Difference between revisions of "Part:BBa K801073"

(→‎Cloning into pSB1C3)
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This BioBrick is the generator for the enzyme xanthosine N-methyltransferase 1 (CaXMT1) and part of the caffeine synthesis pathway. For creating an expression cassette with all three enzymes of the caffeine synthesis pathway [https://parts.igem.org/Part:BBa_K801077 BBa_K801077] based on the substrate Xanthosine different promoters and terminators were assembled to each enzyme. CaXMT1 is regulated by the constitutive promoter Tef2, which is a strong yeast promoter. The used terminator Adh1, is a widely used yeast terminator. The Tef2 promoter was prefered compared to the Tef1 promoter (which is even stronger) in order to limit metabolic stress, which could result in a positive selection of natural mutants (with regard to genome integration).
 
This BioBrick is the generator for the enzyme xanthosine N-methyltransferase 1 (CaXMT1) and part of the caffeine synthesis pathway. For creating an expression cassette with all three enzymes of the caffeine synthesis pathway [https://parts.igem.org/Part:BBa_K801077 BBa_K801077] based on the substrate Xanthosine different promoters and terminators were assembled to each enzyme. CaXMT1 is regulated by the constitutive promoter Tef2, which is a strong yeast promoter. The used terminator Adh1, is a widely used yeast terminator. The Tef2 promoter was prefered compared to the Tef1 promoter (which is even stronger) in order to limit metabolic stress, which could result in a positive selection of natural mutants (with regard to genome integration).
  
 +
===Modifications===
 +
 +
* the 5' UTR and 3' UTR of the original sequences were removed
 +
* the yeast consensus sequence for improved ribosome binding (TACACA) was added 5' of the start codon ATG
 +
* according to N- end rule and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine), to optimize both, protein stability and mRNA translation. This decision was made after proofing the 3D- structure of the enzyme CaDXMT1. Due to the fact, that the first two residues of the amino acid sequence are not shown in the crystalized structure (probably because of high flexibility), we chose to  exchange this amino acid, because it is probably not necessary for the uptake of the ligands ([http://www.uniprot.org/uniprot/Q9AVK0 uniprot] entry further shows, that it is not immediately involved in ligand binding). Because of the high similarity of the enzyme sequences, we also exchanged this amino acid.
 +
* we added a c- terminal strep-tag for purification and detection
 +
* the remaining coding sequence was extended with the standard RFC10 prefix and suffix, respectively
 +
* at last we made an optimization of the coding sequences with respect to the codon usage and mRNA structures
 +
* remove of all critical restriction sites (RFC10 and RFC25)
 +
 +
'''Note: Because of the yeast consensus sequence, this coding part does not start with ATG!'''
  
 
===Cloning into pSB1C3===
 
===Cloning into pSB1C3===
Line 12: Line 23:
  
 
[[Image:TUM12_analyticaldigestCaXMT1cassette.jpg|100px]]
 
[[Image:TUM12_analyticaldigestCaXMT1cassette.jpg|100px]]
 +
 +
To check the successful cloning, we performed an analytical digest with XbaI and PstI.
 +
 +
The expected lengths of the fragments were:
 +
<br/><br/>
 +
* Insert (CaXMT1): ca. 2400bp
 +
* Backbone (pSB1C3): ca. 2050 bp
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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<partinfo>BBa_K801073 parameters</partinfo>
 
<partinfo>BBa_K801073 parameters</partinfo>
 
<!-- -->
 
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=References=
 +
----
 +
 +
*[[http://www.ncbi.nlm.nih.gov/pubmed/18068204 Ashihara et al., 2008]] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. ''Phytochemistry'', 69(4):841–56.
 +
*[[http://www.ncbi.nlm.nih.gov/pubmed/22849837 Franco et al., 2012]] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (''humulus lupulus''), a component of beer, on the activity/rest rhythm. ''Acta Physiol Hung'', 99(2):133–9.
 +
*[[http://www.ncbi.nlm.nih.gov/pubmed/18036626 Kim and Sano, 2008]] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. ''Phytochemistry'', 69(4):882–8.
 +
*[[http://www.ncbi.nlm.nih.gov/pubmed/16925551 Kuranda et al., 2006]] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast ''Saccharomyces cerevisiae'' brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. ''Mol Microbiol'', 61(5):1147–66.
 +
*[[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1914188/ McCarthy and McCarthy, 2007]] McCarthy, A.A., McCarthy, J.G. (2007). The Structure of Two N-Methyltransferases from the Caffeine Biosynthetic Pathway. ''Plant Physiology'', 144(2):879-889.
 +
*[[http://ci.nii.ac.jp/naid/110006323439/ Negishi et al. (1988)]] Negishi O, Ozawa T and Imagawa H (1988). N-Methyl nucleosidase from tea leaves. ''Agric. Biol. Chem.'' 52: 169–175.
 +
*[[http://www.ncbi.nlm.nih.gov/pubmed/12746542 Uefuji et al., 2003]] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. ''Plant Physiol'', 132(1):372–80.
 +
*[[http://www.ncbi.nlm.nih.gov/pubmed/16247553 Uefuji et al., 2005]] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. ''Plant Mol Biol'', 59(2):221–7.

Revision as of 11:13, 23 October 2012

CaXMT1 expression cassette for yeast

Yeast expression cassette for CaXMT1 (BBa_K801070) controlled by the yeast TEF2 promoter (BBa_K801010) and the yeast ADH1 terminator (BBa_K801012).

This BioBrick is the generator for the enzyme xanthosine N-methyltransferase 1 (CaXMT1) and part of the caffeine synthesis pathway. For creating an expression cassette with all three enzymes of the caffeine synthesis pathway BBa_K801077 based on the substrate Xanthosine different promoters and terminators were assembled to each enzyme. CaXMT1 is regulated by the constitutive promoter Tef2, which is a strong yeast promoter. The used terminator Adh1, is a widely used yeast terminator. The Tef2 promoter was prefered compared to the Tef1 promoter (which is even stronger) in order to limit metabolic stress, which could result in a positive selection of natural mutants (with regard to genome integration).

Modifications

  • the 5' UTR and 3' UTR of the original sequences were removed
  • the yeast consensus sequence for improved ribosome binding (TACACA) was added 5' of the start codon ATG
  • according to N- end rule and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine), to optimize both, protein stability and mRNA translation. This decision was made after proofing the 3D- structure of the enzyme CaDXMT1. Due to the fact, that the first two residues of the amino acid sequence are not shown in the crystalized structure (probably because of high flexibility), we chose to exchange this amino acid, because it is probably not necessary for the uptake of the ligands ([http://www.uniprot.org/uniprot/Q9AVK0 uniprot] entry further shows, that it is not immediately involved in ligand binding). Because of the high similarity of the enzyme sequences, we also exchanged this amino acid.
  • we added a c- terminal strep-tag for purification and detection
  • the remaining coding sequence was extended with the standard RFC10 prefix and suffix, respectively
  • at last we made an optimization of the coding sequences with respect to the codon usage and mRNA structures
  • remove of all critical restriction sites (RFC10 and RFC25)

Note: Because of the yeast consensus sequence, this coding part does not start with ATG!

Cloning into pSB1C3

The cloning into pSB1C3 was proved by performing an analytical digest with XbaI and PstI.

TUM12 analyticaldigestCaXMT1cassette.jpg

To check the successful cloning, we performed an analytical digest with XbaI and PstI.

The expected lengths of the fragments were:

  • Insert (CaXMT1): ca. 2400bp
  • Backbone (pSB1C3): ca. 2050 bp

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 138
  • 1000
    COMPATIBLE WITH RFC[1000]


References