Part:BBa_K3633005

Coding sequence for FMO enzyme

Indigo is one of the oldest and most useful dyes globally and is widely used in various areas, such as the food and drug industry. Once before the production of indigo was greatly relied on the extraction of these pigments from plants. Although chemical synthesis of indigo was invented in the 18th century, the method still had lots of drawbacks that it can cause pollution and the substrates for synthesis were harmful to people's health.

With the development of synthetic biology, as early as 1993, pathways of indigo synthesis were found in some bacterias such as Methylophilus and Acinetobacter, and various genes including FMO and sty gene group were discovered useful for bacteria indigo synthesis (Choi et al., 2003, Han, Bang, Lim and Kim, 2010). To achieve our goal of producing natural and harmless hair dyes by engineered bacteria, iGEM20_Shanghai_SFLS_SPBS built the basic part of TnaA, adopted from the composite BioBrick of 2019 Team GreatBay_SZ.

The FMO gene is originally found in M. aminisulfidivorans and is responsible for converting indole that is produced by tryptophanase from L-tryptophan, a common amino acid in E.coli, into indoxyl, which is the most essential part in the process of indigo synthesis. Finally, the product indoxyl is converted into indigo in the presence of oxygen. The FMO gene in the biobrick is successfully expressed in E.coli DH5α in the experiment of iGEM20_Shanghai_SFLS_SPBS, and indigo is successfully produced in the presence of L-tryptophan and oxygen.

Experiments & Results

Successful production in E. coli DH5α

We successfully produced indigo in E. coli DH5α, the bacterial strain used by iGEM19_GreatBay_SZ. We added 100 mg/L L-Tryptophan in one shake flask and no substrate in another. Both shake flasks successfully produced indigo, and the one with the substrate produced more pigments than the other.

Figure 2. (A) Production of indigo in E. coli DH5α, with and without substrate, at 37℃ in 72 h. (B) Production of indigo from 0-60 h.

Quantifying the production of indigo with HPLC-MS

We were honored to have the opportunity to use an HPLC-MS machine from Shimadzu Enterprise Management (China) Co., Ltd. to quantify the levels of production of indigo.

For indigo, we first prepared indigo solutions of different concentrations to acquire a standard curve. Out of solutions of seven concentrations from 1-400 ppb, we acquired a linear standard curve of R^2=0.9955. We then diluted our product and measured its concentration. The concentration of our indigo was 880 μg/mL.

Fig 3. Measurement of indigo standard curve (A) and production level (B) with HPLC-MS.

Sequence & Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1369
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Han, G., Bang, S., Lim, G. and Kim, S., 2010. Bio-indigo production by two types of fermentation systems using recombinant E. coli cells harboring a flavin-containing monooxygenase gene (fmo). Journal of Biotechnology, 150, pp.369-369.

2. Choi, H., Kim, J., Cho, E., Kim, Y., Kim, J. and Kim, S., 2003. A novel flavin-containing monooxygenase from Methylophaga sp. strain SK1 and its indigo synthesis in Escherichia coli. Biochemical and Biophysical Research Communications, 306(4), pp.930-936.

3. "Team:Berkeley/Project/Introduction - 2013.igem.org", 2013.igem.org, 2020. [Online]. Available: http://2013.igem.org/Team:Berkeley/Project/Introduction. [Accessed: Jun-2020].