←Older revision
Revision as of 10:18, 24 October 2012
Line 3:
Line 3:
This BioBrick is the final "caffeine synthesis device". It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made up of the three single expression cassettes for each enzyme (see also [http://partsregistry.org/Part:BBa_K801073 BBa_K801073], [http://partsregistry.org/Part:BBa_K801074 BBa_K801074] and [http://partsregistry.org/Part:BBa_K801075 BBa_K801075]). It can be directly transformed into competent yeast cells or cloned in an adequate yeast genome integration vector.
This BioBrick is the final "caffeine synthesis device". It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made up of the three single expression cassettes for each enzyme (see also [http://partsregistry.org/Part:BBa_K801073 BBa_K801073], [http://partsregistry.org/Part:BBa_K801074 BBa_K801074] and [http://partsregistry.org/Part:BBa_K801075 BBa_K801075]). It can be directly transformed into competent yeast cells or cloned in an adequate yeast genome integration vector.
-
-
The expression of the enzymes CaXMT1 (''Coffea arabica'' xanthosine-N-methyl-transferase 1, BBa_K801070) and CaDXMT1 (''Coffea arabica'' dimethyl-xanthine N-methyl transferase 1, = caffeine synthase, BBa_K801072) is controlled by the TEF2-promoter (BBa_K801010) and the ADH1-terminator (BBa_K801012)
The expression of the enzymes CaXMT1 (''Coffea arabica'' xanthosine-N-methyl-transferase 1, BBa_K801070) and CaDXMT1 (''Coffea arabica'' dimethyl-xanthine N-methyl transferase 1, = caffeine synthase, BBa_K801072) is controlled by the TEF2-promoter (BBa_K801010) and the ADH1-terminator (BBa_K801012)
-
-
The expression of CaMXMT1 (''Coffea arabica'' methyl-xanthine N-methyl transferase 1, BBa_K801071) is controlled by the TEF1 promoter (BBa_K319003) and the ADH1-terminator (BBa_K801012)
The expression of CaMXMT1 (''Coffea arabica'' methyl-xanthine N-methyl transferase 1, BBa_K801071) is controlled by the TEF1 promoter (BBa_K319003) and the ADH1-terminator (BBa_K801012)
Line 16:
Line 12:
+
===Usage and Biology===
+
The enzyme CaXMT1 (xanthosine N-methyltransferase 1 of ''coffea arabica'') catalyses the first reaction step of the caffeine biosynthesis pathway and convertes the sustrate Xanthosine into 7-Methylxanthosine.
+
It uses SAM als methyl-donor and is located in the cytoplasm of the plants.
+
The enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of ''coffea arabica'') catalyses the third reaction step of the caffeine biosynthesis pathway and convertes the sustrate 7-Methylxanthine into 3,7-Dimethylxanthine.
+
It uses SAM (S-Adenosyl-Methionine) als methyl-donor and is located in the cytoplasm of the plants.
+
The enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of ''coffea arabica'') catalyses the fourth reaction step of the caffeine biosynthesis, leading to caffeine.
+
It uses SAM (S-Adenosyl-Methionine) als methyl-donor and is located in the cytoplasm of the plants.
+
Furthermore all three exists as homodimer, being also able to form heterodimers with the other enzymes of the caffeine pathway (see [http://www.brenda-enzymes.info Brenda]).
===Biosynthesis Pathway===
===Biosynthesis Pathway===
Line 22:
Line 26:
-
=
References
=
+
=
Characterization=
-
*
[[
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.
+
===Cloning into pSB1C3==
=
-
*[[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.
+
[[
Image
:
Erste2
.
png|thumb|left|100px|Anal
.
digest
]]
-
*
[[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
.
+
In order to submit the enzyme xanthosine methyltransferase
(
CaXMT1
)
to the partsregistry
,
we cloned the generated sequence into pSB1C3
,
making the system RFC10 compatible
.
However
,
since the sequence does not contain any restriction sites of the RFC25 standard
(
NgoMIV and Age1
)
, RFC25 compatibility can easily be reached without required quick changes by the use of PCR upon usage of appropriate primers
.
-
*[[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
.
+
To check the successful cloning
,
we performed an analytical digest with XbaI
and
PstI
.
-
*[[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.
+
+
The
expected lengths
of the
fragments were
:
+
/
>
/
>
+
*
Insert
(
CaXMT1
):
ca
.
6400bp
+
*
Backbone
(
pSB1C3
):
ca
.
2050 bp
+
/
>
+
The picture on the left shows the analytical digest of BioBrick BBa_K801070 with Xba1
and
Pst1
,
separated by gel
-
electrophoresis on
1
% agarose gel upon usage of ethidium bromide
.
+
/
>
/
>
/
>
/
>
+
+
===Usage and Biology===
===Usage and Biology===
Line 43:
Line 56:
BBa_K801077 parameters
BBa_K801077 parameters
+
+
+
=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.