Team:Paris Saclay/Project/Lemon Scent

From 2014.igem.org

(Difference between revisions)
(Construction of pPS4)
(Countdown)
 
(25 intermediate revisions not shown)
Line 1: Line 1:
{{Team:Paris_Saclay/project_header}}
{{Team:Paris_Saclay/project_header}}
=Lemon Scent=
=Lemon Scent=
-
==Countdown==
 
-
This page is under '''Melanie''''s responsibility
 
-
 
-
* Deadline: 12/oct
 
-
** Final review by Sylvie.
 
-
 
==Summary==
==Summary==
In the part of the project, we want to express the main components of the lemon scent (limonene, β-pinene and citral) in the odor-free ''E. coli'' we constructed. For this, we plan to engineer a plasmid constructed by the team of Taek Soon Lee, bearing a complete mevalonate pathway, a geranyldiphosphate synthase and a (S)-limonene synthase, to express (i) a β-pinene synthase ('''BBa_K517003'''), (ii) a geraniol synthase (GES) and a cinnamyl alcohol dehydrogenase (CAD) to produce β-pinene and citral respectively. We also would like to characterize the limonene synthase biobrick constructed by the Edinburgh team ('''BBa_K762100''') and verify whether it is functional by replacing the intial (S)-limonene synthase by the biobrick.
In the part of the project, we want to express the main components of the lemon scent (limonene, β-pinene and citral) in the odor-free ''E. coli'' we constructed. For this, we plan to engineer a plasmid constructed by the team of Taek Soon Lee, bearing a complete mevalonate pathway, a geranyldiphosphate synthase and a (S)-limonene synthase, to express (i) a β-pinene synthase ('''BBa_K517003'''), (ii) a geraniol synthase (GES) and a cinnamyl alcohol dehydrogenase (CAD) to produce β-pinene and citral respectively. We also would like to characterize the limonene synthase biobrick constructed by the Edinburgh team ('''BBa_K762100''') and verify whether it is functional by replacing the intial (S)-limonene synthase by the biobrick.
-
We managed to construct two plasmids, the plamsid containing the β-pinene synthase gene and the plasmid containing the geraniol synthase gene. Due to lack of time, We could not obtain the other plasmids.
+
We managed to construct two plasmids, the plamsid containing the β-pinene synthase gene and the plasmid containing the geraniol synthase gene. Due to lack of time, we could not obtain the other plasmids.
==Introduction==
==Introduction==
-
The lemon scent is due to many volatile compounds, but the three main components are the limonene, pinene and citral. All these molecules belong the terpene family of metabolites.[1]
+
The lemon scent is due to many volatile compounds, but the three main components are the (S)-limonene, β-pinene and citral molecules. All these molecules belong to the terpene family of metabolites.[1][6]
===Limonene===
===Limonene===
There are two isomeric forms of the limonene molecules:
There are two isomeric forms of the limonene molecules:
-
The odors of these two isomers are  different: the (R)-Limonene has a orangey smell whereas the (S)-Limonene has a strongly lemon scent. [2]
+
Odor of these two isomers are  different: the (R)-Limonene has a orangey smell whereas the (S)-Limonene has a strongly lemon scent. [2]
-
[[File:Paris Saclay project-odor-limonene.jpg|center]]
+
[[File:Paris Saclay project-odor-limonene.png|center]]
===Pinene===
===Pinene===
-
There are also two isomeric forms of pinene: alpha and beta pinene. In the lemon oil, β-pinene represents between 10-17% of the total compounds [3]. In contrast, there is no trace of α-pinene.
+
There are also two isomeric forms of pinene: α- and β-pinene. In the lemon oil, β-pinene represents between 10-17% of the total compounds [3]. In contrast, there is no trace of α-pinene.
-
[[File:Paris Saclay project-odor-pinene.jpg|center]]
+
[[File:Paris Saclay project-odor-pinene.png|center]]
===Citral===
===Citral===
-
This is the principal component of the lemon grass and lemon smell and it is present in many other plants such as orange, verbena... Here again there are two stereoisomeric forms, geranial and neral. The trans isomer, geranial or citral A, has a strong lemon scent. On the contrary, the cis isomer named neral or citral B has sweaker lemon scent. These compounds are often used in the perfume industry to reproduce the smell of lemon.
+
This is the principal component of the lemon grass and lemon smell and it is present in many other plants such as orange, verbena... Here again there are two stereoisomeric forms, geranial and neral. The trans isomer, geranial or citral A, has a strong lemon scent. Whereas, the cis isomer named neral or citral B has sweaker lemon scent. These compounds are often used in the perfume industry to reproduce the smell of lemon.
-
[[File:Paris Saclay project-odor-citral.jpg|center]]
+
[[File:Paris Saclay project-odor-citral.png|center]]
-
All these compounds derive from a common precursor, the geranylpyrophosphate (GPP). This precursor is converted in one or two steps in the molecules we are interested in obtaining.
+
All these compounds come from a common precursor, the geranylpyrophosphate (GPP). This precursor is converted in one or two steps in the molecules we are interested in obtaining.
-
[[File:Paris Saclay project-odor-gpp.jpg|center]]
+
[[File:Paris Saclay project-odor-gpp.png|center]]
The desoxyxylulose 5-phosphate (DXP) parthway is the common pathway used by prokaryote cells to produce GPP.
The desoxyxylulose 5-phosphate (DXP) parthway is the common pathway used by prokaryote cells to produce GPP.
''E. coli'' synthesizes GPP using the DXP pathway but in low quantity [4].
''E. coli'' synthesizes GPP using the DXP pathway but in low quantity [4].
-
However, another pathway, the mevalonate pathway (MEV) exists in eukaryote and some prokaryotes. Moreover, plants have the two pathways to produce GPP in higher quantity.
+
However, another pathway called the mevalonate pathway (MEV) exists in eukaryote and some prokaryotes. Moreover, plants can use both pathways to produce GPP in higher quantity.
-
To copy the plants' strategy, we chose to add the MEV pathway to our engineered ''E. coli'' in order to produce higher quantity of GPP and thereby have a stronger lemon smell.
+
To copy the plants' strategy, we chose to add the MEV pathway to our engineered ''E. coli'' in order to produce higher quantity of GPP and thereby obtain a stronger lemon smell.
-
The figure below illustrates the MEV pathway:
+
The figure below is a scheme of the MEV pathway:
-
[[File:Paris Saclay project-odor-mev-pathway.jpg|center]]
+
[[File:Paris Saclay project-odor-mev-pathway.png|center]]
-
To tackle the problem of the low GPP bioavailability in ''E. coli'', the Taek Soon Lee’s research group designed and constructed a plasmid with a complete biosynthetic mevalonate pathway for the production of GPP [3]. They then expressed the ''Abies grandis'' (S)-limonene synthase (LS) gene in the same plasmid, named pJBEI6409. With this construction, they were able to produce 400 mg/L of (S)-limonene.
+
To tackle the problem of the low GPP bioavailability in ''E. coli'', the Taek Soon Lee’s research group designed and constructed a plasmid with a complete biosynthetic mevalonate pathway for the production of GPP [3]. Then they expressed the ''Abies grandis'' (S)-limonene synthase (LS) gene in the same plasmid, called pJBEI6409. With this construction, they were able to produce 400 mg/L of (S)-limonene.
-
[[File:Paris_Saclay_pJBEI6409_ls.jpg|center|650px]]
+
[[File:Paris_Saclay_pJBEI6409_ls.png|center|650px]]
-
We decided to make use of their construction for the production of (R)-limonene, β-pinene and citral in E. coli. We thank the team of Taek Soon Lee for the gift of the pJBEI6409 plasmid.
+
We decided to use their construction for the production of (R)-limonene, β-pinene and citral in ''E. coli''. We thank the team of Taek Soon Lee for the gift of the pJBEI6409 plasmid.
==Principle of our Constructions==
==Principle of our Constructions==
-
For our project, we need to replace the (S)-limonene synthase (LS) gene by the (R)-limonene synthase gene (BBa_K762100), the β-pinene synthase gene (BBa_K517003) and the geraniol synthase (GES)- cinnamyl alcohol dehydrogenase (CAD) genes.
+
For our project, we need to replace the (S)-limonene synthase (LS) gene by the (R)-limonene synthase gene ('''BBa_K762100'''), the β-pinene synthase gene ('''BBa_K517003''') and the geraniol synthase (GES)- cinnamyl alcohol dehydrogenase (CAD) genes.
-
However, pJBEI6409 was constructed using a 3A assembly method, meaning that there are no restriction sites flanking the LS gene.
+
However, pJBEI6409 was constructed using a 3A assembly method, meaning that no restriction sites flanking the LS gene exist.
-
We therefore decided to replace the (S) Limonene Synthase gene by an apramycin resistance cassette flanked by ''PacI'' and ''SalI'' restriction sites by double homologous recombination. The resulting plasmid (pPS1) will then be used to clone the various genes (limonene synthase – β-pinene synthase – geraniol synthase and cinnamyl alcohol dehydrogenase).
+
We therefore decided to replace the (S)-LS gene by an apramycin resistance cassette surrounded by ''PacI'' and ''SalI'' restriction sites using double homologous recombination. Then, the resulting plasmid (pPS1) will be used to clone various genes (limonene synthase – β-pinene synthase – geraniol synthase and cinnamyl alcohol dehydrogenase).
-
[[File:Paris Saclay project-odor-recap.jpg|center|650px]]
+
[[File:Paris Saclay project-odor-recap.png|center|750px]]
===Construction of pPS1===
===Construction of pPS1===
''Replacement of the LS gene by an apramycin cassette''
''Replacement of the LS gene by an apramycin cassette''
-
The first step of this sub-project is to replace the (S)-limonene synthase (LS) gene by an apramycin-resistant cassette. This cassette is flanked by ''PacI'' and ''SalI'' restriction sites to facilitate the cloning of the different genes.
+
The first step of this sub-project is to replace the (S)-limonene synthase (LS) gene by an apramycin-resistant cassette. This cassette is flanked by ''PacI'' and ''SalI'' restriction sites to make easy the cloning of the different genes.
-
[[File:Paris Saclay project-odor-apramycine-k7.jpg|center]]
+
[[File:Paris Saclay project-odor-apramycine-k7.png|center]]
The (S)-LS starting gene is replaced by the apramycin cassette by PCR targeting.
The (S)-LS starting gene is replaced by the apramycin cassette by PCR targeting.
-
# PCR amplification of the apramycin cassette with oligonucleotides iPS70 and iPS71 that contain the restriction sites ''Pac''I and ''Sal''I. The ends of these oligonucleotides are homologous with the sequence that constitutes the LS gene.
+
# PCR amplification of the apramycin cassette with oligonucleotides iPS70 and iPS71 that contain the restriction sites ''Pac''I and ''Sal''I. The extremities of these oligonucleotides are homologous with the sequence that constitutes the LS gene.
# Transformation of the ''E. coli'' DY330 strain [5] (hyperrecombinant strain) by the pJBEI6409 plasmid.
# Transformation of the ''E. coli'' DY330 strain [5] (hyperrecombinant strain) by the pJBEI6409 plasmid.
# Transformation of the ''E. coli'' DY330/pJBEI6409 with the PCR product after induction of the recombination genes.
# Transformation of the ''E. coli'' DY330/pJBEI6409 with the PCR product after induction of the recombination genes.
Line 74: Line 68:
''Replacement of the apramycin cassette by the (R)- LS gene, '''''Bba_K762100'''''
''Replacement of the apramycin cassette by the (R)- LS gene, '''''Bba_K762100'''''
-
This step consists in replacing the apramycin resistance gene cassette introduced in pJBEI6409 by the (R)-LS gene.
+
This step consists on substituting the apramycin resistance gene cassette introduced in pJBEI6409 by the (R)-LS gene.
# PCR amplification of '''Bba_K762100''' with oligonucleotides iPS66 and iPS67 that contain the restriction sites ''PacI'' and ''SalI''.
# PCR amplification of '''Bba_K762100''' with oligonucleotides iPS66 and iPS67 that contain the restriction sites ''PacI'' and ''SalI''.
# TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (LS Topo plasmid).
# TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (LS Topo plasmid).
Line 88: Line 82:
''Replacement of the apramycin cassette by the β pinene gene, '''BBa_K517003'''''
''Replacement of the apramycin cassette by the β pinene gene, '''BBa_K517003'''''
-
This step consists in replacing the apramycin resistance gene cassette introduced in pJBEI6409 by the β pinene gene.
+
This step consists on substituting the apramycin resistance gene cassette introduced in pJBEI6409 by the β pinene gene.
# PCR amplification of '''Bba_K517003''' with oligonucleotides iPS68bis and iPS69 that contain the restriction sites ''PacI'' and ''SalI''.
# PCR amplification of '''Bba_K517003''' with oligonucleotides iPS68bis and iPS69 that contain the restriction sites ''PacI'' and ''SalI''.
# [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/12 TA-cloning] of the PCR product in the Pcr 4 Blunt-Topo vector (PS Topo plasmid).
# [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/12 TA-cloning] of the PCR product in the Pcr 4 Blunt-Topo vector (PS Topo plasmid).
Line 101: Line 95:
''Replacement of the apramycin cassette by the geraniol synthase gene,  
''Replacement of the apramycin cassette by the geraniol synthase gene,  
-
This step consists in replacing the apramycin resistance gene cassette introduced in pJBEI6409 by geraniol synthase gene -(GS) (obtained from Marc Fischer, from the Grapevine Secondary Metabolism team of the ‘grapevine health and wine quality' research unit (Colmar)).
+
This step consists on substituting  the apramycin resistance gene cassette introduced in pJBEI6409 by geraniol synthase gene -(GS) (obtained from Marc Fischer, from the Grapevine Secondary Metabolism team of the ‘grapevine health and wine quality' research unit (Colmar)).
# PCR amplification of the gene (pcola plasmid) with oligonucleotides iPS81bis and iPS82 that contain the restriction sites ''PacI'' and ''SalI''.
# PCR amplification of the gene (pcola plasmid) with oligonucleotides iPS81bis and iPS82 that contain the restriction sites ''PacI'' and ''SalI''.
# TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (GS Topo plasmid).
# TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (GS Topo plasmid).
# [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/9 Verification] of the clones GS-TOPO by PCR with the oligonucleotides  iPS81bis and iPS82 and sequencing.
# [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/9 Verification] of the clones GS-TOPO by PCR with the oligonucleotides  iPS81bis and iPS82 and sequencing.
-
# Digestion of the GS-TOPO plasmid by ''PacI'' and ''PstI'' to eliminate the topo vector.
+
# Digestion of the GS-TOPO plasmid by ''PacI'' and ''MscI'' to eliminate the topo vector.
# Digestion of pPS1 by ''PacI''.
# Digestion of pPS1 by ''PacI''.
# Ligation of the GS gene into pPS1/''PacI''.
# Ligation of the GS gene into pPS1/''PacI''.
Line 114: Line 108:
''Cloning of the ''CAD'' gene in pPS4''
''Cloning of the ''CAD'' gene in pPS4''
-
This step consists in cloning the Cinnamyl alcohol deshydrogenase (CAD) gene in the ''SalI'' site of pPS4
+
This step consists on cloning the Cinnamyl alcohol deshydrogenase (CAD) gene in the ''SalI'' site of pPS4
# PCR amplification of the truncated CAD gene (synthetic gene with oligonucleotides iPS79 and iPS80 that contain the restriction sites ''PacI'' and ''SalI'').
# PCR amplification of the truncated CAD gene (synthetic gene with oligonucleotides iPS79 and iPS80 that contain the restriction sites ''PacI'' and ''SalI'').
# TA-cloning of the PCR product in the PCR 4 Blunt-Topo vector (CAD Topo plasmid).
# TA-cloning of the PCR product in the PCR 4 Blunt-Topo vector (CAD Topo plasmid).
-
# [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/9 Verification] of the clones LS-TOPO by PCR with the oligonucleotides i iPS79 and iPS80 and sequencing.
+
# [https://2014.igem.org/Team:Paris_Saclay/Notebook/September/9 Verification] of the clones CAD-TOPO by PCR with the oligonucleotides iPS79 and iPS80 and sequencing.
-
# Digestion of the LS-TOPO plasmid by ''SalI'' and ''PvuII'' to eliminate the topo vector.
+
# Digestion of the CAD-TOPO plasmid by ''SalI'' and ''PvuII'' to eliminate the topo vector.
# Digestion of pPS4 by ''SalI''.
# Digestion of pPS4 by ''SalI''.
# Ligation of the CAD gene into pPS4/''SalI''.
# Ligation of the CAD gene into pPS4/''SalI''.
# Transformation of E. coli DH5α with the ligation product
# Transformation of E. coli DH5α with the ligation product
-
# Verification of the clones by PCR with the oligonucleotide
+
# Verification of the clones by PCR
-
==Réferences==
+
==References==
* [1] Marcelo Carnier Dornelas - A genomic approach to characterization of the Citrus terpene synthase gene family - Genet. Mol. Biol. vol.30 no.3 suppl.0 São Paulo  2007
* [1] Marcelo Carnier Dornelas - A genomic approach to characterization of the Citrus terpene synthase gene family - Genet. Mol. Biol. vol.30 no.3 suppl.0 São Paulo  2007
* [2] http://www.societechimiquedefrance.fr/produit-du-jour/limonene-et-monoterpenes.html
* [2] http://www.societechimiquedefrance.fr/produit-du-jour/limonene-et-monoterpenes.html
Line 130: Line 124:
* [4] Jorge Alonso-Gutierrez and al - Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production – metabolique engineering 2013
* [4] Jorge Alonso-Gutierrez and al - Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production – metabolique engineering 2013
* [5] Daiguan Yu and al - An efficient recombination system for chromosome engineering in Escherichia coli – 2000)
* [5] Daiguan Yu and al - An efficient recombination system for chromosome engineering in Escherichia coli – 2000)
-
 
+
* [6] http://www.aromaticplantproject.com/articles_archive/citrus_essential_oils.html
{{Team:Paris_Saclay/default_footer}}
{{Team:Paris_Saclay/default_footer}}

Latest revision as of 06:22, 17 October 2014

Contents

Lemon Scent

Summary

In the part of the project, we want to express the main components of the lemon scent (limonene, β-pinene and citral) in the odor-free E. coli we constructed. For this, we plan to engineer a plasmid constructed by the team of Taek Soon Lee, bearing a complete mevalonate pathway, a geranyldiphosphate synthase and a (S)-limonene synthase, to express (i) a β-pinene synthase (BBa_K517003), (ii) a geraniol synthase (GES) and a cinnamyl alcohol dehydrogenase (CAD) to produce β-pinene and citral respectively. We also would like to characterize the limonene synthase biobrick constructed by the Edinburgh team (BBa_K762100) and verify whether it is functional by replacing the intial (S)-limonene synthase by the biobrick.

We managed to construct two plasmids, the plamsid containing the β-pinene synthase gene and the plasmid containing the geraniol synthase gene. Due to lack of time, we could not obtain the other plasmids.

Introduction

The lemon scent is due to many volatile compounds, but the three main components are the (S)-limonene, β-pinene and citral molecules. All these molecules belong to the terpene family of metabolites.[1][6]

Limonene

There are two isomeric forms of the limonene molecules: Odor of these two isomers are different: the (R)-Limonene has a orangey smell whereas the (S)-Limonene has a strongly lemon scent. [2]

Paris Saclay project-odor-limonene.png

Pinene

There are also two isomeric forms of pinene: α- and β-pinene. In the lemon oil, β-pinene represents between 10-17% of the total compounds [3]. In contrast, there is no trace of α-pinene.

Paris Saclay project-odor-pinene.png

Citral

This is the principal component of the lemon grass and lemon smell and it is present in many other plants such as orange, verbena... Here again there are two stereoisomeric forms, geranial and neral. The trans isomer, geranial or citral A, has a strong lemon scent. Whereas, the cis isomer named neral or citral B has sweaker lemon scent. These compounds are often used in the perfume industry to reproduce the smell of lemon.

Paris Saclay project-odor-citral.png

All these compounds come from a common precursor, the geranylpyrophosphate (GPP). This precursor is converted in one or two steps in the molecules we are interested in obtaining.

Paris Saclay project-odor-gpp.png

The desoxyxylulose 5-phosphate (DXP) parthway is the common pathway used by prokaryote cells to produce GPP. E. coli synthesizes GPP using the DXP pathway but in low quantity [4]. However, another pathway called the mevalonate pathway (MEV) exists in eukaryote and some prokaryotes. Moreover, plants can use both pathways to produce GPP in higher quantity. To copy the plants' strategy, we chose to add the MEV pathway to our engineered E. coli in order to produce higher quantity of GPP and thereby obtain a stronger lemon smell. The figure below is a scheme of the MEV pathway:

Paris Saclay project-odor-mev-pathway.png

To tackle the problem of the low GPP bioavailability in E. coli, the Taek Soon Lee’s research group designed and constructed a plasmid with a complete biosynthetic mevalonate pathway for the production of GPP [3]. Then they expressed the Abies grandis (S)-limonene synthase (LS) gene in the same plasmid, called pJBEI6409. With this construction, they were able to produce 400 mg/L of (S)-limonene.

Paris Saclay pJBEI6409 ls.png

We decided to use their construction for the production of (R)-limonene, β-pinene and citral in E. coli. We thank the team of Taek Soon Lee for the gift of the pJBEI6409 plasmid.

Principle of our Constructions

For our project, we need to replace the (S)-limonene synthase (LS) gene by the (R)-limonene synthase gene (BBa_K762100), the β-pinene synthase gene (BBa_K517003) and the geraniol synthase (GES)- cinnamyl alcohol dehydrogenase (CAD) genes.

However, pJBEI6409 was constructed using a 3A assembly method, meaning that no restriction sites flanking the LS gene exist. We therefore decided to replace the (S)-LS gene by an apramycin resistance cassette surrounded by PacI and SalI restriction sites using double homologous recombination. Then, the resulting plasmid (pPS1) will be used to clone various genes (limonene synthase – β-pinene synthase – geraniol synthase and cinnamyl alcohol dehydrogenase).

Paris Saclay project-odor-recap.png

Construction of pPS1

Replacement of the LS gene by an apramycin cassette

The first step of this sub-project is to replace the (S)-limonene synthase (LS) gene by an apramycin-resistant cassette. This cassette is flanked by PacI and SalI restriction sites to make easy the cloning of the different genes.

Paris Saclay project-odor-apramycine-k7.png

The (S)-LS starting gene is replaced by the apramycin cassette by PCR targeting.

  1. PCR amplification of the apramycin cassette with oligonucleotides iPS70 and iPS71 that contain the restriction sites PacI and SalI. The extremities of these oligonucleotides are homologous with the sequence that constitutes the LS gene.
  2. Transformation of the E. coli DY330 strain [5] (hyperrecombinant strain) by the pJBEI6409 plasmid.
  3. Transformation of the E. coli DY330/pJBEI6409 with the PCR product after induction of the recombination genes.
  4. Verification of the clones by BglII digestion.

We call this plasmid pPS1.

Construction pPS2

Replacement of the apramycin cassette by the (R)- LS gene, Bba_K762100

This step consists on substituting the apramycin resistance gene cassette introduced in pJBEI6409 by the (R)-LS gene.

  1. PCR amplification of Bba_K762100 with oligonucleotides iPS66 and iPS67 that contain the restriction sites PacI and SalI.
  2. TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (LS Topo plasmid).
  3. Verification of the clones LS-TOPO by PCR with the oligonucleotides iPS66 and iPS67 and sequencing.
  4. Digestion of the LS-TOPO plasmid by PacI and by MscI to eliminate the topo vector.
  5. Digestion of pPS1 by PacI.
  6. Ligation of the LS gene into pPS1/PacI.
  7. Transformation of E. coli DH5α with the ligation product.
  8. Verification of the clones by PCR with the oligonucleotides iPS72 and iPS96

Here we failed to obtain an insert in the correct orientation.

Construction of pPS3

Replacement of the apramycin cassette by the β pinene gene, BBa_K517003

This step consists on substituting the apramycin resistance gene cassette introduced in pJBEI6409 by the β pinene gene.

  1. PCR amplification of Bba_K517003 with oligonucleotides iPS68bis and iPS69 that contain the restriction sites PacI and SalI.
  2. TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (PS Topo plasmid).
  3. Verification of the clones PS-TOPO by PCR with the oligonucleotides iPS68bis and iPS69 and sequencing.
  4. Digestion of the PS-TOPO plasmid by PacI and by PstI to eliminate the topo vector.
  5. Digestion of pPS1 by PacI.
  6. Ligation of the PS gene into pPS1/PacI.
  7. Transformation of E. coli DH5α with the ligation product.
  8. Verification of the clones by PCR with the oligonucleotides iPS72 and iPS93.

Construction of pPS4

Replacement of the apramycin cassette by the geraniol synthase gene,

This step consists on substituting the apramycin resistance gene cassette introduced in pJBEI6409 by geraniol synthase gene -(GS) (obtained from Marc Fischer, from the Grapevine Secondary Metabolism team of the ‘grapevine health and wine quality' research unit (Colmar)).

  1. PCR amplification of the gene (pcola plasmid) with oligonucleotides iPS81bis and iPS82 that contain the restriction sites PacI and SalI.
  2. TA-cloning of the PCR product in the Pcr 4 Blunt-Topo vector (GS Topo plasmid).
  3. Verification of the clones GS-TOPO by PCR with the oligonucleotides iPS81bis and iPS82 and sequencing.
  4. Digestion of the GS-TOPO plasmid by PacI and MscI to eliminate the topo vector.
  5. Digestion of pPS1 by PacI.
  6. Ligation of the GS gene into pPS1/PacI.
  7. Transformation of E. coli DH5α with the ligation product.
  8. Verification of the clones by PCR with the oligonucleotides iPS72 and iPS96

Construction of pPS5

Cloning of the CAD gene in pPS4

This step consists on cloning the Cinnamyl alcohol deshydrogenase (CAD) gene in the SalI site of pPS4

  1. PCR amplification of the truncated CAD gene (synthetic gene with oligonucleotides iPS79 and iPS80 that contain the restriction sites PacI and SalI).
  2. TA-cloning of the PCR product in the PCR 4 Blunt-Topo vector (CAD Topo plasmid).
  3. Verification of the clones CAD-TOPO by PCR with the oligonucleotides iPS79 and iPS80 and sequencing.
  4. Digestion of the CAD-TOPO plasmid by SalI and PvuII to eliminate the topo vector.
  5. Digestion of pPS4 by SalI.
  6. Ligation of the CAD gene into pPS4/SalI.
  7. Transformation of E. coli DH5α with the ligation product
  8. Verification of the clones by PCR

References