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

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               Laccase TVEL5 in RFC25 BBa_K1615067
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               Laccase TVEL5 in RFC25 (BBa_K1615067)
 
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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 <i>Escherichia coli</i><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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            Laccases are glycosylated polyphenol oxidases and have four copper ions per molecule<sup>1</sup>. This allows them to catalyse the reduction of O<sub>2</sub> to 2H<sub>2</sub>O while oxidising an aromatic substrate<sup>2</sup>. This laccase is coded by the structural gene <i>lcc</i> in <i>Trametes versicolor</i>, a species of white rot fungus<sup>3</sup>.  
 
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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.
+
To increase the functions of laccase, we took the sequence from iGEM12_Bielefeld-Germany laccase BBa_K863030 and codon optimised it for the chassis <i>Escherichia coli</i>. We then made it RFC25 compatible by adding the prefix and suffix and removing all illegal restriction sites. This laccase can now be used in protein fusions as there is an in-frame 6-nucleotide scar.
 
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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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<br>
 
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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.  
+
<sup>1</sup>Lontie, R. (1984). <i>Copper proteins and copper enzymes</i> (Vol. 2). CRC.
−
 
+
<sup>2</sup>Thurston, C. F. (1994). The structure and function of fungal laccases. <i>Microbiology</i>, 140(1), 19-26.
−
<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.  
+
<sup>3</sup>Collins, P. J., & Dobson, A. (1997). Regulation of laccase gene transcription in Trametes versicolor. <i>Applied and Environmental Microbiology</i>, 63(9), 3444-3450.
  
 
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo">
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             Morphine-6-Dehydrogenase BBa_K1615000
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             mRFP (BBa_K1615089)
 
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                 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>.
+
                 RFP is important in synthetic biology as a visualisation tool. While multiple versions of RFP exist in the registry, only one is RFC25 compatible. This RFP (BBa_K1351021) contains a Shine-Dalgarno sequence in the RFC25 prefix which precludes it from using the common lac expression cassette (BBa_K314103) which already contains a ribosome binding site and only produces RFC10 fusions. By adding the RFC25 prefix without the Shine-Dalgarno sequence we hope to improve the utility of this RFP.
 
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We visualised RFP using multiple methods to show that it was folding and expressing.
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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.
+
 
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+
 
−
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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.
+
−
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+
−
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<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.
+
 
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
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               Monoamine oxidase A BBa_K1615022
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               CBDcipA (BBa_K1615111)
 
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                Monoamine oxidase A is coded by the gene <i>maoA</i> and is subject to catabolite and ammonium ion repression<sup>1</sup>. Amine oxidases that contain copper/topaquinone (TPQ), like monoamine oxidase A, convert primary amines into their corresponding aldehydes, hydrogen peroxide and ammonia<sup>2</sup>.  
+
              Cellulose binding domains (CBDs) mediate the binding of enzymes to cellulose<sup>1</sup>. CBDs are divided into over a dozen families based on their sequence homology <sup>2</sup>. Family III of CBDs is divided into a, b and c with CBDCipA belonging to family III a; the clostridial scaffoldin CBDs<sup>3</sup>. CBDCipA was identified in <i>Clostridium thermocellum</i> and is capable of binding to crystalline cellulose in a reversible manner<sup>4</sup>. CBDCipA includes endogenous linker sequences at both the N and C-terminals which help to prevent the CBD from interfering with the folding of any other protein it may be fused to.
 +
 
 +
To characterise the binding of CBDCipA to Whatman 54 we fused it to RFP (BBa_K1615089) at both the N and C terminals and looked at dissociation of the CBD over time.
 +
 
 +
 
 +
<b>Design:</b> In an effort to improve the CBDCipA existing in the registry from iGEM14_Imperial we used the sequence from BBa_K1321014 and made it RFC25 compatible by removing the illegal EcoRI site at 148.
 
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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.
 
 
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Design: This monoamine oxidase A sequence was found in <i>Klebsiella pneumoniae</i><sup>3</sup> 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.
+
<sup>1</sup>Ferreira, L. M., Durrant, A. J., Hall, J., Hazlewood, G. P., & Gilbert, H. J. (1990). Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase. <i>Biochem. J</i>, 269, 261-264.
−
<br>
+
<br><sup>2</sup>Tomme, P., Warren, R. A. J., Miller, R. C., Kilburn, D. G. & Gilkes,
−
<br>
+
N. R. (1995). Enzymatic Degradation of Insoluble Polysaccharides, edited by J. M. Saddler & M. H. Penner, pp. 142±161. Washington, DC: American Chemical Society
−
<sup>1</sup>Oka, M., Murooka, Y., & Harada, T. (1980). Genetic control of tyramine oxidase, which is involved in derepressed synthesis of arylsulfatase in Klebsiella aerogenes. <i>Journal of bacteriology</i>, 143(1), 321-327.
+
<br><sup>3</sup>Shimon, L. J., Belaich, A., Belaich, J. P., Bayer, E. A., Lamed, R., Shoham, Y., & Frolow, F. (2000). Structure of a family IIIa scaffoldin CBD from the cellulosome of Clostridium cellulolyticum at 2.2 Å resolution. <i>Acta Crystallographica Section D: Biological Crystallography</i>, 56(12), 1560-1568.
−
<br><sup>2</sup>McIntire, W. S., & Hartmann, C. (1993). Copper-containing amine oxidases. <i>Principles and applications of quinoproteins</i>, 97-171.
+
<br><sup>4</sup>Bayer, E. A., Chanzy, H., Lamed, R., & Shoham, Y. (1998). Cellulose, cellulases and cellulosomes. <i>Current opinion in structural biology</i>, 8(5), 548-557.
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<br><sup>3</sup>Sugino, 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. <i>Journal of bacteriology</i>, 174(8), 2485-2492.
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Revision as of 00:45, 19 September 2015

Improved Parts

Laccases are glycosylated polyphenol oxidases and have four copper ions per molecule1. This allows them to catalyse the reduction of O2 to 2H2O 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 Escherichia coli. We then made it RFC25 compatible by adding the prefix and suffix and removing all illegal restriction sites. This laccase can now be used in protein fusions as there is 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.

RFP is important in synthetic biology as a visualisation tool. While multiple versions of RFP exist in the registry, only one is RFC25 compatible. This RFP (BBa_K1351021) contains a Shine-Dalgarno sequence in the RFC25 prefix which precludes it from using the common lac expression cassette (BBa_K314103) which already contains a ribosome binding site and only produces RFC10 fusions. By adding the RFC25 prefix without the Shine-Dalgarno sequence we hope to improve the utility of this RFP.
We visualised RFP using multiple methods to show that it was folding and expressing.

Cellulose binding domains (CBDs) mediate the binding of enzymes to cellulose1. CBDs are divided into over a dozen families based on their sequence homology 2. Family III of CBDs is divided into a, b and c with CBDCipA belonging to family III a; the clostridial scaffoldin CBDs3. CBDCipA was identified in Clostridium thermocellum and is capable of binding to crystalline cellulose in a reversible manner4. CBDCipA includes endogenous linker sequences at both the N and C-terminals which help to prevent the CBD from interfering with the folding of any other protein it may be fused to. To characterise the binding of CBDCipA to Whatman 54 we fused it to RFP (BBa_K1615089) at both the N and C terminals and looked at dissociation of the CBD over time. Design: In an effort to improve the CBDCipA existing in the registry from iGEM14_Imperial we used the sequence from BBa_K1321014 and made it RFC25 compatible by removing the illegal EcoRI site at 148.



1Ferreira, L. M., Durrant, A. J., Hall, J., Hazlewood, G. P., & Gilbert, H. J. (1990). Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase. Biochem. J, 269, 261-264.
2Tomme, P., Warren, R. A. J., Miller, R. C., Kilburn, D. G. & Gilkes, N. R. (1995). Enzymatic Degradation of Insoluble Polysaccharides, edited by J. M. Saddler & M. H. Penner, pp. 142±161. Washington, DC: American Chemical Society
3Shimon, L. J., Belaich, A., Belaich, J. P., Bayer, E. A., Lamed, R., Shoham, Y., & Frolow, F. (2000). Structure of a family IIIa scaffoldin CBD from the cellulosome of Clostridium cellulolyticum at 2.2 Å resolution. Acta Crystallographica Section D: Biological Crystallography, 56(12), 1560-1568.
4Bayer, E. A., Chanzy, H., Lamed, R., & Shoham, Y. (1998). Cellulose, cellulases and cellulosomes. Current opinion in structural biology, 8(5), 548-557.