Difference between revisions of "Part:BBa K3633010"

Latest revision as of 10:13, 27 October 2020

A composite part to express TnaA and FMO, responsible for indigo production

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. (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 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
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 2889
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1317
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].

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3633010 short</partinfo>
-=Description=
+==Description==
-<!-- insert image -->
+[[File:T--Shanghai_SFLS_SPBS--Indigo Synthesis--Pathway.png|600px|center|thumb|Fig 1. Indigo Synthesis Pathway]]
-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.<br>
+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.<br>
-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(Hack Sun Choi. et. al. 2003, Gui Hwan Hana.et. al. 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.<br>
+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.<br>
-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 DH5&#945; in the experiment of iGEM20_Shanghai_SFLS_SPBS, and indigo is successfully produced in the presence of L-tryptophan and oxygen.<br>
+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.<br>
-=Experiment=
+==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.<br>
+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.<br>
-=Sequence and Features=
+[[File:T--Shanghai SFLS SPBS--indigo.png|600px|center|thumb|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. ]]
-<!-- Add more about the biology of this part here
+===Quantifying the production of indigo with HPLC-MS===
-===Usage and Biology===
+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.
+[[File:T--Shanghai_SFLS_SPBS--Indigo HPLC.png|600px|center|thumb|Fig 3. Measurement of the indigo standard curve (A) and production level (B) with HPLC-MS.]]
+==Sequence & Features==
-<!-- -->
 <span class='h3bb'></span>
 <partinfo>BBa_K3633010 SequenceAndFeatures</partinfo>
 <!-- Uncomment this to enable Functional Parameter display
@@ Line 33: / Line 37: @@
 <partinfo>BBa_K3633010 parameters</partinfo>
 <!-- -->
+==References==
+. 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.
+. 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.
+. "Team:Berkeley/Project/Introduction - 2013.igem.org", 2013.igem.org, 2020. [Online]. Available: http://2013.igem.org/Team:Berkeley/Project/Introduction. [Accessed: Jun-2020].