Part:BBa_K1658003

RBS + CAD1 + Terminator

Figure1: Here, we have obtained the double digested CAD1 enzyme coding gene with RBS and Terminator at approximately 1300 basepairs and after cloning on pSB1C3, double digestion gave us lanes at approximatle 3300 base pairs.

Design Notes

Composite part was obtained by combining Ribosome Binding Side(B0034), Cinnamyl alcohol dehydrogenase1 (K1658006) and Terminator(B0015). After having a proper ligation, we have cloned it into pSB1C3 plasmid.The enzyme CAD1 belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is cinnamyl-alcohol:NADP+ oxidoreductase. Other names in common use include cinnamyl alcohol dehydrogenase, and CAD. This enzyme participates in phenylpropanoid biosynthesis. Chemical Reaction of CAD: Cinnamyl alcohol + NADP+ = cinnamaldehyde + NADPH. Cofactor :Zn2+ Note: Binds 2 Zn2+ ions per subunit. Involved in lignin biosynthesis. Catalyzes the final step specific for the production of lignin monomers. Catalyzes the NADPH-dependent reduction of coniferaldehyde, 5-hydroxyconiferaldehyde, sinapaldehyde, 4-coumaraldehyde, caffeyl aldehyde and cinnaml alcohol to their respective alcohols Functional expression of the Ocimum CAD1in E.coli PCR was used to introduce EcoRI , Xbal, Spel, Notl and Pstl at side ends of CAD1 coding region. The amplified product was single and double digested with EcoRl and Notl and the resultant fragment was cloned into the same sides ofthe puC57 vector. The engineered plasmid was transformed into E.coli BL 2l (DE3) cells which has lack of nudeases and proteinoses and ideal for expression.

Source

Ocimum basilicum is sweet basil (Eukaryota, Viridiplantae, Streptophyta, Embryophyta, Tracheophyta, Spermatophyta, Magnoliophyta, eudicotyledons, Gunneridae, Pentapetalae, asterids, lamiids, Lamiales, Lamiaceae,Nepetoideae, Ocimeae, Ocimum)

Since the sequence of CAD1 was obtained as mRNA of Ocimum Basilicum, we have obey the codon bias rule in order to express the enzyme in Escherichia Coli BL21(DE3) properly. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

Iijima, Yoko, and Guodong Wang. "Analysis of the Enzymatic Formation of Citral in the Glands of Sweet Basil." Archives of Biochemistry and Biophysics 21 July 2005: 145. Print Ma, Q.(2010). Functional analysis of a cinnamyl alcohol dehydrogenase involved in biosynthesis in wheat. Journal of Experiment Botany, 2735-2744.