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

Man is a tool-using animal. Without tools he is nothing, with tools he is all.
Thomas Carlyle Signs of the Times (1829)

Overview

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BioBrick plasmid

Introduction

A key part of synthetic biology is to streamline the process of engineering biological systems, by standardizing parts and methods ^{"reference number"}. 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

The pUC19 part and the gBlock fragments was amplified using primers with USER tails, and fused using USER cloning (See Figure XX).

*07072016_pBBaYL_uncut_restrictiondigestion_PCR_onegel_2* picture

The USER reactions was transformed into chemically competent E. coli DH5alpha cells, and purified. The identity of the product was checked using PCR, restriction analysis (see Figure 4) and sequencing (data not shown).

*Gel Pic*

Testing

After having confirmed the identity of the plasmid, we set out to test its functionality. This was done in three steps: 1. Testing the plasmids replicability and selectivity in Y. Lipolytica, 2. Testing the plasmids cloning capabilities in E. coli and finally 3. Combining the two first tests by cloning a construct in E. coli and which is expressed when transformed into Y. Lipolytica.

1. Replicability and selectability in Yarrowia lipolytica

The pSB1A8YL plasmid was purified from E. coli DH5alpha, and transformed in Y. Lipolytica PO1f cells. The transformants was selected on selective dropout media not containing uracil, thus only yielding uracil autotroph transformants. A negative control was included substituting the plasmid for MQ water. The transformations only yielded colonies on the plated containing the cells which were transformed with the plasmid. To ensure that these results indeed meant that our plasmid was stably replicating in the Y. Lipolytica cells, a few colonies were subjected to colony PCR (see Figure X). *Colony PCR* These results confirm that pSB1A8YL replicates in Y. Lipolytica, and the chosen uracil selection marker allows for selection of transformants. To further assess the functionality of pSB1A8YL, the possibility of counter selection was investigated. This was done by growing colonies containing pSB1A8YL on plates containing 5-FOA. Colonies appearing on these plates were then transferred to selective dropout media not containing uracil. As no growth was observed on the latter plate, this proved that pSB1A8YL supports counter selection.

2. Cloning capabilities in E. coli

In order to test this, we decided to produce a device using BioBricks from the distribution kit which would allow us to easily assess whether the cloning were successful. When deciding BioBricks that would allow this, we received inputs from the SDU iGEM team. We ended up choosing the strong Andersson promoter (BBa_K880005) and pair this with three chromoproteins: amilCP (K592009), amilGFP (K592010) and mRFP (E1010), which would allow us to easily pick transformants and visually inspect if the cloning was successful. The cloning flow is shown in Figure 5. *Cloning flow* The BioBricks were retrieved from the distribution kit, and assembled with our plasmid as carrier backbone using standard 3A assembly and transformed into chemically competent E. coli DH5alpha cells. The transformants yielded colored colonies, and the identity of the constructs were confirmed using restriction analysis and PCR (data not shown). These results confirm that pSB1A8YL can be used for cloning in E. coli and supports the BioBrick standard.

3. Combining construct cloning in E. coli and expression in Y. Lipolytica

CRISPR