Difference between revisions of "Part:BBa K3633014"

Revision as of 05:28, 27 October 2020

A composite part to express Tyr1 induced by IPTG

Description

Fig 1. Melanin Synthesis Pathway

In this part, the gene of Tyr1 is added to promoter pTac-RBS.

Tyr gene provides instructions for making enzyme tyrosinase, which is located in melanocytes in our skin. Melanocytes are responsible for producing melanin. Melanin is responsible for giving our skin and hair color. It includes eumelanin and pheomelanin. Eumelanin usually shows darker color than pheomelanin. As shown below, tyrosinase is responsible for the first step in melanin production.

Tyrosine is one of the 20 standard amino acids present in the body and is used by cells to synthesize proteins. This is a non-essential amino acid, meaning that it is also synthesized by the body when it is not only ingested from the diet. Tyrosine is found in casein, a protein in milk and other milk-based products (like cheese). Tyrosine is also present in non-dairy foods that are aged, including some types of meats and red wine.

Experiments & Results

Successful production in E.coli BL21(DE3) and vibrio natriegens

We constructed the plasmid pTac-Tyr1 and successfully transformed the plasmid into E. coli BL21(DE3) and Vibrio natriegens ATCC 14048. We added 2.5 mg L-DOPA and 0.4 mg L-Tyrosine as the substrates and added 0.1 mM IPTG for induction. The bacteria were cultured at 220 rpm, at 37℃. The result shows that melanin was produced. However, the effect of the promoter was not as good as the constitutive promoter J23102.

Fig 2.Production of melanin in E. coli BL21(DE3) and Vibrio natriegens at 37℃ in 78 h. The horizontal axis is time (hours), and the vertical axis is the absorbance of the bacterial solution at 400 nm.

Sequence & Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 376
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

References

1. 赵飞龙, 徐亚军, "微生物黑色素的研究", 中国调味品, vol. 34, no. 9, 2009.

2. Z. Wang et al., "Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application", Applied and Environmental Microbiology, vol. 86, no. 5, 2019. Available: 10.1128/aem.02749-19.

3. M. Pavan, N. López and M. Pettinari, "Melanin biosynthesis in bacteria, regulation and production perspectives", Applied Microbiology and Biotechnology, vol. 104, no. 4, pp. 1357-1370, 2019. Available: 10.1007/s00253-019-10245-y.

4. "Team:NYMU-Taipei/Pigments - 2017.igem.org", 2017.igem.org, 2020. [Online]Available: http://2017.igem.org/Team:NYMU-Taipei/Pigments. [Accessed: Jun-2020].

@@ Line 9: / Line 9: @@
 In this part, the gene of Tyr1 is added to promoter pTac-RBS.
-Tyr gene provides instructions for making enzyme tyrosinase which is located in melanocytes in our skin. Melanocytes are responsible for producing melanin. Melanin is responsible for giving our skin and hair color. It includes eumelanin and pheomelanin. Eumelanin usually shows darker color than pheomelanin. As shown below, tyrosinase is responsible for the first step in melanin production.
+Tyr gene provides instructions for making enzyme tyrosinase, which is located in melanocytes in our skin. Melanocytes are responsible for producing melanin. Melanin is responsible for giving our skin and hair color. It includes eumelanin and pheomelanin. Eumelanin usually shows darker color than pheomelanin. As shown below, tyrosinase is responsible for the first step in melanin production.
-Tyrosine is one of the 20 standard amino acids present in the body and used by cells to synthesize proteins. This is a non-essential amino acid, meaning that when it is not only ingested from the diet, it is also synthesized by the body. Tyrosine is found in casein, a protein in milk and other milk-based products (like cheese). Tyrosine is also present in non-dairy foods that are aged, including some types of meats and red wine.
+Tyrosine is one of the 20 standard amino acids present in the body and is used by cells to synthesize proteins. This is a non-essential amino acid, meaning that it is also synthesized by the body when it is not only ingested from the diet. Tyrosine is found in casein, a protein in milk and other milk-based products (like cheese). Tyrosine is also present in non-dairy foods that are aged, including some types of meats and red wine.
 ==Experiments & Results==
 ===Successful production in E.coli BL21(DE3) and vibrio natriegens===
-We constructed the plasmid pTac-Tyr1 and successfully transformed the plasmid into E. coli BL21(DE3) and Vibrio natriegens ATCC 14048. We added 2.5 mg L-DOPA, 0.4 mg L-Tyrosine and 1mM IPTG as substrates and cultured the bacteria in shakers (220 rpm) at 37℃. The result shows that melanin was produced. However the effect of pTac promoter is not as good as the constitutive promoter J23102.
+We constructed the plasmid pTac-Tyr1 and successfully transformed the plasmid into E. coli BL21(DE3) and Vibrio natriegens ATCC 14048. We added 2.5 mg L-DOPA and 0.4 mg L-Tyrosine as the substrates and added 0.1 mM IPTG for induction. The bacteria were cultured at 220 rpm, at 37℃. The result shows that melanin was produced. However, the effect of the promoter was not as good as the constitutive promoter J23102.
-[[File:T--Shanghai_SFLS_SPBS--Melanin Result 7.png|600px|center|thumb|Fig 2.Production of melanin in E. coli BL21(DE3) and Vibrio natriegens at 37℃ in 78 h. The horizontal axis is time (hours), and the vertical axis is absorbance of the bacterial solution at 400 nm. ]]
+[[File:T--Shanghai_SFLS_SPBS--Melanin Result 7.png|600px|center|thumb|Fig 2.Production of melanin in E. coli BL21(DE3) and Vibrio natriegens at 37℃ in 78 h. The horizontal axis is time (hours), and the vertical axis is the absorbance of the bacterial solution at 400 nm. ]]