Difference between revisions of "Team:Edinburgh/Basic Part"
| Line 175: | Line 175: | ||
<h2>Materials</h2> | <h2>Materials</h2> | ||
<ul> | <ul> | ||
| − | The structural gene morphine-6-dehyrogenase (<i>morA</i>) was first isolated from <i>Pseudomonas putida</i> M10 as it is capable of growth with morphine as its sole carbon | + | The structural gene morphine-6-dehyrogenase (<i>morA</i>) was first isolated from <i>Pseudomonas putida</i> M10 as it is capable of growth with morphine as its sole carbon source<sup>1</sup>. Morphine dehydrogenase catalyses the oxidation of both morphine and codeine to produce morphinone and codeinone. During this process NADP<sup>+</sup> is reduced to NADPH which means that it is frequently used to detect morphine and codeine enzymatically<sup>2</sup>. |
<br> | <br> | ||
<br> | <br> | ||
| − | To | + | To test the morphine dehydrogenase activity it can be coupled with codeine and NADP<sup>+</sup> to produce codeinone and NADPH. The amount of NADPH produced can be measured at x nm. |
<br> | <br> | ||
<br> | <br> | ||
| − | + | Design: To make this gene standardised it was codon optomised for the chassis <i>Esherichia coli</i> as well as making it RFC25 compatible which required getting rid of all illegal restriction sites in the gene sequence. | |
| − | <br> | + | <br> |
| + | <br> | ||
| + | <sup>1</sup>Bruce, 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. <i>Archives of microbiology</i>, 154(5), 465-470. | ||
| + | <br><sup>2</sup>Rathbone, 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. <i>Applied and environmental microbiology</i>, 63(5), 2062-2066. | ||
| + | |||
</ul> | </ul> | ||
</p> | </p> | ||
Revision as of 13:03, 17 September 2015
Materials
Heroin esterase, an acetylmorphine carboxylesterase, was isolated from Rhodococcus erythropolis strain H1 in 1994 from the garden soil at Cambridge and is able to use heroin as its sole carbon and energy source1. The gene her encodes this enzyme and has the ability to be expressed in the chassis Escherichia coli2. The pH optimum for this enzyme to function is in 8.5 in bicine buffer1.The activity of heroin esterase can be tested using 4-nitrophenyl acetate which is hydrolised(?) by heroin esterase to form 4-nitrophenol + actetate. This produces a yellow colour as well as being able to be read at 410 nm.
Design: The sequence for our enzyme used the original sequence from Rathbone, et al., and was then codon optimised for E. coli. The RFC25 prefix and suffix were added along which required all illegal sites (EcoRI, SpeI, AgeI, NotI, NgoMIV and XbaI) to be removed. As this was a difficult sequence to make as a gBlock, it was ordered as a gene in an ampicillin backbone where it was then digested and ligated into the pSB1C3 backbone.
1Cameron, G. W., Jordan, K. N., Holt, P. J., Baker, P. B., Lowe, C. R., & Bruce, N. C. (1994). Identification of a heroin esterase in Rhodococcus sp. strain H1. Applied and environmental microbiology, 60(10), 3881-3883.
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. 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
- 10ml Luria Broth (LB)
- 10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
- Loop (for picking colony)
- Ethanol
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.