Part:BBa_K3633004

Coding sequence for TnaA in E. coli

Description

Fig 1. Indigo Synthesis Pathway

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 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 product indole is the initial substrate for the synthesis of indigo. Subsequently, The FMO gene that is originally found in M. aminisulfidivorans is responsible for converting indole produced by tryptophanase into indoxyl. Finally, the product indoxyl will be converted into indigo in the presence of oxygen. The TnaA gene in the biobrick is successfully expressed in E.coli DH5α in the experiment, 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 a 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.

Figure 2. (A) Production of indigo in E. coli DH5α, with and without substrate, at 37℃ in 72 h. The horizontal axis is time (hours), and the vertical axis is the absorbance of the bacterial solution at 605 nm. (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.

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 the 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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1234
  
• 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].

The following is from QHFZ 2021

Condition

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. Produce a marked effect with FMO and sty gene group 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).

Method

Bacterial strains and growth conditions. Strains SP850 and JW5503 served as sources for the ΔcyaA : : kan and ΔtolC : : kan alleles, respectively (Baba et al., 2006; Shah & Peterkofsky, 1991). Kanamycin (50 µg ml−1), chloramphenicol (10 µg ml−1) or ampicillin (100 µg ml−1) was added to growth media as required. Strains were grown overnight in Luria–Bertani (LB) medium supplemented with 0.4 % glucose. Overnight cultures were diluted 1 : 100 into 2 ml LB medium with or without 0.2 % glucose and incubated with shaking at 37 °C. When applicable, various amounts of sodium cAMP (Sigma-Aldrich) were added after 1 h, and the cultures were incubated for an additional 3 h. To induce β-galactosidase, cells were incubated with IPTG (1 mM final concentration) and cAMP (0–10 mM) for 75 min, as described previously (Hantke et al., 2011).

Genetic manipulations and plasmid construction. Chromosomal genes were deleted by using λ-Red recombination (Datsenko & Wanner, 2000). The appropriate primers are listed in Table 2. To construct GL348 (Δcrp : : kan), GL350 (ΔcpdA : : kan), GL617 (Δcrr : : kan) and GL736 (ΔptsN : : kan), the kan cassettes were amplified from the chromosome of GL38 and inserted into the chromosome of MG1655, replacing the crp, cpdA, crr and ptsN genes, respectively. To construct GL500 (ΔcpdA : : frt tolC : : kan), the ΔtolC : : kan fragment was amplified from the chromosome of JW5503 and inserted into the chromosome of GL491 (ΔcpdA : : frt), replacing the entire tolC gene. Note that the kan cassette in GL500 could not be removed because cpdA and tolC are separated by only two genes, and attempts to remove the kan cassette always resulted in removal of the region between cpdA and tolC, as well. Plasmid pTacTolC was constructed by amplifying the tolC gene from the chromosome of MG1655, after which the product was ligated between the BamHI and HindIII sites of pACT3 (Ptac lacIq CamR) (Dykxhoorn et al., 1996), thus placing the gene downstream of an IPTG-inducible tac promoter (Dykxhoorn et al., 1996). All constructs were confirmed by DNA sequencing or diagnostic PCR.

Produce indigo. 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 product indole is the initial substrate for the synthesis of indigo. Subsequently, The FMO gene that is originally found in M. aminisulfidivorans is responsible for converting indole produced by tryptophanase into indoxyl. Finally, the product indoxyl will be converted into indigo in the presence of oxygen. The TnaA gene in the biobrick is successfully expressed in E.coli DH5α in the experiment, and indigo is successfully produced in the presence of L-tryptophan and oxygen. Then produce indigo in E. coli DH5α, the bacterial strain used by iGEM19_GreatBay_SZ. Surprisingly, the tubes showed a 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.

Reference

[1] Li G, Young KD. A cAMP-independent carbohydrate-driven mechanism inhibits tnaA expression and TnaA enzyme activity in Escherichia coli. Microbiology (Reading). 2014 Sep;160(Pt 9):2079-2088. doi: 10.1099/mic.0.080705-0. Epub 2014 Jul 24. PMID: 25061041.