Thursday, July 14, 2016

Green Flourescent Protein(GFP) Beer, a short story

Yeast is used in alot of food and drink. It is used to make some of my favorite drinks, beer and whiskey.

I started engineering Yeast about 1.5 years ago. I thought it would be a useful skill to have in general and as I moved more towards running The ODIN fulltime I wanted to be able to have some yeast strains and experiments on the site.

My first ever attempt to genetically engineer yeast was a success(I used the Lithium Acetate method). I don't know how? because I didn't use SS DNA and probably used like 400ng of plasmid.

My 2nd - 10th attempts I think failed, hah.

I eventually figured out how incubation time at 42-43C matters much more than it does in bacteria and many more things and had it working again. YAY! I don't mind yeast much and almost prefer it to bacteria now.

The other part of this story is Nick Moench, who runs Inoculum Ale Works. He sent me some emails(they were loooonnggg) about how he ran his own brewery in Florida and was interested in Engineering yeast to do cool things. At the time it was just me doing ODIN stuff so I didn't have much time or money to do anything and I think we had a few emails. He almost appeared on our now maybe defunct video cast.

I guess I half-hearted thought about making beers and stuff but as I preferred to drink with the least effort possible, i.e. purchase the alcohol. I didn't think much about it. Eventually some other people started helping out with The ODIN and we ran a few preliminary experiments on fermentation using Saccharomyces cereveisiae BY4742. To us non-brewers, we were excited when we could grow GFP yeast in grape juice and still see the fluorescence, days or weeks later.

Nick and I had been chatting on facebook and I brought up how we had grown some genetically engineered labs strains of yeast and that they glow green in grape juice even after weeks. He was interested and excited. He wanted to brew some beer. Unfortunately, as non-brewers we didn't really know what went into brewing beer. We sent Nick the strain and it failed, miserably. I think Nick thought it was his fault, only for us to discover that one of the most abundant sugars in beer(maltose) couldn't be processed by most yeast lab strains(S288C derivatives have knock-outs for enzymes that import(I think) and degrade maltose, this includes BY474(1|2), W303, &c). Also, there are other things that are important like attenuation and flocculation of the yeast that helps make a good beer that brewing strains are selected for.

Sad. But not heart broken. There are plenty of brewers strains of yeast that can process maltose. Problem is that they are not auxotrophic so normal yeast plasmids would be difficult to use and select for.

We tried a bunch of different  engineering methods with a bunch of failures. Nick tried his best to nurse the lab strains in hopes that he could coax them into making a beer, almost. We both run businesses and have many other projects but it was cool having someone who actually wants to DO stuff and not just talk about it.

GFP beer like GFP yogurt is one of those fabled things that Biohackers have been talking about since Biohacking started and you can find many blog posts, writings or articles about people's theorizing about it. It's funny because at UChicago(where I did my Ph.D.), one of the many unofficial mottos is "That's all well and good in practice but how does it work in theory."(Scientists and scholars love to theorize, especially at UChicago). That is an interesting and sometimes fun part of Science but in the end if we all just theorize nothing will actually ever be accomplished. Why I hate business meetings and shit also. Let's stop talking about it and just do it. Show me what you got, I want to see what you got. Even if something is not completely planned out I have learned that sometimes you just need to say "Fuck it!" and try something out and see if it works.

We already knew that the GFP could fluoresce in sedimented yeast cells at low pH for weeks(months maybe) at a time. So I read and read some more and I know more about yeast than I wanted to(my brain-drive is already pretty full).

We manage to engineer yeast and Nick, had us run a bunch of experiments and we started working with the yeast and then he created some beer.

During fermentation you can see the glow of Green Fluorescent Protein (GFP) especially when the yeast flocculates or sediments. It is more difficult but still possible to see in bottled beer but it will require some more testing to make it a more consumer product if we eventually go that route.

People ask what it tastes like. Mostly, it just tastes like whatever you are brewing it to taste like. Nick's Brewery brews sours. So it has had a sours taste to it. We try and have some yeast sediment in there meaning there is a more yeasty taste than normal to it but otherwise there is not much in terms of flavours to discern the GFP! We debuted it at the New Harvest Conference, yesterday(July 13, 2016) and everyone was excited to try it and was excited about it!

I guess GFP yogurt is next.

Tuesday, July 5, 2016

I transplanted someone else's microbiome in(on)to my body and it was so surreal - Results - Part III

This is a case study of a 35 year old caucasian Male of European ancestry living in the United States, a maternal Haplogroup of H1e1a and a paternal haplogroup of I1*. NOD2 Genotype SNP(rs2066844) CC indicating decreased risk of Crohn’s disease. The subject presented with increased bowel movements(3+ times a day) Bristol type stool 5-7, mainly 6. Blood in stool more than 2 but less than 5 times a month. Experiences nausea, abdominal cramps and pain 2 or more days a week with no correlated inducers besides stress. Diet consists mainly of rice, vegetables and meat protein(chicken or pork). Eats out 1-2 times per week. Average kilocalories consumed per day ~2100. Height 5’9”(1.75 m) weight before experiment 167 lbs (76 kg). Exercises 1-2 days per week. Subject also presents with type II bipolar disorder and takes clonazepam as needed to help sleep. Subject has chronic sinusitis with no clear cause and has been tested for allergies and nasal polyps both coming back negative or inconclusive. Subject has been diagnosed with chronic prostatitis and was treated with ciprofloxacin and then bactrim in Dec. 2013(also last time antibiotics were taken) which did not relieve symptoms. Acute symptoms resolved to mild pain during urination.

The subject attempted a full body microbial transplant from a healthy donor Male caucasian ~30 year of age using fresh stool samples, skin, mouth and nasal swabs. Bacterial swabs were taken from, skin, mouth, nose, poop and environment before and throughout the experiment using sterile swabs and stored in 150mM NaCl and 0.01% Tween.

The subject self treated with 500 mg Tetracycline and 500 mg Ciprofloxacin, four doses over 2.5 days. Subject also performed a complete body scrub with soap and tetracycline, including a nasal rinse. The subject proceeded to stay in a precleaned hotel room using new untouched sheets. The subject did not touch another person during the course of the transplant without the use of nitrile gloves. The subject stayed in the hotel room for 3 days and 3 nights during which he ingest 3-6 grams of donor feces enclosed in gelatin pills. He coated himself with 20-50mL saline solution containing swabs from the donors skin. He also inoculated his mouth and nasal passages no less than 6 times with the donors swabs. Patient returned home and attempted to clean and sterilize ~700 sq ft (65 sq m) apartment and inoculate it with donor skin bacterial cultures.

Within one week of the experiment the subject’s bowel movements were consistently reduced to 1 time per day. Stomach pains and cramps reduced almost completely within 2 weeks. Subjects weight reduce to an average of ~ 160 lbs(73 kg) 2 months after the experiment. Diet has remained very similar(rice, veg, meat) except subject notices more meat(no craving) and a newfound craving for sugary foods. Prostatitis resolved completely. Symptoms from post nasal drip seem reduced but uncertain, symptoms still flare up at least 2-5 times a month. Bipolar disorder not affected.

A total of 77 samples were collected before, during and after experiment. DNA extraction, 16s amplicon library prep using 515f and 806r and Illumina MiSeq 151x151 sequencing was done by Argonne National Lab in Batavia Illinois. Of the 77 samples 73 had counts.

Data Analysis
QIIME 1.9.1 was used for data analysis

Samples 65(storage buffer) and 66(storage buffer and sterile swab) were control samples and used to filter out contamination using standard QIIME workflow. Afterward sample #55 had below 1000 counts and so was removed from the rest of the study.

Beta diversity was calculated for poop samples using a jacknifed subset of 5000 sequences. PCoA plots of weighted UniFrac are displayed below.

Less than two weeks after the transplant the microbiota in the gut of the subject became more closely related to the donors gut microbiota than to the subjects gut microbiota before the experiment.

Observing the different types of bacteria in the samples both on the Class and Family levels, the subjects gut had increased diversity before the transplant (#9 and #11) as compared to after the transplant (50, 51, 52, 53) and had more similar diversity to the donor’s samples (59, 60). Diversity was insinuated by the portion of a sample belonging to species other than those the top 10-15 samples, Shown by "Other".