Difference between revisions of "Part:BBa K3570003"

Revision as of 18:23, 25 October 2020

Lemon taste induction system in S. cerevisiae

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 3900
Illegal XbaI site found at 3238
Illegal XbaI site found at 3629
Illegal PstI site found at 137
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 3900
Illegal PstI site found at 137
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 3900
Illegal BglII site found at 524
Illegal BamHI site found at 3246
Illegal XhoI site found at 5695
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 3900
Illegal XbaI site found at 3238
Illegal XbaI site found at 3629
Illegal PstI site found at 137
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 3900
Illegal XbaI site found at 3238
Illegal XbaI site found at 3629
Illegal PstI site found at 137
Illegal NgoMIV site found at 3519
Illegal AgeI site found at 979
1000
COMPATIBLE WITH RFC[1000]

Introduction

The purpose of this biobrick is to give to S. cerevisiae a lemon taste. This is achieved by expressing the limonene synthase, which produces limonene, a lemon odorant monoterpenoid. This biobrick can be directly integrated into the yeast genome thanks to the YPRCtau3 homology sequences. The experimentator will be able to counter-select the cells that have integrated the construction since they are meant to acquire a prototroph character for methionine.

Design

Fig. 1: LimoneneSynthase-pUC19. The integrative locus used is YPRctau3. The selective marker is MET17. The gene coding for limonene synthase comes from C. limon. We used the terminator PGK and the promoter Gal1/10.

Limonene is one of the most common compounds found in the essential oils of aromatic plants and is widely used in the flavor and fragrance industries[1]. Limonene is produced by limonene synthase which uses geranyl pyrophosphate (GPP) which is the universal precursor of monoterpenoids. Saccharomyces cerevisiae produces geranyl pyrophosphate via the mevalonate pathway where it occurs as an intermediate[2]. It has been shown that S. cerevisiae has enough free GPP to be used by exogenous monoterpene synthases to produce monoterpenes such as limonene[2],[3].

The GAL1/10 bidirectional promoter was chosen based on its multiple properties (similar activities of GAL1 and GAL10 promoters, induction by the many version of the GAL4 transcriptional factor[4]. The sequence was identified from Dr. Gilles Truan and taken from here [5]. PGK1 terminator was chosen because of their large usage in yeast biotechnological manipulations[6]. The sequence was identified from personal communication with Dr. Anthony Henras.

YPRctau3 upstream and downstream homology sequences(BBa_K3570016 and BBa_K3570017) are used to target a functional yeast integration locus. This will result in homologous recombination within a region of type 4 long terminal repeat (Ty4 LTR) in chromosome XVI of the S. cerevisiae's genome. This sequence was identified from a personal communication with Dr. Jean-Marie Francois.

MET17 selection marker (BBa_K3570017) is a gene commonly used as a selection marker for yeast. Only the cells that have integrated the biobrick (and the LEU2 gene in it) would be able to grow without methionine addition in the medium. The sequence was taken from pRS401 plasmid [8].

Experiments

Team iGEM Toulouse 2020 did not have sufficient time to complete the cloning and hence, to test this part functionality.

References

[1]- Erasto, P., & Viljoen, A. M. (2008). Limonene - a Review: Biosynthetic, Ecological and Pharmacological Relevance. Natural Product Communications, 3(7), 1934578X0800300. https://doi.org/10.1177/1934578x0800300728
[2]- Oswald, M., Fischer, M., Dirninger, N., & Karst, F. (2007). Monoterpenoid biosynthesis in Saccharomyces cerevisiae. FEMS Yeast Research, 7(3), 413–421. https://doi.org/10.1111/j.1567-1364.2006.00172.x
[3]- Herrero, Ó., Ramón, D., & Orejas, M. (2008). Engineering the Saccharomyces cerevisiae isoprenoid pathway for de novo production of aromatic monoterpenes in wine. Metabolic Engineering, 10(2), 78–86. https://doi.org/10.1016/j.ymben.2007.11.001
[4]- Elison, G. L., Xue, Y., Song, R., & Acar, M. (2018). Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter. Cell Reports, 25(3), 737-748.e4. https://doi.org/10.1016/j.celrep.2018.09.050
[5]- MRI-34 plasmid
[6]- Curran, K. A., Karim, A. S., Gupta, A., & Alper, H. S. (2013). Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metabolic Engineering, 19, 88–97. https://doi.org/10.1016/j.ymben.2013.07.001
[7]- Bai Flagfeldt, D., Siewers, V., Huang, L., & Nielsen, J. (2009). Characterization of chromosomal integration sites for heterologous gene expression in Saccharomyces cerevisiae. Yeast, 26(10), 545–551. https://doi.org/10.1002/yea.1705
[8]- pRS401 plasmid

@@ Line 6: / Line 6: @@
 <h2>Introduction</h2>
 <p style="text-indent: 40px">
-The purpose of this biobrick is to give a <I>S. cerevisiae</I> a lemon taste. This is achieved by expressing the limonene synthase, which produces limonene, a lemon odorant monoterpenoid. This biobrick can be directly integrated into the yeast genome thanks to YPRCtau3 homology sequences. The experimentator will be able to counter-select the cells that have integrated the construction since they are meant to acquire a prototroph character for methionine.</p>
+The purpose of this biobrick is to give to <I>S. cerevisiae</I> a lemon taste. This is achieved by expressing the limonene synthase, which produces limonene, a lemon odorant monoterpenoid. This biobrick can be directly integrated into the yeast genome thanks to the YPRCtau3 homology sequences. The experimentator will be able to counter-select the cells that have integrated the construction since they are meant to acquire a prototroph character for methionine.</p>
 <h2>Design</h2>
-[[File:T--Toulouse_INSA-UPS--2020_BBa_K3570003.png|600px|thumb|center|Fig. 1: LimoneneSynthase-pUC19. The integrative locus used is YPRctau3. The selective locus used is MET17. The gene coding for limonene synthase comes from <i>C. limon.</i> We used the terminator PGK and the promoter Gal1/10. ]]
+[[File:T--Toulouse_INSA-UPS--2020_BBa_K3570003.png|600px|thumb|center|Fig. 1: LimoneneSynthase-pUC19. The integrative locus used is YPRctau3. The selective marker is MET17. The gene coding for limonene synthase comes from <i>C. limon</i>. We used the terminator PGK and the promoter Gal1/10. ]]
 <p style="text-indent: 40px">