Difference between revisions of "Team:DTU-Denmark/molecular toolbox"
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| − | + | A key part of synthetic biology is to streamline the process of engineering biological systems, by standardizing parts and methods [Engineering BioBrick vectors from BioBrick parts]. Perhaps the most versatile standards available is the BioBrick standard, in part due to the contributions made during the annual iGEM competition. The BioBrick registry currently has over 20,000[http://parts.igem.org/Collections], and by creating BioBrick plasmid backbones compatible with a new organism, one is effectively unlocking the entire BioBrick registry available for that specific organism. Realizing this, it was decided to develop a plasmid that supports the BioBrick standard and replicates in <i>Y. Lipolytica</i>. Due to the convenience of manipulating <i>Escherichia</i> coli it was determined to develop a shuttle vector that allows for cloning and confirmation of the construct in E. coli, before the construct is transformed in <i>Y. Lipolytica</i>. Additionally, as the only replicative plasmids currently available for <i>Y. Lipolytica</i> is low copy yeast chromosomal plasmids (YCp) [Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function] this allows for high amounts of DNA to easily be propagated in E. coli, before the the plasmid is purified and transformed into <i>Y. Lipolytica</i>. Figure 1 shows a suggested workflow for the proposed BioBrick plasmid. | |
| + | </p> | ||
| + | <p> | ||
| + | *Cloning workflow* | ||
| + | </p> | ||
| + | <h3 class="h3">Design</h3> | ||
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| + | For the design of the plasmid, we decided to incorporate a high copy <i>E. coli</i> part for cloning and propagation DNA. The design was based on the pUC19 vector as it fulfils the criteria of being high copy [Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors], while perhaps being one of the most widely used cloning vectors for <i>E. coli</i>. To support the BioBrick standard, we only used the ampicillin resistance and replication origin elements of the plasmid. It was found that the sequence in and between these elements did not contain any restriction sites of any current BioBrick assembly standard, thus no further modification of the sequence was needed. | ||
</p><p> | </p><p> | ||
| − | + | For the <i>Y. Lipolytica</i> part of the plasmid we decided to base the design on the pSL16-CEN1-1(227), as it has found to exhibit high transformation efficiency compared to similar plasmids [Dissection of Centromeric DNA from Yeast Yarrowia lipolytica and Identification of Protein-Binding Site Required for Plasmid Transmission], and perhaps for this reason this plasmid and its derivatives are utilized in many recent studies. [Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function][Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach][Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR–Cas9-Mediated Genome Editing in Yarrowia lipolytica]. Again only the sequence of the replicative and selective elements were chosen. Although, the original sequence was not BioBrick compatible, and thus it was decided to order the sequence as a gBlock. This also introduced the added benefit of being able to incorporate the BioBrick prefix, suffix and a 5’ terminator in the gBlock and exchange the original leucine autotrophy marker with a uracil autotrophy marker allowing for negative selection of the plasmid with 5-FOA [Identification of the UMP synthase gene by establishment of uracil auxotrophic mutants and the phenotypic complementation system in the marine diatom Phaeodactylum tricornutum]. In order to comply with the iGEM plasmid nomenclature [http://parts.igem.org/Help:Plasmid_backbones/Nomenclature], the plasmid was dubbed “pSB1A8YL”, YL was added in the end to emphasize that the plasmid is used for <i>Y. Lipolytica</i>. Figure 2 shows a graphical representation of the sequence map of pSB1A8YL. | |
| + | </p><p> | ||
| + | *Sequence map* | ||
</p> | </p> | ||
| − | <h3 class="h3"> | + | <h3 class="h3">Cloning</h3> |
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Revision as of 12:45, 26 August 2016
Molecular toolbox
In the substrate section we established that Yarrowia Lipolytica constitutes a great platform utilizing waste streams. In order to unlock the potential of Y. Lipolytica, we developed a molecular toolbox allowing us to efficiently engineering Y. Lipolytica. In this section we present the theory and results of the development of a BioBrick backbone and CRISPR tools for Y. Lipolytica
Overview
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BioBrick plasmid
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Introduction
A key part of synthetic biology is to streamline the process of engineering biological systems, by standardizing parts and methods [Engineering BioBrick vectors from BioBrick parts]. Perhaps the most versatile standards available is the BioBrick standard, in part due to the contributions made during the annual iGEM competition. The BioBrick registry currently has over 20,000[http://parts.igem.org/Collections], and by creating BioBrick plasmid backbones compatible with a new organism, one is effectively unlocking the entire BioBrick registry available for that specific organism. Realizing this, it was decided to develop a plasmid that supports the BioBrick standard and replicates in Y. Lipolytica. Due to the convenience of manipulating Escherichia coli it was determined to develop a shuttle vector that allows for cloning and confirmation of the construct in E. coli, before the construct is transformed in Y. Lipolytica. Additionally, as the only replicative plasmids currently available for Y. Lipolytica is low copy yeast chromosomal plasmids (YCp) [Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function] this allows for high amounts of DNA to easily be propagated in E. coli, before the the plasmid is purified and transformed into Y. Lipolytica. Figure 1 shows a suggested workflow for the proposed BioBrick plasmid.
*Cloning workflow*
Design
For the design of the plasmid, we decided to incorporate a high copy E. coli part for cloning and propagation DNA. The design was based on the pUC19 vector as it fulfils the criteria of being high copy [Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors], while perhaps being one of the most widely used cloning vectors for E. coli. To support the BioBrick standard, we only used the ampicillin resistance and replication origin elements of the plasmid. It was found that the sequence in and between these elements did not contain any restriction sites of any current BioBrick assembly standard, thus no further modification of the sequence was needed.
For the Y. Lipolytica part of the plasmid we decided to base the design on the pSL16-CEN1-1(227), as it has found to exhibit high transformation efficiency compared to similar plasmids [Dissection of Centromeric DNA from Yeast Yarrowia lipolytica and Identification of Protein-Binding Site Required for Plasmid Transmission], and perhaps for this reason this plasmid and its derivatives are utilized in many recent studies. [Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function][Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach][Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR–Cas9-Mediated Genome Editing in Yarrowia lipolytica]. Again only the sequence of the replicative and selective elements were chosen. Although, the original sequence was not BioBrick compatible, and thus it was decided to order the sequence as a gBlock. This also introduced the added benefit of being able to incorporate the BioBrick prefix, suffix and a 5’ terminator in the gBlock and exchange the original leucine autotrophy marker with a uracil autotrophy marker allowing for negative selection of the plasmid with 5-FOA [Identification of the UMP synthase gene by establishment of uracil auxotrophic mutants and the phenotypic complementation system in the marine diatom Phaeodactylum tricornutum]. In order to comply with the iGEM plasmid nomenclature [http://parts.igem.org/Help:Plasmid_backbones/Nomenclature], the plasmid was dubbed “pSB1A8YL”, YL was added in the end to emphasize that the plasmid is used for Y. Lipolytica. Figure 2 shows a graphical representation of the sequence map of pSB1A8YL.
*Sequence map*
Cloning
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Testing
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