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

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               <h2>Materials</h2>
 
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               Heroin esterase, an acetylmorphine carboxylesterase,  was isolated from <i>Rhodococcus erythropolis</i> strain H1 in 1994 from the garden soil at Cambridge and is able to use heroin as its sole carbon and energy source<sup>1</sup>. The gene <i>her</i> encodes this enzyme and has the ability to be expressed in the chassis Escherichia coli<sup>2</sup>. The pH optimum for this enzyme to function is in 8.5 in bicine buffer<sup>1</sup>.
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                <li>1g Agarose
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                <li>100ml 1X TAE buffer
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                <li>5µl GelRed stain
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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.
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Design: The sequence for our enzyme used the original sequence from Rathbone, et al., and was then codon optimised for <i>E. coli</i>. 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.
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<sup>1</sup>Cameron, 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.
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<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.
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            Monoamine Oxidase A
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              <h2>Materials</h2>
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                <li>Buffer QG
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                <li>10µl 3M sodium acetate
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                <li>Isopropanol
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                <li>750µl Buffer PE
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                <li>25µl Buffer EB
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              <h2>Procedure</h2>
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              All centrifuge steps are carried out at 13,000 rpm.
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                <li>1. Excise the region of gel containing the DNA fragment using a scalpel.  Cut close to the DNA to minimise the gel volume.
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                <li>2. Place the gel slice in a 1.5ml tube and weigh it. Record the volume of the gel.
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                <li>3.  Add 300µl of Buffer QG for each 100mg of gel.
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                <li>4. Incubate at 50°C for 10 minutes or until the gel has completely dissolved. Mix by vortexing the tube every 2 minutes during the incubation.
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                <li>5. Once the gel is completely dissolved, the mixture should be yellow. If the mixture is orange or violet add 10 µl of 3M sodium acetate and mix until it turns yellow. Yellow colour indicates the solution is the optimum pH for DNA binding to the QIAquick membrane.
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                <li>6. Add 1 gel volume of isopropanol to the solution and mix (1:1 volumes of isopropanol to gel slice).
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                <li>7. Place a QIAquick spin column in a 2ml collection tube.
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                <li>8. Pipette the sample onto the QIAquick column and centrifuge. Discard flow-through.
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                <li>9. Place column back in same collection tube. Add 500µl of Buffer QG to the column and centrifuge for 1 minute to remove all traces of agarose.
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                <li>10. Wash column by adding 750µl buffer PE. Let it stand for 2-5 min and then centrifuge for 1 minute.
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                <li>11. Discard the flow-through. Centrifuge for 1 minute to remove the residual buffer PE.
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                <li>12. Then place the column in a clean, labelled 1.5ml Eppendorf tube.
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                <li>13. To elute the DNA, add 25µl of Buffer EB to the centre of the column membrane, let it stand for 1 minute and then centrifuge for 1 minute.
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                <li>14. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 minute and then centrifuge for 1 minute. The DNA will now be in the flow-through.
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Revision as of 13:02, 17 September 2015

Basic Parts

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 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.