Being part way through the awesome book Experiment Eleven has inspired me to get out into the field more as Schatz did and take more soil samples. The Streptomyces are slow growing as compared to other bacteria and the production of secondary metabolites can be influenced heavily by growth media. The plan is to start with selective or semi-selective growth media in the hopes of minimizing the unwated growth of gram negative bacteria, yeasts, and ascomycete fungi (molds). Once Streptomyces are isolated, they will be subcultured onto different media like Soya Flour Mannitol, Tryptic Soy Agar, and Starch Casein Agar. I have propionic acid and nalidixic acid to help with selection.
Actinomycetes Isolation Agar w/ Glycerol
Sodium Caseinate ……………..……………………………….2.0 g
Sodium Propionate…..………..………..………..………..….4.0 g
Dipotassium Phosphate………..………..………..………....0.5 g
Magnesium Sulfate …………..………..………..………..…..0.1 g
Ferrous Sulfate …………………………………………………..1.0 mg
The pictures from a few weeks ago were of Streptomyces coelicolor and Streptomyces avermitilis on both PDA and NA (potato dextrose agar and nutrient agar, as those were the only plates I had ready that day). I re-streaked them because I was going to do some inhibition testing but instead found myself taking pictures of their beautiful pigmentation and enjoying the smell of geosmin.
I did end up using a spare plate of the coeliclor for a small streak test against Staph. The interesting thing you might note is the color change to a more reddish hue, which is due to a decrease in the pH of the local medium probably due to waste/metabolites. The blue colored actinorhodin is interesting to read about, and pretty to look at!
I’m also quite excited because the brand new UPLC-MS should be online within 2 weeks which will send natural product discovery as well as synthesis verification into light-speed. I also spent $500 of my own money to finally get an iGEM kit, and am laying the seeds for the first iGEM team at SSU before I leave to grad school next year. Lots of cool new things to do some hardcore science with!
We’re attempting our first multi-step synthesis and have finally gotten off the chalkboard, out of the notebook, and into the lab. We’re starting with a small 500mg pilot reaction to make sure everything is in working order before we scale up. As all of the reagents are being paid for out of pocket, mistakes and wasted reagents can become costly!
Acylation is a standard orgo 1 or 2 lab experiment, but doing something on paper is different than in a round bottom flask. The arrival of our acyl halide made moving forward with the (hopefully) 5′ acylation of Meldrum’s acid a possibility. Not wanting to dump our entire 25g ($40) of Meldrum’s acid in a RBF and go with the literature procedure, we decided to try it out ourselves on a small scale.
We started with 500mg of the recrystalized (from acetone) Meldrum’s acid dissolved in 2mL of DCM which had previously been dried over sodium sulfate. We followed that with 4 eq of pyridine which were added in 200uL batches over a period of about 10 minutes. Since everything was done on such a small scale, we did not use addition funnels, as adding 700uL of a reagent over 10 minutes would be futile using large glassware. Instead, we opted for small additions using a p1000 micro pipette, which seemed to work ok.
Our Schlenk line was not setup, and the nitrogen gas wasn’t delivered on time, so we weren’t able to run the reaction under nitrogen like we wanted to. While not absolutely necessary, it could theoretically improve yields by keeping moister out of the reaction container, which would quickly eat up our acyl halide.
An equimolar equivalence of our acyl halide was added to the reaction after being diluted 4x in dry DCM. Upon addition of the first small batch of acyl halide, the reaction turns yellowish orange as expected. Small partitions of 200uL of the acyl halide in DCM are slowly added to the reaction mixture over a period of about 45 minutes, and the solution starts to turn darker orange, then deep red/purple.
After the acyl halide was completely added, the reaction mixture stirred for another 30 minutes on ice, then was removed from the ice bath and allowed to stir at room temperature for another 30 minutes. The reaction mixture was then quenched with 20mL of 2N HCl and the organic layer was removed in a sep funnel. The aqueous layer was extracted 2x with DCM and the organic layer and DCM extracts were combined. The combined organic layers were washed with 2N HCl and then with saturated NaCl solution.
They were dried over anhydrous sodium sulfate to yield an orange solution, and excess solvent was removed by rotary evaporation to yield a deep red/orange oil.
TLC was done throughout the reaction, though trying to take pictures of them under UV light was kind of a pain. We’ll need to develop them with iodine in the future, especially since the acyl halide might not be visible with just UV as any unreacted acyl halide will probably convert to the carboxylic acid upon touching the silica or just atmospheric moisture. Hopefully when the NMR is up and running we can make use of though. I’d like to trust the FTIR we have but…I’m unsure of how accurate it is.
The big dot all the way to the left is the Meldrum’s acid standard. The 2nd column is half way through the reaction with 0.5eq Acyl halide added, you can see a dot slightly above the Meldrum’s acid and then a dot town towards the bottom. The 3rd column is the final extracted and rotovapped product. Since we’re adding a pretty decent sized alkyl chain, I was thinking the product would be more soluble in the mobile phase, and give a higher spot on the TLC. You can maybe see that the large dot in the final column is indeed higher than the Meldrum’s acid standard…but I’m not that comfortable in determining the success of the reaction via TLC. I’ll be happy with a decent NMR, though I’d really like to just be able to fly it in an LCMS.
A lot learned and a lot to learn, I’ll try to make picture quality and video quality better in the future, but I was just in a hurry to get going as I’m sick of waiting around for things. Take action every day!
So the reagents for the synthesis are finally almost all here, last one should arrive today. While we’ve been waiting for things to arrive we’ve been trying to get the NMR at school back up and running. A lack of nitrogen for the high pressure tank kept me from running any samples today though sadly, so will have to wait until next Tuesday to see how well it works when the gas is delivered. It’s sad to see such a nice machine (Varian 300mhz) sit in a dark back room so we’re trying to give it a purpose to come back to life!
From a granular yellow/white powder to pretty nice crystals, the first pass will be enough to test the next step which is acylation of the Meldrum’s acid with an acyl halide. The conditions call for slow addition of pyridine to the Meldrum’s acid and acyl halide in DCM under inert conditions. We’ll see how that goes…
Just a quick update that our donated strain samples are growing! We don’t have an incubator so we’re making due with a room temperature dark cabinet, so while not optimal, it gets the job done.
We plated our samples of Streptomyces coelicolor and Streptomyecs avermitilis about a week ago, and it seems they have finally undergone formation of aeriel hyphae alongside production of the pigmented polyketide antibiotic actinorhodin (in the case of coelicolor). One of the plates of coelicolor does not look the same as the other colonies though. A purple color is being produced by the organism, and they have a much smoother/slimier look to the colonies. Perhaps they are a day or two away of forming the fuzzy aeriel mycelia, or perhaps they are a mutant. We will keep an eye on them.
We will be using them in our future work as part of a bio-assay for the compounds we are making. The one picture of the brown pigment diffusing from the two colonies are those of S. avermitilis, the maker of avermectin.
So while we’re waiting for some reagents to come in for the synthesis of our first chemical product, I’ve been helping to run a class on the natural products of fungal endophytes. Most of the work we’ve been doing the past few years on our own has been surrounding bio-active secondary metabolites originating from fungal endophytes, inspired mainly by the work of Dr. Gary Strobel of Montana State University. One of our professors liked the idea enough to actually make it the main focus of one of her courses. In exchange for our help and the use of some of our equipment, we get access to a University lab space and the associated perks that come with that, like consumables and access to machines we could never afford on our own like a UPLC, NMR, GC-MS and soon an LCMS.
It’s been rewarding, but also stressful at times, as trying to do molecular biology and microbiology in a chemistry lab is…difficult. We’ve basically had to buy or borrow all the necessary equipment, and sometimes had to get creative.
Nevertheless, with a few setbacks like our -20C freezer failing and ruining all our reagents, we’ve been able to push forward to obtain some sequence data from the ITS (internal transcribed spacer) region of a few isolates the students have made.
The gel above is from students using a quick lysis method which entails a toothpick tip full of mycelia in water followed by incubation at 95C for 3 minutes, then ice until ready for use. The ladder used is a 1kb ladder from NEB. The first two lanes are +controls comparing previous DNA preps of a basidiomycete, followed by student samples.
Only three PCR products were observed on the gel, one being quite faint, in addition to the two controls. The student who prepped the reaction in lane 7 and 8 seemed to have forgotten template DNA while overloading primer, and forgetting it in the 8th tube…which potentially explains the extremely bright primer cloud and absence of it in lane 8.
Nevertheless, we were still able to get some decent reads and contigs out of the data.
For the band in well 5 – it was a white filamentous ascomycete isolated from garlic.
It appears to be from Fusarium Proliferatum which, upon a quick morphological check and Google, seems to check out. I guess that particular species is present as a plant pathogen in garlic bulbs according to google. With few mycologists around, it’s been hard to positively ID via morphology many of the species. I can only pick out with confidence Penicillium and Aspergillus species. I have a better picture on my phone of the spores, but they look like long stretched out footballs and seem to match the spore shape of many fusarium.
95C 3 min initial denature
95C 30s denatration
52C 45s annealing
72C 1 min elongation
72C 5 min final elongation
1 uL crude template
2uL ITS1 / ITS4 10uM primer master mix
12.5 uL 2x Taq NEB Master Mix
9.5 uL water
5uL PCR rxn load onto a 1% agarose gel stained with GelGreen
So the past two weeks have been pretty crazy, in a good way. I’ve managed to meet up with people from MIT, BOSSLab, and a few local biotechs on the North Shore. It’s been strange not having to wake up to go to a 9-5. I wouldn’t say it’s good, because distractions abound, but I was able to meet more people in two weeks in regards to pure science and business development than I ever had in a year prior. I suppose a 9-5 for someone else is equal to a 9-midnight job you do for yourself. Being able to meet at any time and anywhere is a good thing when it comes to progressing an entrepreneurial endeavor. Not being payed while you meet these people, though, is the downside. I can live with raman noodles and rainwater for now though.
Some people can pull off starting a side-business while working their main job, but I found that starting DNA extractions and PCR’s at 8 pm just wasn’t fun, couple that with spending the weekends in the lab and you felt like you weren’t even living life for someone in their twenties. Hard work is important, yes, but hard work and torture are two different things. We only get a finite amount of time on this planet and it can’t all be spent reading journal articles and running gels…human interaction is important to happiness and sanity.
I’d venture to say that starting a biotech company is not something that is easy to moonlight. Wittling soap into gnomes for beer money might be something you can pull off in your spare time, but multi-step organic synthesis is not.
We managed to acquire from T.K. an Eppendorf 5417C centrifuge along with some unique bacterial strains for use in a current project. I have no idea how people feel about their name being used on the internet so I will bypass naming and use just initials. We also managed to acquire from A.L. the parts for the Lightbulb PCR machine which had been developed over the past year at http://tequals0.wordpress.com/ and http://bosslab.org/
K.G. hooked it up with some E. Coli strains including a ccDB toxin/antitoxin containing strain.
All in all it has been a good two weeks. Met a lot of really interesting people all doing really inspiring work. I hope one day we’ll all make it big, and if only one of us make sit big, they’ll help the rest of us out!
But in all seriousness, the point of this post wasn’t just a quick update, but it was to mention the importance of human interaction in achieving what it is you want to achieve. Humans, by nature, want to help each other. You’d think if natural selection had its way, everything would be out to kill everything else, but that’s not the way it turned out. People feel good helping one another, and rightly so. We are all in this struggle together on Earth, each awkwardly living our own life trying to make it better for ourselves, our friends, and our family…and hopefully strangers once in a while.
So if there is one piece of advice I could give you if you are trying to start your own company, or even just living your life day to day, it would be to cultivate friendship and cultivate kindness. Random acts of kindness go a long way. Do something because it is the right thing to do, not because you hope for a reward.
Also attached are random pictures of us making some growth media for transformation experiments, followed by my strain cultivation of three different kinds of oyster mushrooms; king oyster, golden oyster, blue oyster…and last but not least the boxes that contained our $1,200 set of reagents from NEB for our future experiments. SCIENCE TIME BABY
So after years upon years of talk, planning, more talk, and more empty promises to myself and others, I finally took a leap of faith. At a certain point, you realize that there isn’t anymore time for planning and writing down ideas, action needs to be taken. Many people, myself included, have let this unattainable goal of “perfection” completely ruin pursuing dreams and business opportunities. I’ve come to the conclusion that nothing will ever be perfect. A product will never be perfect, the timing for entering a market will never be perfect, and perhaps execution of a well written business plan will never be perfect, but that should not deter one from trying.
Many people fear leaving their comfort zone, I know I did, and still do. It’s easy to become complacent in the comfort of stability and normalcy. You go to work, get a paycheck, maybe get to travel for a week or two a year, have the weekends off, and everything seems fine. Some people are perfectly happy living like that, I’m not. I’d rather sleep in a van and eat raman noodles for a year if it meant a shot at achieving my dreams and goals.
Certain sacrifices no doubt will be made, nights out with friends will now be nights in spent with Excel calculating profit margins and shipping expenses. That weekend canoe trip with your buddies will be that weekend of experiments in a cramped lab space with no air conditioning, praying for a product yield above 10%. While your girlfriend sips from a $5 bottle of triple distilled water, you sip from the rainwater you collected last night in an empty cup you found in the dumpster behind Arby’s.
But someday, when you look back at your life in one year, ten years, or fifty years, you’ll be able to say without regret that you took a shot, and that’s more than a lot of people can say.
I guess many people question what it is they want to do in life after finishing college and working for a year or two. All of a sudden this giant weight which is reality starts to rest on your shoulders, and you realize as each year passes that you haven’t done 1% of all the cool things you had hoped to be doing when you were planning your life for your future self in middle school or high-school. That trip across the country never happened, backpacking through Europe became backpacking in the woods behind your house with a 30-pack, and winning the Nobel prize while simultaneously becoming a billionaire before 30 doesn’t look like it’s going to happen.
I still have a few years left for that last challenge though…
I plan on documenting the journey via a series of videos, as well as keeping track of how I spend my time, in the hopes that one day it might be of use for someone else. There are plenty of stories out there of people’s successes, and even failures, but not many people talk about the nitty gritty in between time. That period of purgatory where you are in limbo between becoming a successful validated business, or disappearing into nothingness.
We’ll see how far the science gets us in our journey, so for now stay tuned for more content, including videos and write-ups about what it is we are actually working on and what we hope to accomplish.
A few samples were selected for hyphal tipping and pure culture isolation. Shown, in no particular order, are some of the samples to be taken and re-plated on fresh PDA. Small pieces of the mycelia were excised with a sterile scalpel and placed in the center of fresh plates. In some instances, like the dark red pigmented colony growing within a few others, hope of a perfectly pure inoculum wasn’t expected, and instead we were just interested to see what might grow on the plate.
There were instances like the first picture, in which a very homogeneous mycelial mass was obtained, which looks like a pure culture isolation. We will perform DNA isolation and ITS amplification on some of the select organisms, as well as observe some of them under the microscope. This week, agar plugs of pure strains will be put into liquid broth to ferment, with the downstream goal of processing the fermentation broth to isolate a crude extract of secondary metabolites to be used for bio-assay guided fractionation.
We’ve done enough PCR on fungal cultures and samples to know that our PCR program, enzymes, and primers are working as intended. In order to give us confidence in the ITS amplification of unknown endophytic fungal samples, we wanted to amplify a known species and confirm that we can get clean sequencing reads.
We performed a DNA extraction using our homemade buffers and an isopropanol precipitation followed by a 50uL PCR reaction.
PCR reaction conditions are as follows:
2 uL ITS master mix (ITS1F, ITS4R – see older posts for primer sequences)
We highly recommend them! They went above and beyond in order to assure we got quality sequencing data as well as went out of their way to personally call us and discuss sequencing parameters after our primers did not arrive, even with a sample order size of two. The company is run by fantastic people and is well worth supporting.
Our sequencing chromatograms looked good, with one giving a read of over ~700 bases, while the other was cut short at ~400 bases. Nevertheless, for a first try the very crude attempt at sequencing went better than expected, and enough data was retrieved to give a greater than 99% confirmation on the sample sequenced via BLAST. We did indeed do ITS amplification of Agaricus Bisporus, and the sequencing data confirms that our protocols for DNA isolation, amplification, and purification are working as intended, and lend confidence to future endeavors in the identification of unknown fungal endophytes.
In the quest to isolate new endophytic fungi and build a strain library for screening, one of the main issues we face is still being unable to afford a laminar flow hood. Sterility is a big concern in endophyte isolation, as it is very important to assure that any microorganisms found in the petri dish are coming from within the plant tissue, not on it, or outside of it. A quick google search will return many home-built laminar flow hoods as well as more simple versions such as a “sterile hood” which consists of a fish tank or plastic container turned on its side and cleaned thoroughly with bleach/isopropyl alcohol.
In this experiment, we wanted to test the bare minimum (ie, no laminar flow hood, no make-shift sterile hood) and see if we could get a control with no growth and samples that looked like endophytic fungi were growing from them.
Samples were washed for 30s under tap water, then cut into small sections and placed in 5% sodium hypochlorite for 30s, followed by 70% ethanol for 30s, followed by two sterile water washes for 30s each. 100uL of the final water wash was plated as the control.
Afterwards, samples were plated on PDA (potato dextrose agar) supplemented with chloramphenicol to inhibit bacterial growth,and left in a dark cabinet at room temperature after being sealed with parafilm.
Fast forward a week and here are how the plates look
We’ll work on a follow up post on how the samples were re-plated via hyphal tipping to try to get pure cultures, but the good news is, the control doesn’t have any growth! Also, on the Rhododendron leaf stem (the last two pictures) you can see the white mycelium growing out of the vascular tissue, pretty cool! In some of the cases, like leaving the outer bark on the ginko stem, contamination from epiphytes/ectophytes will occur, but it is still interesting observing the organisms grow.
There was at least one yeast colony on one of the ginko leaves, and a few samples were selected to re-plate to try to get pure cultures. Ideally, we’d be observing the growth of the organisms daily and taking note of their growth rates, coloration, and other features, but we both have 9-5’s and sometimes cannot always get to the lab every day. The Ivy sample was interesting to look at, but mostly discarded for further isolation because of the density of growth of the organisms.
Needless to say, it seems that with a little bit of practice and a very crude aseptic technique, you can isolate some endophytes of your own.