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We have successfully shown that we are able to barcode an unknown fungal sample, but the question now is, can we reproduce it? We have gathered the necessary tools and now face the task of making sure that our protocols are thorough, easy to follow and most importantly, reproducible. Over the past week we have ran ~28 reactions (DNA extraction, PCR, isolation). Unfortunately, what we have found is that our protocol is not as robust as we were hoping.

Samples 1 to 3 are DNA extractions from the same fungal colony using 3 different DNA extraction solutions. All used ITS primers (see below for primer sequences).

Sample 1 used an extraction solution of Tris-HCl, EDTA and SDS.
Sample 2 used 20mM NaOH
Sample 3 used 20mM KOH.

Samples 4 and 5 are DNA extractions from the same plant sample using Epoch Life Sciences Plant Genomic DNA Extraction Kit (Cat # 1560050).

Sample 4 used primer set LEP
Sample 5 used primer set rbcLA

Sample 6 used primer set rbcLA and a plant template extracted using 80mM NaOH

Sample 7 used fungal DNA extracted using 80mM NaOH and ITS primers

So what do we think is going on?

As for Sample 1 , we initially thought our extraction solution would be effective for lysing cells and extracting gDNA. Unfortunately, at the time we were not thinking of the impact SDS and EDTA would have on the polymerase/overall PCR reaction. SDS is a strong detergent that is often used to lyse cells and extract DNA, but is also very good at unfolding proteins. That is not good news for our polymerase. The other problem with sample 1 was the EDTA. Metal ions (specifically Mg2+) are essential co-factors for our polymerase to work correctly. Having too little or too much free Mg2+ can be devastating to a PCR reaction (as we are finding). In our case, the presence of excess EDTA and SDS rendered our reaction, unreactive…

Sample 2 and 3 seemed to work very well with 20mM NaOH and KOH, respectively. The simple colony PCR protocol was a tip given to us by an experienced molecular biologist. We figured that the base would be aggressive enough (but not too aggressive) to lyse cells and make DNA available for extraction. We were right! It felt good to know that that method worked since it’s so cheap and fast.

Sample 4 was expected to fail as the primers were designed for insect DNA amplification.

Sample 5 worked as anticipated. Again, this sample used the Epoch kit, so started with clean plant genomic template DNA.

Sample 6 was a plant prep using 80mM NaOH. It was encouraging to see enough plant DNA could be prepared by such a simple, fast, and cheap method and give comparative amplification to Sample 5, which was prepared using a spin kit costing ~$1.50 per prep.

Sample 7 was a failed ascomycete fungal sample prepared by grinding the sample in 80mM NaOH.

Standard 25uL reaction

1uL template DNA
0.5uL 10uM forward primer
0.5uL 10uM reverse primer
12.5uL NEB Taq 2x Master Mix
10.5uL water

Overall, these 7 samples showed us that out procedure was somewhat reproducible, but we do have some variables to consider looking into. Primer design and PCR conditions for each primer set are definitely worth checking out. Also, maybe our Taq is starting to go bad; we have taken it in and out of the freezer dozens of times, same goes for our primers. We will definitely look into this further.

This is a simple disc diffusion assay that we conjured up. The aim here is to see the effects an antibiotic has on a bacterial colony. In this case, we plated E.coli on PDA plates with no antibiotics in the media. We made a stock solution of 1mg/mL chloramphenicol in absolute ethanol and soaked small pieces of filter paper in various amounts (0ug, 5ug, 10ug, 20ug and 50ug) and placed them on plates that were freshly coated with E.coli. We incubated at 37C overnight.

The plate on the left is the before shot, prior to incubation. On the right is the plate after incubating at 37C overnight. You can clearly see the growth of E.coli around the 0ug disc (containing no chloramphenicol) and the lack of growth around the discs that do contain chloramphenicol. You can also see that the inhibition of growth is larger next to the 50ug disc versus the 5ug disc, as expected.

It’s also worthy to note that chloramphenicol is a bacteriostatic antibiotic, meaning that it does not kill bacteria, but prevents them from reproducing. If we took a plate that already had a full lawn of E.coli, we wouldn’t see complete absence of colonies after incubation, just less growth from those colonies exposed to the antibiotic. On the other hand, a bactericidal antibiotic (such as penicillin) will in fact kill bacterial colonies.

Overall, this nicely demonstrates how a disc diffusion assay works. In the future, we plan to isolate our own compounds and use this same setup to test for bioactivity and potency of our isolates. Stay tuned…

Below you can see another plate which had no antibiotics in the media and was not exposed to a disc diffusion assay.

We’ve finally acquired enough equipment from ebay, suppliers, and junk piles to conduct our own full scale test, so to speak, of our current makeshift DIY laboratory. The whole experiment involved DNA isolation, PCR, and a gel run with visualization in order to confirm that our equipment and reagents worked as intended. We managed to complete the entire experiment using (mostly) all of our own equipment, with great results.

Our equipment/reagents we were testing:

Idaho Tech RapidCycler PCR machine
Eppendorf 5415C high speed microcentrifuge
Epoch Life Sciences plant genomic DNA isolation kit
GelGreen, agarose, TAE
Gel Electrophoresis box purchased from IO Rodeo
Electrophoresis power supply from junk pile
Fungal ID primers
Taq Mastermix

Purpose of experiment – To isolate genomic DNA from a fungus, then amplify a barcoding sequence in the ITS region of its genome via PCR, followed by verification with a gel and ultimately resulting in the PCR product being sequenced and having the results BLAST searched in the NCBI database to confirm phylogeny/species ID.

Experimental overview – A button mushroom (Agaricus Bisporous) was purchased from the store for 16 cents, and a plant genomic DNA prep kit from Epoch was used on a small sample from both the cap and the stem of the mushroom (freshly cut in half).

We used two different pairs of primers: ITS1 / ITS4 and NLB4 / NSI1

These primers target an area of DNA (known as rDNA) which codes for ribosomal RNA comprising the large and small ribosomal sub-units (LSU, SSU), and the internal transcribed spacer (ITS) which has become a “standard” for barcoding and species identification of fungi.

NSI1 (forward) GAT TGA ATG GCT TAG TGA GG
NLB4 (reverse) GGA TTC TCA CCC TCT ATG AC
ITS1 (forward) TCC GTA GGT GAA CCT GCG G
ITS4 (reverse) TCC TCC GCT TAT TGA TAT GC

We expected products of around ~700 for ITS and ~900-1kb for NLB/NSI but these can vary for different mushroom varieties (ie. basiodiomycetes vs ascomycetes etc)

We tested our PCR machine, an Idaho Technologies Rapid Cycler vs a peltier effect based machine. The Idaho Tech machine uses a halogen lamp and a tornado-like air circulation effect for fast ramp times. Neither of the machines had heated lid capabilities, so mineral oil was used in each PCR reaction to avoid evaporation.

After gDNA isolation, PCR reactions were setup as follows for a final reaction volume of 25 uL in 200uL thin walled plastic PCR tubes.

10uM forward primer 0.5uL
10uM reverse primer 0.5uL
Template DNA 2uL
OneTaq 2x Master mix 12.5 uL
ddH2O 9.5 uL

Our PCR program was as follows:
94C for 30s
54C for 45s
72C for 45s

30 cycles (we wanted 35 but time didn’t allow for it)

Even at 30 cycles and those program times, the Idaho Tech machine finished ~30-45 minutes before the peltier machine, boosting confidence in the PCR machine purchased off ebay for $50.

10uL of our PCR product was run on a 1% agarose gel pre-stained with GelGreen. Product sizes were compared to 1kb NEB ladder, and single bands were observed for all wells, minus the presence of some primer dimers.

Optimization of PCR is needed, but this is a great first step which proves that with a little ingenuity and some patience in acquiring decent equipment on ebay, one can do some decent molecular biology on short funds.

The samples are being prepped for sequencing and another post will follow giving our findings.

There are a few companies whose names get tossed around when it comes to getting decent equipment, glassware, and chemicals as a DIY’er;

United Nuclear http://www.unitednuclear.com/

Elemental Scientific http://www.elementalscientific.net/

HMS Beagle http://www.hms-beagle.com/

…and today I think Home Science Tools http://www.homesciencetools.com/ can be added to the list.

Though I have only ordered from Home Science Tools, I think it’s important to at least spread the word about smaller companies that ship to individuals or non-businesses/institutions and still provide good quality equipment and customer service.  Do your own research on each of these companies (as I have heard mixed reviews in regards to Elemental Scientific’s shipping times) and compare prices as you will, but I was very happy with the HST order I made.  They also have a really great deal on shipping, and I’m not sure if its because of the small quantities they ship, or if they eat the cost.

Either way, being able to get reagents in smaller quantities is great because it offers enough material for a few experiments, but without the hastle or worry of having to store hundreds of grams of acid, oxidizers, or heavy metal salts you won’t use often.

So far, Home Science Tools was the only place I found smaller quantity reagents at really good prices, and for that reason the order was placed with them.

For anyone that cares to see the unboxing video, check out the video below.

Natural product discovery is such an attractive field because it offers someone the chance to discover a new compound, not yet known to science, with potentially life saving medicinal properties.  Even most non-scientists will know about penicillin, its use as an antibiotic during World War II, and the numerous lives it has saved since then.  There are guaranteed to be many as yet undiscovered antimicrobial or antifungal compounds out there somewhere in nature, and our goal is to one day be able to find them.  The process of identifying and purifying an unknown compound can be a daunting task, so to gain experience, we’ve decided to start with isolating a well known compound.

The basic goals are to isolate a Penicillium strain, culture it, do a liquid/liquid extraction to try to isolate penicillin, then do disc diffusion assays and check for rings of inhibition.  If all goes well and we can get a really pure sample with some separation techniques, it’d be great to run an NMR or LC-MS and see if we could elucidate its structure, pretending it was an “unknown new bio-active compound”.

The lemon piece may have been sampled too late, as the normally olive green Pencillium strain was replaced and covered with vegetative white mycelia.  Time will tell if it is a different species or not.

Above are some pictures taken at different stages of the experiment.  The lemon covered in white is the end of the experiment, the olive green was half way through.  The microscope pictures were taken from the olive green mold sample, and show Penicillium’s tell-tale fan shaped conidia.

Read more about Penicillium at  http://en.wikipedia.org/wiki/Penicillium

Below are some videos of me finally transferring some samples into liquid growth flasks, aka plastic coffee cups, to see if we can get more growth, perhaps of a semi-pure culture – and the secondary metabolites of said strain.

Although the goal was to get a conventional thermal cycler for PCR, this unique machine was too interesting to pass up doing a little more detective work on.  After reading a bit more about the RapidCycler system designed by Idaho Technology, it seemed to perfectly match what we wanted to do, amplify target sequences of under 1,000 base-pairs.   The machine is supposed to be able to do a 30 cycle amplification in about 15 minutes, pretty fast when compared to the hour or more a conventional machine might take.

The difference is, this machine uses a high power halogen lamp and a fan for rapid temperature ramping and cooling.  It can drop from a 94C denature temperature down to a 55C annealing temperature in about three seconds. Pretty fast!

It sounds good on paper, and seems to cycle well after a basic test, but it won’t be until we actually run some samples then visualize them on a gel that we can know for sure it works.

If anyone has any experience using a rapid cycler, or experience using glass/plastic capillary tubes to hold samples instead of the traditional plastic PCR tubes, let us know!  The one thing I am still a little unsure on is…how exactly to get our PCR mixes and reagents into the little capillary tubes.  I asked an Idaho Tech rep and they mentioned there is an “aspirator”, or the equivalent of a mini micro-pipettor, specifically designed to add and remove things to the capillary tubes.

We managed to snag this off ebay, so always be on the lookout for cool machines you can get to add to your lab for a decent price!

Here is a video of it in action right out of the box, we’ll keep you updated as we get some capillary tubes and an aspirator and can actually test it.

Video can go up to 720p, so change the resolution if you want a little clearer image.

Our next step for some of these fungal cultures is to be able to positively ID them by using DNA extraction methods along with PCR to amplify conserved regions of their genome. A couple of weeks ago, I was able to synthesize some common primers used for fungal barcoding. In general, some common regions that are highly conserved in fungal DNA are 15s, 5.8s, ITS1 and ITS4. We were able to come across a nice paper (Genomic DNA Extraction and Barcoding of Endophytic Fungi) that gave a solid procedure for DNA extraction, amplification and some commonly used primer sequences for fungal barcoding.

Specifically what we would like to do is culture a know fungal sample, such as Penicillium, extract its DNA, PCR a conserved region mentioned above and successfully ID it by sending the amplified region to be sequenced. If everything goes smoothly, we should be able to BLAST search the sequence we get back and confirm that we successfully isolated DNA from our sample and confirm that it is in fact Penicillium.

In doing this, we can assure that our protocol is robust enough to move on to unknown fungal samples. Hopefully from there we can move on to figuring out how to isolate secondary metabolites such as penicillin from the Penicillum culture and configure a procedure to use on unknown fugal samples.

Been a while since I’ve updated.  We have a bunch of photos we need to upload of what happened to all the fungal cultures, so hopefully that will come within the next week.  In the meantime, I happened upon a pretty neat new piece of software, thanks to the diybio google group found at www.diybio.org called Genome Compiler.

The founder and CEO of Genome Compiler was nice enough to give me permission to use some of the artwork on the website, so I’ve done a small update to provide an image which links to the company’s website.  In addition, he has made himself available on the DIYbio group, sharing lots of information, taking feedback, and offering help, which in my mind says many good things about the people working on this software.

The software is in the alpha testing stage, but it is still fun to play around with, cutting genes out of one organism and pasting them into a plasmid or another organism.   I spent a few hours shuffling genes around and reading up on BioBricks, so there is still a lot you can do even in this early stage of the software’s development.  I imagine when it is finally released it will be a pretty decent tool for synthetic biologists.  Give it a download and a try.

This is a follow up to this post, looking at what things are growing on our plates (and hopefully out of our samples).  We didn’t use antibiotics in our PDA growth media, so we expected some bacterial growth as chances are high that they would outgrow the fungi.

We were not disappointed, many of our samples showed both bacterial and fungal growth, unfortunately they also showed a lot of contamination.  We didn’t have access to a laminar flow hood during the plating phase so we just used a still air hood (basically a clean chemical hood with the blower turned off to minimize air flow.

We are chemists, remember, not really microbiologists, though we are expanding our skill-set and knowledge fast!

Either way, not all of our samples were contaminated, and our surface sterilization protocol seemed to work very well on leaves, but not on the bark of stems.  This makes more sense, as bark is much rougher and has more nooks and crevices for spores or bacteria to hide and escape the bleach or ethanol, especially since a wetting agent was not used.

Notice here in the control plate (sorry for the glare) that the leaf press after surface sterilization showed no contamination, whereas the press of the bark sample even after surface sterilization showed a few fungal colonies.  This gave some credit to our sterilization protocol, but we have to re-think how we will deal with stem and bark samples in the future.  The most likely way will be more agitation, a longer soak time, and a wetting agent.

Above are my two stem samples, the only samples I plated that showed some fungal growth on or around the excised piece of plant matter.  The leaf samples I plated have only shown yeast/bacteria.  I admit, having never done this before, I was quite happy to see anything growing at all, but I was especially excited to see what looked like two distinctly colored mycelia growing out of the sample on the right.  That third one with the green center looks to be contamination.  As for the sample on the left, that may also be growing from something that wasn’t killed during surface sterilization.

One thing I have learned is…no longer am I going to plate the brown bark/stem itself, but rather remove that and plate the soft woody tissue right below it.

Here are the samples a few days later.

I cannot say for sure whether anything that has grown was endophytic, which is what I was hoping for, or if everything was just contamination or unsterilized surface organisms. Either way, I am amazed at how beautiful some of these fungal colonies look, as well as the brilliant colors produced by some of the bacterial or yeast colonies on some other plates.

Take for example my control plate.  After a while whatever was growing on the “stem” side decided to spread across to the “leaf” side, leaving behind what looks like a sun exploding with some planets nearby and a sprinkling of stars in the background.

Anyone can pick up a book or search the internet and figure out that the diversity of microorganisms in  our environment (and on us) is immense, but there is something about actually growing the organisms yourself, and finding things you never intended to find, which made the whole process really gratifying and exciting.

I may not switch to becoming a full-time microbiologist, but I am definitely starting to look at the world through new eyes, because now I want to sample and culture every plant, leaf, flower, doorknob & cellphone I see – to figure out what is growing on or in it!

Here are a few extra photos of some leaf samples that showed no growth, and one that showed a lot of growth…of something