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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


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, 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)3 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.

DESCRIPTION
Figure 1: The suggested workflow using pSB1A8YL described above. The workflow is broken down to three distinct steps: cloning, confirmation and expression. Cloning is the assembly of the construct, which is conducted in E. coli using typical cloning methods. Confirmation is where the identity of the construct is confirmed, using analytical methods such as PCR, analytical restriction enzyme digestion and/or sequencing. Expression includes the work done in the final host organism, Y. lipolytica, and includes inserting the construct and optimization of the expression and cultivation conditions.

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 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.

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 plasmids5, and perhaps for this reason this plasmid and its derivatives are utilized in many recent studies.6,7,8 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-Fluoroorotic Acid (5-FOA)9. In order to comply with the iGEM plasmid nomenclature10, 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.

DESCRIPTION
Figure 2: Sequence map of pSB1A8YL. The colored blocks represents the following: Orange: pUC19 part, Blue modified pSL16-CEN1-1(227) part, pink: BioBrick prefix, purple: BioBrick suffix, red: terminator, green selection markers, grey: origin of replication. The full annotated sequence can be found HERE.

Cloning

The pUC19 part and the gBlock fragments was amplified using primers with USER tails, and fused using USER cloning (See Figure 3).

DESCRIPTION
Figure 3: PCR fragments with USER tails, which were fused to construct pSB1A8YL. The fragment lengths can be seen on the ladder. The primers used and the theretical fragment length can be seen under the bands.

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).

DESCRIPTION
Figure 4: pSB1A8YL undigested and linearized. Analytical PCRs are also included in the right of the gel picture. The fragment lengths can be seen on the ladder. The restriction enzymes and primers used and the theretical fragment length can be seen under the bands

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 5).

DESCRIPTION
Figure 5: Colony PCR of pSB1A8YL in Y. lipolytica. The fragment lengths can be seen on the ladder. The primers used and the theretical fragment length can be seen under the bands

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/strong RBS BioBrick (BBa_K880005) and pair this with three chromoproteins: amilCP (BBa_K592009), amilGFP (BBa_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 6.

DESCRIPTION
Figure 6: Cloning flow of the test of pSB1A8YL in E. coli. The expression of the chromoproteins should yield a color ouput detectable by visual inspection.

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 (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.

DESCRIPTION
DESCRIPTION
Figure 7: Actual results from the test of pSB1A8YL in E. coli. Top: A color output was visible both in liquid culutres, pellets and on plates. Bottom: Restriction analysis of the constructs. The fragment lengths can be seen on the ladder. The restriction enzymes used and the theretical fragment length can be seen under the bands
3. Combining construct cloning in E. coli and expression in Y. Lipolytica

To test if our plasmid would support cloning of a construct in E. coli, which would be expressed, and ultimately prove that our plasmid works as intended, we chose to develop our own BioBricks. Namely a constitutive TEF promoter (BBa_K2117000) and a hrGFP gene (BBa_K2117003) previously used successfully in Y. lipolytica7. By combining these two parts in a device (BBa_K2117004), the expression should, in theory, be easily detected due to the fluorescence signal produced. The cloning flow can be seen in Figure 8.

DESCRIPTION
Figure 8: Cloning flow of the expression test of pSB1A8YL in Y. lipolytica. The expression of the GFP should yield a flourescent ouput detectable by fluorescence microscopy or fluorometer measurements.

The parts were ordered as gBlocks and assembled in E. coli using A3 assembly. The assembly was confirmed using PCR, restriction analysis and sequencing. Afterwards the construct was transformed into Y. lipolytica PO1f and grown on plates containing selective media. Single colonies were picked and grown in liquid selective media, and subjected to fluorescence microscopy and measured in a fluorometer. Unfortunately we did not observe any fluorescence signal, despite the sequencing confirmed the identity of the construct. We hope that a future team will debug the construct, and unlock the true potential of pSB1A8YL.

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 and expression in E. coli and replication, selection and counterselection 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. Unfortunately, we were not able to detect any expression from the constructs we were able to clone. The plasmid was submitted to the registry, and we hope that future teams will debug the construct, facilitating the use of Y. lipolytica to a larger extend in iGEM and general research context. 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.

CRISPR

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References

  1. Shetty, R. P., Endy, D., & Knight, T. F. (2008). Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering, 2(1), 5. article. http://doi.org/10.1186/1754-1611-2-5
  2. iGEM collections
  3. Liu, L., Otoupal, P., Pan, A., & Alper, H. S. (2014). Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function. Fems Yeast Research, 14(7), 1124–1127. doi:10.1111/1567-1364.12201
  4. Yanisch-Perron, C., Vieira, J., & Messing, J. (1984). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene, 33(33), 103–119. doi:10.1016/0378-1119(85)90120-9
  5. Yamane, T., Sakai, H., Nagahama, K., Ogawa, T., & Matsuoka, M. (2008). Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission. Journal of Bioscience and Bioengineering, 105(6), 571–578. doi:10.1263/jbb.105.571
  6. Liu, L., Otoupal, P., Pan, A., & Alper, H. S. (2014). Increasing expression level and copy number of a Yarrowia lipolytica plasmid through regulated centromere function. Fems Yeast Research, 14(7), 1124–1127. doi:10.1111/1567-1364.12201
  7. Blazeck, J., Liu, L., Redden, H., & Alper, H. (2011). Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach. Applied and Environmental Microbiology, 77(22), 7905–7914. doi:10.1128/AEM.05763-11
  8. Schwartz, C. M., Hussain, M. S., Blenner, M., & Wheeldon, I. (2016). Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica. Acs Synthetic Biology, 5(4), 356–359. doi:10.1021/acssynbio.5b00162
  9. Sakaguchi, T., Nakajima, K., & Matsuda, Y. (2011). Identification of the UMP Synthase Gene by Establishment of Uracil Auxotrophic Mutants and the Phenotypic Complementation System in the Marine Diatom Phaeodactylum tricornutum. Plant Physiol, 156(1), 78–89.
  10. iGEM nomenclature

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