When choosing an organism to help us produce acetaminophen and vitamin B-12 we sought out an FDA approved microbe; however, we resorted to using a non-toxic cyanobacteria as a model for spirulina. Spirulina is generally recognized as safe organism and our genes were chosen from bio-safety level 1 (BSL-1) organisms.

L A B SAFETY

Proper PPE for BSL-1 organisms are worn in our laboratory. Within the lab space, every person is expected to be wearing gloves, long pants, close-toed shoes, a lab coat, and protective goggles. All personnel also must be accompanied with one other person so that there is no one alone in lab.

P R O J E C T SAFETY

We are growing two different strains of S. elongatus: 7002 and 7942. While PCC 7942 will act as our host organism due to its metabolic similarity to A. platensis, we plan to use PCC 7002, which requires external B12 for survival, as a growth reference for the detection of vitamin B12. PCC 7942 was obtained from the University of California, San Diego (UCSD) iGEM team and PCC 7002 was obtained from Dr. Donald Bryant at Pennsylvania State University.

Vitamin B-12 Safety

G E N E S U S E D:

I. ssuE => produces FMN reductase

Organism: Synechoccocous elongatus strain PCC 7002
Biosafety Level I

The synthesis of 5,6-DMB first requires the presence of reduced flavin mononucleotide (FMNH2). FMNH2 is reduced from flavin mononucleotide (FMN) by FMN reductase, which is encoded with the ssuE gene [19,20].

FMNH2 + NADP+ = FMN + NADPH

II. bluB => 5,6-DMB synthase

Organism: Bacillus subtilis
Biosafety Level I

Acetaminophen Safety

G E N E S U S E D:

I. 4ABH => 4-aminophenol

Organism: Agaricus bisporus
Biosafety Level I

II. nhoA => converts 4-aminophenol into acetaminophen

Organism: Escherichia coli
Biosafety Level I

T O X I T Y T E S T S

In order to judge whether our project would possibly be safe for consumption, we performed toxicity tests for the acetaminophen pathway in PCC 7942. Varying amounts of acetaminophen were added to liquid culture and the OD of each sample was observed. Liquid culture samples containing 1.32 mM, 6.62 mM, and 13.23 mM of acetaminophen were left to oxidize overnight and the color change was compared with liquid cultures samples containing similar concentrations of 4-aminophenol.