The structural gene morphine-6-dehydrogenase (morA) was first isolated from Pseudomonas putida M10 as it is capable of growth with morphine as its sole carbon source¹. Morphine dehydrogenase (MDH) catalyses the oxidation of both morphine and codeine to produce morphinone and codeinone respectively. During this process NADP⁺ is reduced to NADPH which means that this enzyme is frequently used to detect morphine and codeine².





To test the morphine dehydrogenase activity it can be coupled with morphine and NADP⁺ to produce morphinone and NADPH. The amount of NADPH produced can be measured at 340nm. Morphine dehydrogenase with t7 promoter characterised by Edinburgh iGEM team was provided by Prof Chris French. Michealis Menten kinetic analysis was performed giving values of Vmax and Km, 61.22 and 140.5 uM respectively.


Following table summarises the kinetic analysis and statistics of the measurment.

Design: To make this gene standardised it was codon optimised for the chassis Escherichia coli as well as making it RFC25 compatible which required removing all illegal restriction sites in the gene sequence.



¹Bruce, N. C., Wilmot, C. J., Jordan, K. N., Trebilcock, A. E., Stephens, L. D. G., & Lowe, C. R. (1990). Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Archives of microbiology, 154(5), 465-470.
²Rathbone, D. A., Holt, P. J., Lowe, C. R., & Bruce, N. C. (1997). Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. Applied and environmental microbiology, 63(5), 2062-2066.
²WALKER, E., et al. "Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation." Biochem. J 345 (2000): 687-692.

Monoamine oxidase A is coded by the gene maoA and is subject to catabolite and ammonium ion repression¹. Amine oxidases that contain copper/topaquinone (TPQ), like monoamine oxidase A, convert primary amines into their corresponding aldehydes, hydrogen peroxide and ammonia².

To test the activity of monoamine oxidase A, tyramine can be used as a substrate and its corresponding aldehyde as well as ammonia and hydrogen peroxide will be produced. When the hydrogen peroxide is coupled with horseradish peroxidase and Amplex red, resorufin, a red colour, will be produced.

Design: This monoamine oxidase A sequence was found in Klebsiella pneumoniae³ and was codon optimised for the chassis Escherichia coli as well as made RFC25 compatible with the corresponding prefix and suffix and illegal restriction sites were removed.

¹Oka, M., Murooka, Y., & Harada, T. (1980). Genetic control of tyramine oxidase, which is involved in derepressed synthesis of arylsulfatase in Klebsiella aerogenes. Journal of bacteriology, 143(1), 321-327.
²McIntire, W. S., & Hartmann, C. (1993). Copper-containing amine oxidases. Principles and applications of quinoproteins, 97-171.
³Sugino, H., Sasaki, M., Azakami, H., Yamashita, M., & Murooka, Y. (1992). A monoamine-regulated Klebsiella aerogenes operon containing the monoamine oxidase structural gene (maoA) and the maoC gene. Journal of bacteriology, 174(8), 2485-2492.