over a 24 hour period – hopefully I’ll be able to test out a full on UV lamp

Blue shift DNA mutagenesis test

Quick update on the day’s activity before the weekend. Growth comparison of the halomedia fungi contamination against YPD media yielded a somewhat disappointing result. The fungi seems to thrive in YPD with far slower growth rate in regular and acidic halomedia we have in stock.

Quick update on the day’s activity before the weekend. Growth comparison of the halomedia fungi contamination against YPD media yielded a somewhat disappointing result. The fungi seems to thrive in YPD with far slower growth rate in regular and acidic halomedia we have in stock. Whatever the contamination is, it’s merely very halotolerant and not a halophile organism we could use as a model.

Halomedia and East River screening update

I ran out of the original iteration of the kitchen media built around PPES-II using store bought milk and soy protein powder. The original media performed above expectations for the East River environmental sampling, but not quite so with the regular propagation of our in-house model organisms, notably brewer’s yeast and Deinococcus radiophilus.

Improving the microbio kitchen media

With yesterday’s test PCR successful, we immediately got to Q5 (high fidelity enzyme) PCR for Sanger sequencing of the final genome fragment. After this, we can finally update the Nanopore genome and finalize the figures for our publication.

Last PCR round 2

For the past couple of weeks, I’ve been part of an exciting class on frugal science (https://www.frugalscience.org/) – the class consists of lecture-workshop portions and student-led research projects to be wrapped up and presented by the end of the course.

For the past couple of weeks, I’ve been part of an exciting class on frugal science (https://www.frugalscience.org/) – the class consists of lecture-workshop portions and student-led research projects to be wrapped up and presented by the end of the course. The lecture portions are interesting enough, with emphasis given to presenting tidbits of knowledge to broaden the perspective more so than detailed execution of projects.

Diving into Frugal Science course

A quick report on the East River water sample struck out on home-made PPES-II type media. Two days were enough for the new plates to propagate, though the initially white colonies will take some time to accrue whatever the pigment they were originally producing on the raw East River water inoculation plate.

A quick report on the East River water sample struck out on home-made PPES-II type media. Two days were enough for the new plates to propagate, though the initially white colonies will take some time to accrue whatever the pigment they were originally producing on the raw East River water inoculation plate. The original inoculation plate continues to grow. I’m still quite impressed by the controlled growth of the river microbes.

East River screening III

I’ve been reading into bacteriochlorophyll a utilizing alphaproteobacteria recently, prompted by the question of selecting among wildtype microbes and studying their evolution long-term. Chances are, whatever the sample I’ll be able to screen from the East River would be carotenoid producing aerobic photoheterotroph, likely from the upper sediment layer.

Halobacteria carotenoid extraction

Results of Deinococcus radiophilus soft agar run from last week is finally out – and it is about as I expected, as disappointing as it might be. While I was hoping for a confirmation of the sometimes bizarre growth pattern of Deinococcus radiophilus (observed across multiple different labs!), at least as far as our current […]

Results of Deinococcus radiophilus soft agar run from last week is finally out – and it is about as I expected, as disappointing as it might be.   While I was hoping for a confirmation of the sometimes bizarre growth pattern of Deinococcus radiophilus (observed across multiple different labs!), at least as far as our current soft agar irregularity is concerned, the results are far more pedestrian than I might have expected.

Commonplace Lab