Cobalamin is a vitamin involved in the metabolism of every cell in the human body. It is needed for development and maintenance of the nervous system and brain. Vitamin B12 is involved in DNA synthesis and regulation of the transcription. Neither humans nor animals are able to synthesize this vitamin. Foods of animal source are the only natural source of cobalamin in human diet and Cobalamin deficiency results in heavy symptoms including brain damage. It makes it the cause of one of the major vitamin deficiency in developing countries.

We tackled this issue by the re-engineering the food microbiome of idli which is a very popular dish used as primary food source throughout India. Since the microbial ecosystem of idli doesn't produce vitamin B12, we introduced Propionibacterium freudenreichii, a bacterium that has been reported as a Cobalamin producer (Takahashi-Iñiguez et al. 2012) and assessed its fitness in the idli environment.

Propionibacterium freudenreichii is a bacterium that has the Generally Recognized As Safe (GRAS) status, and is commonly found is cheese such as Emmental. It is a natural big producer of vitamin B12, conferring high amounts of this vitamin on cheese. This is why we decided to engineer the microbiome of idli by directly adding this bacterium to the batter, in the hope of increasing its vitamin B12 content.

As a matter of fact, P. freudenreichii was found to grow well in idli (see figure on the right).

Background

In order to avoid a costly and labor intensive HPLC to analyze production of cobalamin (B12) by our strains, we wanted to develop a reliable assay which would be cheap, easy to use, precise, specific and sensitive. We chose to develop a riboswitch-based biosensor, as others have been developed for B12 which we could adapt or improve.

To test the functionality of our construct, we measured the fluorescence level emitted by our strain in the presence of increasing levels of Adocobalamin. Cells were grown in M9-glucose; fluorescence of mRFP1 and Venus and the OD600 were measured on a TECAN plate reader. Overall, we do observe a slight decrease in RFP expression as the adocobalamin concentration is increased, as would be expected if the riboswitch functions properly.

Normalizing the mRFP1 expression to the Venus expression gives you the power to get rid of any noise coming from the bacterial metabolism, or it’s environment.

We were waiting our solution containing the E. coli sensor to reach an OD of 0.1 and dilute it 1:20 with M9-Glucose. Then this diluted solution containing our bacterial sensor was inoculated (75uL) with increasing concentrations of AdoCbl diluted in M9-Glucose (75uL) in plate reader wells covered by 50uL mineral oil. M9 was used because it is free of B12 and thus their is no interference in our measure.