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A B C

The observation of three consecutive cell cultures grown in BG-11(N+) showed a selection for cells that grew faster in BG-11(N+). A.) The average growth curves of a third passage inoculated in early August at OD₆₀₀, OD₆₅₀, OD₇₃₀, and at OD₇₅₀ respectively. The cell culture is seen to enter log phase at day 7. B.) The average growth curves of a fourth passage inoculated in late August at OD₆₀₀, OD₆₅₀, OD₇₃₀, and at OD₇₅₀ respectively. The cell culture is seen to enter log phase at day 6. C.) The average growth curves of a fourth passage inoculated in late October at OD₆₀₀, OD₆₅₀, OD₇₃₀, and at OD₇₅₀ respectively. The cell culture is seen to enter log phase at day 4.

2c. Toxicity Tests

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 1.32mM, 6.62mM, and 13.23mM of acetaminophen were left to oxidize overnight and the color change was compared with liquid cultures samples containing similar concentrations of 4-aminophenol.

Acetaminophen toxicity tests

4-aminophenol toxicity tests

Cell culture toxicity tests were performed with 4-aminophenol. Cell cultures were observed after the addition of 1.8µM, 9.1µM, and 18.3µM respectively. All samples with 4-aminophenol added turned from dark green to dark brown overnight.

A cell culture toxicity test suggested 13.32mM (10mg) of acetaminophen to be detrimental to cell growth. While the amount of time at which the cell culture showed discoloration from green to nearly clear to brown varied in duplicate samples, both concluded with cell death and discoloration of cell sample. Cell culture containing 6.6mM (5mg) of acetaminophen showed inconsistent discoloration and cultures containing 1.8mM (1mg) of acetaminophen showed no significant color change. Cultures were also tested with 1.8µM and 2.9µM of acetaminophen (to align with the expected amount based on a patent for the biosynthesis of acetaminophen in E. coli^[1]. No significant discoloration was observed. The discoloration and subsequent color change of the culture is suspected to be due to the degradation of acetaminophen into its precursor 4-aminophenol. Further tests to determine the contents of the sample following the observed color change is recommended.

VI. Riboswitch

A detection mechanism for vitamin B₁₂, specifically the molecule cobalamin, would allow for an instantaneous assay of its presence. The biobrick part, BBa_K1913011, from the Wageningen 2016 iGEM team was modified and tested for its affinity to 5,6-DMB-cobalamin (DMB B₁₂) or adenine-cobalamin (B₁₂ analogs). The part consist of a riboswitch, btub, that binds specifically to cobalamin, sequestering the ribosomal binding site (RBS) upstream of the TetR gene, thereby hindering the expression of the repressor. In the absence of cobalamin, the TetR gene is constitutively expressed producing the Tet repressor which trans-regulates the Tet operator, TetO. The operator was designed upstream of the reporter gene, mRFP. Therefore, detection of the reporter RFP implies the presence of vitamin B₁₂.

Plasmid EN15 was constructed through Gibson Assembly. Gene block fragments consisting of the riboswitch/reporter parts, NSII homologous sequences, and an ampicillin resistance were ligated onto an E. coli specific sequence, pBR322. The novel plasmid was designed for recombination into Neutral Integration Site 2 (NSII) within the chromosomal genome of S. elongatus PCC 7942. The riboswitch system was modified for expression within S. elongatus PCC 7942. A light induced psbAI promoter, documented to be constitutively expressed in cyanobacteria such as S. elongatus PCC 7942, was placed upstream of the biobrick parts, producing two separate mRNA strands.

Expression of the plasmid EN15 produces two strands of mRNA. In the absence of cobalamin, the Tet repressor is expressed hindering the expression of the reporter gene. In the presence of cobalamin, mRFP thus becomes expressed.

The riboswitch system was modified to be expressed in E. coli as a proof that all parts work as expected. E. coli produces vitamin B₁₂ analogs which bind onto the riboswitch and express the mRFP reporter gene. Once the assembly of the plasmid EN15 was confirmed, it was then transformed into E. coli which further validated that all parts worked accordingly. Plasmid EN15 was confirmed via sanger sequencing.

P R O J E C T

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HOMEPAGE

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ACETAMINOPHEN

VITAMIN B₁₂

MODELING

TARGET ORGANISM

PARTS

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[1] Anderson, J. C., HSIAU, T., Srivastava, S., RUAN, P., KOTKER, J. P. I., BODIK, R., & Seshia, S. A. (2016). Method for biosynthesis of acetaminophen. Google Patents.