BioBrick Plasmid

Introduction

A key part of synthetic biology is to streamline the process of engineering biological systems, by standardizing parts and methods¹. 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 20,000² BioBricks, 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 Yarrowia 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 are low copy yeast chromosomal plasmids (YCp)³ our plasmid allows for high amounts of deoxyribonucleic acid (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.

Design

For the design of the plasmid, we decided to incorporate a high copy E. coli part for cloning and propagation of DNA. The design was based on the pUC19 vector as it fulfills the criteria of being high copy⁴, 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.

Cloning

The USER reactions was transformed into chemically competent E. coli DH5Α cells, and purified. The identity of the product was checked using PCR, restriction analysis (see Figure 4) and sequencing (data not shown).

Testing

After having confirmed the identity of the plasmid, we set out to test its functionality. This was done by: 1. Testing the plasmids replicability and selectivity in Y. Lipolytica and 2. testing the plasmids cloning capabilities in E. coli.

1. Replicability and selectability in Yarrowia lipolytica

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 clonings 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/strong RBS BioBrick (BBa_K880005) and pair this with three chromoproteins: amilCP (BBa_K592009), amilGFP (BBa_K592010) and mRFP (BBa_E1010), which would allow us to easily pick transformants and visually inspect if the cloning was successful. The cloning flow is shown in Figure 6.

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 DH5Α cells. The transformants yielded colored colonies (See Figure 7), 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.

CRISPR

Introduction

By creating a modular plasmid that is compatible with the BioBrick standards and that is able to replicate in both E. coli and Y. lipolytica, the entire biobrick registry has been made available for use in Y. lipolytica. However, one important aspect of industrial-scale protein expression is the stable and homogenous expression of relevant pathway genes. In production organisms with multi-enzyme pathways there is often a need for accurate control of the expression levels of the associated genes, both in terms of promoter strength and copy numbers¹¹. Plasmid based systems for the expression of heterologous genes are widely used, but offer limited control of copy number and in many cases show notable segregational instability even during growth in selective media. Therefore, it is often preferred to integrate the genetic pathway of interest into the genome of the production organism of choice to ensure long-term strain stability and homogenous expression of pathway genes¹¹.

The CRISPR-Cas9 has revolutionized the way in which model organisms can be engineered. In addition, it has made genetic engineering applicable to a wide range of organisms that were previously considered to be uninteresting for cell factory purposes, largely because the genetic engineering tools for these organisms were under characterized. The CRISPR-Cas9 system is ideally suited for the integration of heterologous genes in production organisms, as it relies on Double Strand Break (DSB) induction and its subsequent repair by homologous recombination (HR). In addition, almost any sequence of interest can be targeted with the most commonly used Cas9 from Streptococcus pyogenes¹². The basic mechanism of CRISPR-Cas9 is illustrated in Figure 9, for a more detailed overview the reader is referred to Nødvig et al.¹²

We adapt the CRISPR-Cas9 system for use in Y. lipolytica in order to obtain a proof-of-concept of the construction of a stable production strain. We do so by a proof-of-concept integration of a gene in Y. lipolytica. In addition, we obtain a proof-of-concept of the deletion of a native Y. lipolytica gene. With these proof-of-concepts, we envision that the metabolic flux towards a possible compound of interest could be increased, leading to tailored Y. lipolytica strains for cell factory engineering purposes.

Experimental design

The CRISPR-Cas9 system has previously been successfully applied to Y. lipolytica by Schwartz et al.⁸ As this was a proof-of-concept study, the native Y. lipolytica PEX10 gene was targeted for disruption, which allowed for an easily selectable phenotype. This is because PEX10 codes for Peroxisome biogenesis factor 10, a protein that is involved in peroxisome biogenesis. Consequently, PEX10 disruption results in an inability to catabolize long-chain fatty acids. We set out to confirm the observed PEX10 deletion. In addition, we set out to integrate URA3, a commonly used yeast auxotrophic marker gene which codes for Orotidine 5’-phosphate decarboxylase, an enzyme involved in pyrimidine biosynthesis.

We had obtained both a commonly used Y. lipolytica laboratory strain (PO1f, MATA URA3-­302 LEU2-­270 XPR2-­322 AXP2 ­ΔNU49 XPR2::SUC2; obtained from Schwartz et al.⁸) and also this same strain with a knockout of the Non-Homologous End-Joining (NHEJ) DNA repair pathway gene Ku70 (PO1fΔKu70, MATA URA3-­302 LEU2-­270 XPR2-­322 AXP2 ­ΔNU49 ΔKu70 XPR2::SUC2; obtained from Schwartz et al.⁸). The NHEJ pathway for DNA repair is commonly disrupted in laboratory strains to improve the efficiency of CRISPR-Cas9 induced HR integration of heterologous DNA. However, an intact NHEJ pathway can be utilized to disrupt genes without the need for an DNA repair template, as this DNA repair pathway is intrinsically more error-prone than HR. Thus, in order to disrupt the native Y. lipolytica PEX10 gene, we used a CRISPR-Cas9 plasmid (pCRISPRyl) in the PO1f strain. For the URA3 insertion, we co-transformed the same CRISPR-Cas9 plasmid and a linearized HR donor plasmid (pIW501) in the PO1fΔKu70 strain. An overview of our workflow is provided in Figure 10. Overviews of the pCRISPRyl and pIW501 are provided in Figures 11 and 12, respectively.

Results & Discussion

1. URA3 insertion

In order to integrate the URA3 gene into the genome of the Y. lipolytica PO1fΔKu70 strain, we first constructed a pCRISPRyl-derived plasmid which contained a protospacer that targets the PEX10 locus. This protospacer was used previously for successful disruption of PEX10⁸. The pCRISPRyl-derived plasmid (pIW357) was constructed through the Gibson assembly of a 60 bp fragment containing the protospacer of interest into the AvrII linearized pCRISPRyl. Subsequently, pIW357 was purified from selected E. coli DH5ɑ transformants and a double restriction analysis was performed with PstI and AvrII (Figure 13). As the purified pIW357 was no longer digested with AvrII, the protospacer had been correctly inserted. In addition, the protospacer insertion was confirmed through Sanger sequencing (data not shown).

Next, we tested pIW357 in the Y. lipolytica PO1fΔKu70 strain. In order to account for Cas9 activity of pIW357, we also transformed the pCRISPRyl as a positive control. The results of these transformations are depicted in Figure 14. With an intact NHEJ repair pathway and/or a template for HR, the induced DSB should easily be repaired. However, as this is the PO1fΔKu70 strain and as there is no HR donor template available, the induced DSB should be lethal. Based on the difference in transformant counts between the pIW357 and pCRISPRyl transformations, pIW357 is functioning properly. The observed residual growth in the pIW357 transformation can be attributed to either mutations in pIW357 (for example in Cas9 or in the the sgRNA/tRNA promoter) or to possible repair by the Microhomology-Mediated End-Joining (MMEJ) pathway¹⁴.

With a functioning pIW357, we then co-transformed pIW357 and pIW501 (linearized) into the Y. lipolytica PO1fΔKu70 strain (Figure 15). The observation of growth on SC-ura media indicates that the URA3 gene had been successfully integrated. Growth was also observed when solely the linearized pIW501 was transformed. In addition, transformants from the co-transformation of pIW357 and pIW501 were screened for URA3 integration through gap-check colony PCR (Figure 16). The observed shift in band size between non-pIW501 transformants and pIW357/pIW501 co-transformants corresponds to the 1700 bp size of the URA3 promoter+CDS+terminator sequence that is present on pIW501.

The pIW501 is linearized prior to transformation in order to increase HR efficiency and in order to reduce the amount of false positive transformants (a gene cannot be expressed from a linear DNA fragment). However, growth was also observed when solely the pIW501 is transformed, indicating that CRISPR-Cas9 DSB induction is not necessarily required for the integration of an auxotrophic marker gene. We did not have time to compare the efficiencies of co-transformation of pIW357 and pIW501 as compared to solely pIW501 transformation. However, it has been shown previously that co-transformation with a CRISPR-Cas9 plasmid increases the HR efficiency¹². We thus hypothesize that the integration of the URA3 gene was facilitated by pIW357. We also did not have time to test the integration of a non-marker gene (which would still allow for an easily selectable phenotype such as GFP or lacZ). This might have been a better option, as the efficiency of non-CRISPR-Cas9 induced HR is lower for a non-marker gene. This would have allowed us to more easily distinguish between CRISPR-Cas9-mediated and non-CRISPR-Cas9-mediated gene integration.

2. PEX10 deletion

In order to disrupt PEX10, we used the same pCRISPRyl-derived plasmid (pIW357) as described above. However, instead of using the Y. lipolytica PO1fΔKu70 strain, we used the Y. lipolytica PO1f strain, as we were aiming for PEX10 disruption through error-prone NHEJ (Figure 10). We thus transformed pIW357 into our selected strain and screened transformants for the inability to grow on a medium that contained solely long-chain fatty acids as a carbon source (SC-oleic acid). In turn, the same transformants were screened on a rich YPD medium which would allow ample growth. However, although we have confirmed that pIW357 functions appropriately (Figure 14), we could not identify transformants that were unable to grow on SC-oleic acid (data not shown). Nonetheless, following several rounds of restreaking, transformants were observed that had a distinct morphology (Figure 17). As PEX10 disruption can also be the cause of this morphology change (C.M. Schwartz, personal communication, October 18, 2016), transformants with an extraordinary morphology were selected for further analysis by colony PCR and subsequent Sanger sequencing of the PCR product. The results of these experiments have not yet been obtained.

Conclusion

We successfully developed a shuttle vector that allows the user to harvest the efficiency and accessibility of cloning in E. coli, while still allowing for replication in Y. lipolytica. We extensively tested the plasmid, and showed that it allows for replication, selection in E. coli and replication, selection and even counterselection in Y. lipolytica. The plasmid was shown to be compatible with BioBricks which, to our knowledge, makes it the first tool that allows for the use BioBricks in Y. lipolytica. It is our hope that other actors in iGEM community will use this tool in order harvest the great potentials of Y. lipolytica as a chassis for biorefineries of the future.

As a proof-of-concept, we have shown that we can integrate a gene of interest into Y. lipolytica and also disrupt a native gene of interest (still awaiting confirmation by sequencing). These proof-of-concept experiments validate the use of Y. lipolytica for future cell factory engineering purposes.

Here, we have focused on the integration of an auxotrophic marker gene. However, the CRISPR-Cas9 system is ideally suited for markerless gene integrations¹³. Markerless genetic modifications are advantageous as they do not affect host cell physiology and allow for iterative genetic modification cycles of the production organism of interest. In addition, this iterative process can be sped up as the modular nature of the CRISPR-Cas9 system makes it ideally suited for simultaneous multiplex genome editing^13,15.

References

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2. iGEM collections
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10. iGEM nomenclature
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