Difference between revisions of "Part:BBa K2906014"
 
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===Background:===
 
===Background:===
  
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'''Figure 1.''' Vanillin biosynthesis pathway from L-tyrosine consisting of five enzymes.
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===Characterisation:===
 
  
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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.
 
  
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'''Figure 1.''' Vanillin biosynthesis pathway from L-tyrosine consisting of five enzymes.
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===Characterisation:===
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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).
  
  
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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.
  
 
[[File:Caffeic acid and ferulic acid 2.png|750px|center]]
 
[[File:Caffeic acid and ferulic acid 2.png|750px|center]]

Latest revision as of 15:21, 20 October 2019


T7 promoter- RBS- Sam5- T7 terminator

sam5 gene of Saccharothrix espanaensis encodes a 4-coumarate 3-hydroxylase (C3H). It converts p-coumaric acid to caffeic acid by hydroxylation of the benzene ring. It is the second enzyme required in the vanillin biosynthesis pathway. This gene was codon optimised by GeneArt GeneOptimiser for expression in E. coli. A His-tag and a TEV protease cleavage site were added to it for purification and functional analysis of the protein. The T7 promoter, RBS and T7 terminator come from the pETM-11 plasmid after cloning.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 435
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 205
    Illegal AgeI site found at 382
    Illegal AgeI site found at 748
    Illegal AgeI site found at 1030
    Illegal AgeI site found at 1132
  • 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).


SDSandWestern.png

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.

Caffeic acid and ferulic acid 2.png

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