Difference between revisions of "Team:Edinburgh/Improved Part"

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Revision as of 23:07, 18 September 2015

Improved Part

Materials

Laccases are glycosylated polyphenol oxidases and have four copper ions per molecule1. This allows them to catalyse the reduction of O2 to 2H2 while oxidising an aromatic substrate2. This laccase is coded by the structural gene lcc in Trametes versicolor, a species of white rot fungus3.

To increase the functions of laccase, we took the sequence from iGEM12_Bielefeld-Germany laccase BBa_K863030 and codon optimised it for the chassis Esherichia coli. We then made it RFC25 compatible by added the prefix and suffix and removing all illegal restriction sites. This laccase can now be fused to other protein genes with an in-frame 6-nucleotide scar.

1Lontie, R. (1984). Copper proteins and copper enzymes (Vol. 2). CRC.
2Thurston, C. F. (1994). The structure and function of fungal laccases. Microbiology, 140(1), 19-26.
3Collins, P. J., & Dobson, A. (1997). Regulation of laccase gene transcription in Trametes versicolor. Applied and Environmental Microbiology, 63(9), 3444-3450.

Procedure

  • 1. Mix the agarose with the 1X TAE buffer in a flask.
  • 2. Heat the mixture until all the agarose is dissolved.
  • 3. Swirl the flask under cold running water to cool the mixture.
  • 4. Add the gel stain.
  • 5. Pour into an assembled gel tray and let it cool.

Materials

    The structural gene morphine-6-dehyrogenase (morA) was first isolated from Pseudomonas putida M10 as it is capable of growth with morphine as its sole carbon source1. Morphine dehydrogenase catalyses the oxidation of both morphine and codeine to produce morphinone and codeinone. During this process NADP+ is reduced to NADPH which means that it is frequently used to detect morphine and codeine enzymatically2.

    To test the morphine dehydrogenase activity it can be coupled with codeine and NADP+ to produce codeinone and NADPH. The amount of NADPH produced can be measured at x nm.

    Design: To make this gene standardised it was codon optomised for the chassis Esherichia coli as well as making it RFC25 compatible which required getting rid of 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.

Procedure

  • 1. Place gel tray into the electrophoresis apparatus.
  • 2. Pour 1X TAE so that the gel is covered by buffer.
  • 3. Prepare the samples by adding the appropriate amount of loading dye.
  • 4. Load samples and DNA ladder into wells on the gel.
  • 5. Run the gel at roughly 100V for around an hour

Materials

    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.

Procedure

  • 1. Pour 10ml of LB into a 50ml Falcon tube.
  • 2. Pipette 10µl of antibiotic into the broth.
  • 3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
  • 4. Incubate at 37°C overnight in a shaking incubator.