January2016/Experimental design
We're basing these experiments on the [https://www.ncbi.nlm.nih.gov/pubmed/16078069 Kim2015 paper.
The plan is to grow our strain in a bioreactor. We'll need high aeration throughout the experiment.
The starting conditions for flask cultures used in the paper are:
- 2% yeast extract (we'll need defined media with no uracil)
- 2% glucose
- 3% galactose
This is likely fine for our initial proof of concept. Figure 2b from the Kim2005 the paper is especially helpful in understanding the flask cultures growth.
Bioreactor measurements
Biomass
The cell concentration was measured by a spectrophotometer at 660 nm (Lambda 20, Perkin-Elmer, USA). -- Kim2015 paper
We have a working spectrophotometer that can handle 660 nm at both CCL and Biocurious :)
Glucose / Galactose concentration
Glucose, galactose, glycerol, and alcohols in the culture supernatant were analyzed at 50°C using a high-per-formance liquid chromatograph (1100 series, Agilent Technologies, USA) equipped with a Shodex-SH1011 packed column (/ 8 mm·300 mm, Showa Denko K.K., Japan) and a refractive index detector. -- Kim2015 paper
This equipment would cost around $15,000 so not within our price range.
We could get a GlucCell Glucose Monitoring System which is the same type of device used by diabetics but created especially for cell cultures. I wonder if there is a real difference or if it's just a marketing gimmick. If we buy one we should test it against a normal drug-store blood glucose meter. It can be bought for $360 and the test strips are just over $1 each and sold in packs of 50.
On its website the GlucCell is compared to the NOVA BioProfile Biochemical Analyzer so that's likely an industry standard analyzer. An older 200 series NOVA can be found on ebay for $400 in allegedly working condition and with a 6 month warranty, but it is unclear if it will need reagents/chemistry to function. I expect that it will since the product page for the newer 400 series lists the glucose test methodology as enzyme/amperometric. It is possible that it is using probes with fixated enzymes but I wonder what the lifetime on such probes would be. Also the manual is not available anywhere that I can find. Someone could contact the company and ask if they still sell reagents fro the 200 and which reagents/supplies we'd need. If it _doesn't_ need reagents then that would be a very useful piece of equipment!
Galactose would be cool to measure but it's not absolutely necessary. There are definitely galactose colorometric kits out there that will allow us to use our spectrophotometer to measure galactose.
High yield batch-fed culture
To get high concentrations Kim et al. used a batch-fed bioreactor and in the paper they detail the specifics of how they initialize it but the most important part is probably this:
During the production phase, the glucose concentration was maintained below 1 g/l and the galactose concentration at approximately 15 g/l. The temperature and pH were maintained at 30°C and 5.0 – 5.5, respectively, throughout the bioreactor operation. -- Kim2015