Part:BBa_K3633010
A composite part to express TnaA and FMO, responsible for indigo production
Description
Indigo is one of the oldest and most useful dyes in the world and is widely used in various areas including food and drug industries. 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). In order to achieve our goal of producing natural and harmless hair dyes by engineered bacteria, iGEM20_Shanghai_SFLS_SPBS adopted the composite BioBrick from 2019 Team GreatBay_SZ, which consists of the common J23102 promoter, ribosome binding site, TnaA gene and FMO gene.
The TnaA gene codes for tryptophanase in E.coli, which helps convert L-tryptophan, the common amino acid, into indole and is common in the metabolic pathway of tryptophan in E.coli. The FMO gene is originally found in M. aminisulfidivorans and is responsible for converting indole that is produced by tryptophanase into indoxyl, which is the most essential part in the process of indigo synthesis. And finally, the product indoxyl will be subsequently converted into indigo in the presence of oxygen. The Biobrick is successfully expressed in 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 decided to try producing indigo in E. coli DH5α, the bacterial strain used by iGEM19_GreatBay_SZ. Surprisingly, the tubes showed blue color. We then used shake flasks and E. coli DH5α to produce indigo. 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. The production in the flask with substrate is estimated to be 41.6 g/L.
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.
Sequence & Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2889
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1317
- 1000COMPATIBLE 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].
//function/biosynthesis
| biology | Escherichia coli |