Difference between revisions of "Part:BBa K2906016"

Latest revision as of 15:22, 20 October 2019

T7 promoter- RBS- COMT- T7 terminator

comt is from the plant Arabidopsis thaliana and is codon optimised by GeneArt GeneOptimiser for expression in E.coli.. A His-tag is fused to it after cloning into pETDuet-1 plasmid. It encodes catechol O-methyltransferase (COMT), which converts caffeic acid to ferulic acid, the third step in the vanillin biosynthesis pathway.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 102
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 141
Illegal AgeI site found at 324
1000
COMPATIBLE WITH RFC[1000]

Background:

Vanillin biosynthesis pathway from tyrosine.png

Figure 1. Vanillin biosynthesis pathway from L-tyrosine consisting of five enzymes.

Characterisation:

To show the functionality of the gene after cloning into pETM-11, we performed SDS-PAGE and Western blot to prove expression of the protein C3H (Figure 2).

Figure 2. SDS-gel (a) and western blot (b) of TAL, C3H and COMT, the first three enzymes in the vaniliin biosynthesis pathway encoded by sam8, sam5 and comt. The expected sizes of the proteins are 57.4 kDa, 60.49 kDa and 41.39 kDa for TAL, C3H and COMT respectively. The negative control (-ve) is the pETM-11 (empty vector) and the positive control (+ve) is the pETM-11-ABU58587 expressing a 36 kDa His-tagged protein.

As sam5 and comt were cloned to separate incompatible plasmids and due to the low stability of caffeic acid, we decided to make a pETM-11-sam5 and pETDuet-1-comt BL21 (DE3) co-culture (1:1) to detect and quantify caffeic acid and ferulic acid by UPLC-MS/MS providing 3 mM p-coumaric acid as a substrate for C3H (Figure 3). A 10 mL culture (1/100) of the respective strain in 2xYT media, was induced by 100 µM IPTG and grown for 72 hours at 26°C and 180 rpm which was then processed for analysis by UPLC-MS/MS; 1.207 µM and 3,914 µM of caffeic acid and ferulic acid were produced.

Figure 3. UPLC–MS/MS chromatograms of caffeic acid (a) and ferulic acid (b), the products of C3H and COMT respectively, from pETM-11-sam5 (c) and pETDuet-1-comt (d) BL21 (DE3) bacterial providing 3 mM of the substrate p-coumaric acid. X-axis show retention time.

References:

Gruz, J., Novák, O. and Strnad, M. (2008). Rapid analysis of phenolic acids in beverages by UPLC–MS/MS. Food chemistry, 111(3), pp.789-794. DOI: https://doi.org/10.1016/j.foodchem.2008.05.014.

Ni, J., Tao, F., Du, H. and Xu, P. (2015). Mimicking a natural pathway for de novo biosynthesis: natural vanillin production from accessible carbon sources. Scientific reports, 5: 13670. DOI: https://doi.org/10.1038/srep13670.

Kim, Y.H., Kwon, T., Yang, H.J., Kim, W., Youn, H., Lee, J.Y. and Youn, B. (2011). Gene engineering, purification, crystallization and preliminary X-ray diffraction of cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis membrane protein. Protein expression and purification, 79(1), pp.149-155. DOI: https://doi.org/10.1007/s10811-013-0113-5.

@@ Line 18: / Line 18: @@
 ===Background:===
-[[File:Vanillin biosynthesis pathway from tyrosine.png|500px|center]]
+[[File:Vanillin biosynthesis pathway from tyrosine.png|500px|left]]
 '''Figure 1.''' Vanillin biosynthesis pathway from L-tyrosine consisting of five enzymes.
@@ Line 25: / Line 48: @@
 ===Characterisation:===
-To show the functionality of the gene after cloning into pETM-11, we performed SDS-PAGE and Western blot to prove expression of the protein C3H (Figure 2). Due to the low stability of caffeic acid, we decided to make pETM-11-''sam5'' and pETDuet-1-''comt'' BL21 (DE3) co-culture to detect and quantify caffeic acid and ferulic acid by UPLC-MS/MS upon addition of 3 mM p-coumaric acid as a substrate for C3H (Figure 3). From this co-culture, 1.207 µM and 3,914 µM of caffeic acid and ferulic acid were produced, as quantified by UPLC-MS/MS.
+To show the functionality of the gene after cloning into pETM-11, we performed SDS-PAGE and Western blot to prove expression of the protein C3H (Figure 2).
@@ Line 33: / Line 55: @@
 '''Figure 2.''' SDS-gel (a) and western blot (b) of TAL, C3H and COMT, the first three enzymes in the vaniliin biosynthesis pathway encoded by ''sam8'', ''sam5'' and ''comt''. The expected sizes of the proteins are 57.4 kDa, 60.49 kDa and 41.39 kDa for TAL, C3H and COMT respectively. The negative control (-ve) is the pETM-11 (empty vector) and the positive control (+ve) is the pETM-11-ABU58587 expressing a 36 kDa His-tagged protein.
+As sam5 and comt were cloned to separate incompatible plasmids and due to the low stability of caffeic acid, we decided to make a pETM-11-''sam5'' and pETDuet-1-''comt'' BL21 (DE3) co-culture (1:1) to detect and quantify caffeic acid and ferulic acid by UPLC-MS/MS providing 3 mM p-coumaric acid as a substrate for C3H (Figure 3). A 10 mL culture (1/100) of the respective strain in 2xYT media, was induced by 100 µM IPTG and grown for 72 hours at 26°C and 180 rpm which was then processed for analysis by UPLC-MS/MS; 1.207 µM and 3,914 µM of caffeic acid and ferulic acid were produced.