Summary
Our Troygenics are designed to aim at their target cell in a highly specific manner whilst leaving every other organism unaffected. To make the system as safe as possible, we split the Troygenic genome on two plasmids. The Assembly Plasmid codes for every protein necessary to replicate the Troygenic apart from the minor coat protein pIII, while the Application Plasmid codes for a shortened pIII, a modified pVIII and the gene of interest which should be delivered into the target cell. By splitting up the essential genes into two plasmids, we can ensure that our Troygenics cannot be built with only one plasmid.
To get our system inside the target cell, we fused fraction of the major coat proteins to target-specific ligands that are known to induce receptor mediated endocytosis.
After entering the cell via endocytosis, the DNA carried by the Troygenic integrates into the host cell’s genome, so a gene of interest, in some cases Cas13a and some host specific guide RNAs as our CeDIS, are expressed. As soon as our CeDIS detects any of its host specific targets, it starts collateral RNA cleavage, making the target unable to produce any protein which ultimately results in cell death.
The two plasmids Assembly Plasmid and Application Plasmid and their respective coded proteins, as well as the
correctly built Troygenic as a product of these two plasmids. The Assembly Plasmid codes for all coat proteins except of
pIII. This plasmid is only for the packing of the DNA in the protein coat. The Application Plasmid codes for a truncated
version of pIII and a fusion protein of pVIII and mCherry (most times fused to an endocytotic ligand), as well as it is replicated as single stranded DNA which will
be packed in the Troygenic as its DNA
Troygenic Assembly
In this part of our project – the Troygenic Assembly – we aim to build a basic Troygenic without any of its
natural or later added functions. For the assembly of our Troygenic we have to build two plasmids – one for
the Assembly Plasmid and another one for our Application Plasmid.
Assembly Plasmid
Assembly Plasmid containing genes II-VIII, the terminator and genes III-IV from the M13K07 helperphage
One important part of the Troygenic assembly is the Assembly Plasmid which contains the required protein coding genes
for the Troygenic assembly, such as the genes for coat proteins (VI-IX), for assembly proteins (I, IV, XI), and for the
single stranded DNA binding protein (V). The plasmid is constructed to feature all desired genes, except of gene III. We set primers around
genes II-VIII, the terminator and genes III-IV and cloned these three fragments into the iGEM backbone pSB3K3, which contains
a kanamycin resistance. E. coli DH5α was transformed with Application Plasmid and successfully transformed clones were
selected on an agar plate containing kanamycin antibiotics.
Application Plasmid
Application Plasmid containing f1 origin of replication, mCherry and genes VIII and III from M13K07 helperphage
The second plasmid - the Application Plasmid - consists of an f1 origin of replication for E. coli, the fluorescence
marker mCherry, the genes coding for pVIII and pIII from the M13K07 helperphage and the iGEM backbone pSB1C3. We put the
genes VIII and III on this plasmid because these are the two genes we modified during our project. As a result of having
these genes on the Application Plasmid, we only needed to vary one of our
two plasmids and the other one could stay the same over the whole project. We shortened gene III because we did not want
to keep the N-terminus, which is required for the binding to a bacterium. We needed the f1 ori because only with the ori
the single stranded DNA would be constructed in E. coli. mCherry is a marker to check if our Troygenics have been built.
It is fused to gene VIII encoding the major coat protein. So, mCherry is placed outside on our Troygenics. We constructed
the Application Plasmid by Gibson assembly and plated the transformed E. coli DH5α cells on chloramphenicol agar.
Truncated pIII
truncated pIII, the N1 and N2 domains are not present on the Troygenics, because they are required for the binding to a bacterium
A main difference between the natural pIII protein of the M13 bacteriophage and the one from our Troygenics is a truncated
pIII protein. For this, we set the PCR primers to remove the N1 and N2 domains as well as the linkers and kept the residues
275-425 which express the C-Terminus of the pIII protein. The Assembly Plasmid, which contains almost all genes for the
Troygenic construction, contains only the residues 369-425 of gene III, which cannot build the protein III in any form, but it is placed on this plasmid because
in this sequence the promoter for gene VI is located.
Assembly
pVIII is represented on both of our plasmids because on the one hand we needed the native one from the M13K07 helperphage and on the other hand we needed the pVIII fused
to an mCherry. This separation is due to the steric hindrance that would result, if only the fusion protein pVIII-mCherry would
be incorporated into the Troygenics coat. So, we expressed the native pVIII on the Assembly Plasmid and the one fused to
mCherry on the Application Plasmid. We expressed our working Troygenics by transforming both plasmids together in E. coli.
In this way all genes the Troygenics need to operate were nearby and could be combined to a functional Troygenic genome.
E. coli ER2566 containing both of our plasmids – the Assembly Plasmid and the Application Plasmid
For cases like ours, where you need two or more different plasmids in one producing organism, we designed split antibiotic
resistances as our basic and
improved part. With this split resistances,
you can use one antibiotic resistance for two plasmids. Only if both plasmids are in the cell, the organism gets resistant to
the antibiotic. You can look at our part collection
to see, which split resistances could be a possible benefit for your project.
Endocytosis
To specifically attack our target organisms, we searched for specific surface proteins of Saccharomyces
cerevisiae and Aspergillus niger. Ligands for those proteins will be presented on a fraction of
the major coat protein pVIII of M13 to enhance specific endocytosis.
Saccharomyces cerevisiae
Ligand 1: Mating Factor α
Modes of action of Mating factor a, α and a/α
One possible target is the transmembrane G-protein coupled receptor Ste2. The almost 10 000 Ste2 receptors
on the cell surface are involved in the mating pathway of S. cerevisiae (Manfredi et al., 1996).
Three different types of S. cerevisiae exist: a, α and a/α (Siliciano & Tatchell, 1984). While
a/α is the diploid form, a and α are haploids (Siliciano & Tatchell, 1984). The haploid yeast cells can mate
with a second haploid cell of the other mating type. This mating results in a diploid yeast cell and requires
cell-cell communication via mating pheromones (Bardwell, 2004). Cells from the a type secrete the mating factor
a which can be recognized by α-type cells. α-type cells, in contrast, secrete the tridecapeptide mating factor
α (Kurjan & Herskowitz, 1982) which binds to the pheromone receptor Ste2 on a-type cells (Bardwell, 2004).
Ligand-bound pheromone receptor is endocytosed to regulate the induced answer inside the cell (Bardwell, 2004).
Mating type is regulated via the mating type locus. In diploid cells containing the a- and the α-locus the proteins
of the mating pathway are negatively regulated. The gene products of the a-locus suppress expression of the α-type
mating proteins and vice versa (Kurjan & Herskowitz, 1982). So usually diploid cells do not express pheromone
receptors. However, the yeast strain we use is homozygous for the a-locus, the expression of pheromone receptor
Ste2 is not suppressed and can be used as a target for receptor mediated endocytosis.
Ligand 2: Opy2p
The second S. cerevisiae-specific ligand we try out is Opy2p. Opy2p is a protein involved in the
yeast high osmolarity glycerol pathway (Tatebayashi et al., 2015). S. cerevisiae has to face different
environmental conditions, so the cell expresses several sensing proteins for many parameters. The osmolarity of
the surroundings are very important for unicellular eukaryotes like yeast. As one adaptation to react properly
to changes in osmolarity, S. cerevisiae expresses the osmo-sensor Sho1p. This transmembrane protein
does not only sense the osmolarity of the environment but also serves as a scaffold for the assembly of a multicomponent
signaling complex made of several Sho1p, Hkr1 and Opy2p (Tatebayashi et al., 2015). In-between the different
transmembrane proteins of this complex there are several interactions. While Sho1p and Opy1p interact through
their transmembrane domains, Hkr1 and Opy2p interact extracellularly (Tatebayashi et al., 2015). The extracellular
cysteine-rich domain of Opy2p binds to the extracellular domain of Hkr1 and the Hkr1-Msb2-homology domain of the
signalling mucin Msb2p on the surface of S. cerevisiae (Adhikari, Caccamise, Pande, & Cullen, 2015;
Tatebayashi et al., 2015; Yamamoto, Tatebayashi, & Saito, 2016). Like many other receptors Msb2p and Hkr1 are internalized
by endocytosis constantly so binding to them via the fusion of the cysteine-rich domain of Opy2p to the major coat
protein pVIII of the M13 phage could enable us to enter S. cerevisiae cells.
Ligand 3: Flo11p
Last but not least we would like to use an abundant cell surface protein of S. cerevisiae, Flo11p (Gianvito,
Tesnière, Suzzi, Blondin, & Tofalo, 2017). Flo11p is a 1367 amino acid cell wall protein that mediates calcium dependent
cell-cell adhesion, so called flocculation (Karunanithi et al., 2010; Lo & Dranginis, 1997, p. 11, 1998). The protein
consists of a C-terminal glycosy-phosphatidyl-inositol-(GPI) anchored domain, a central domain of Serin and Threonine
repeats and the N-terminal domain, which is required for its homotypic interaction (Douglas, Li, Yang, & Dranginis, 2007;
Goossens & Willaert, 2012; Karunanithi et al., 2010). It has been shown that the N-terminal domain of Flo11p is O-glycosylated
but this glycosylation is not required for binding of other Flo11p proteins (Meem & Cullen, 2012). Since the homotypic
aggregation of Flo11p N-terminal domains is very stable (Douglas et al., 2007; Moreno-García et al., 2018, p. 5), binding
our Troygenic to the S. cerevisiae target via homotypic interactions of Flo11p N-terminal domains could be
stable enough to coat the cell with our particle. Constitutive endocytosis could then be a possible way to enter the target cell.
Aspergillus niger
As fungi, like every living organism, communicate with their environment, they have specific receptors on their surface to sense
the environment (Braunsdorf et al. 2016).
Aspergillus niger is communicating via pheromones.
Prof. Zeisinger-Migsich
Institute for Microbiology, Innsbruck
Unfortunately A. niger is an asexual fungus which means, that the possibility to use a mating pheromone like Mating factor
alpha for S. cerevisiae is not given (Swart et al. 2001). Furthermore the communication systems of A. niger are
less investigated than other model organisms such as S. cerevisiae.
To get our troygenics internalized by A. niger despite the fact that there are no known pheromone receptors on its surface,
we talked to several experts.
You can use an Aspergillus-specific Proline transporter to get inside the target cell.
Prof. Diallinas
Department of Biology, National and Kapodistrian University of Athens
This method is also used by several viruses. Cell entry is a critical step for all enveloped and non enveloped viruses and is enabled
through target cell specific proteins in the envelope or the capsid of the virus (Thorley et al. 2010). While enveloped viruses can
entry the target cell via membrane fusion, non enveloped viruses disrupt the plasma membrane or rely on cellular mechanisms like
macropinocytosis or endocytosis (Thorley et al. 2010). To get actively internalized by the target cell viruses first have to attach to
the plasma membrane. For this purpose many viruses like ecotropic murine leukemia virus (E-MuLV) or feline leukemia virus subgroup A (FeLV-A)
bind to transporter proteins on the cell surface (Olah et al. 1994; Fujisawa und Masuda 2007; Tailor et al. 1999).
Inspired by the virus‘ way we searched for mycoviruses for A. niger.
Even though there are many mycoviruses for Aspergillus, none of them infects Aspergillus through an extracellular phase.
Prof. Ulrich Schaffrath
Institute for Plantphysiology from RWTH Aachen
Further research confirmed this argument. Most mycoviruses are transmitted via hyphal fusion or asexual sporulation (van Diepeningen et al. 2006;
Kotta-Loizou und Coutts 2017). But there is a known virus for Sclerotinia sclerotiorum which has an extracellular phase and has even
successfully been used as a protective agent for plants exposed to for S. sclerotiorum. (Liu et al. 2016) For our Troygenics we used
the same approach many viruses use, exploiting an Aspergillus-specific proline transporter called PrnB.
This transporter was characterized on A. nidulans but is also present on the surface of A. niger.
Prof. Diallinas
Department of Biology, National and Kapodistrian University of Athens
We found the same information during our research (Novodvorska et al. 2013). PrnB is the major proline transport system in Aspergillus nidulans
(Sophianopoulou and Scazzocchio 1989) and structurally similar to S. cerevisiae amino acid transporters. (Sophianopoulou and Scazzocchio 1989).
Since PrnB usually binds and imports free L-Proline specifically (Gournas et al. 2015), we designed a Proline-Glycine-Sequence and fused it to our Troygenics.
This should give the N-terminal proline the flexibility needed to fit into PrnB even though it is bound to a complex protein. Since most proteins in
E. coli lack the first formyl-methionine due to the activity of the endogenous methionine aminopeptidase (Wingfield 2017) we postulated
that the following L-proline will be exposed to PrnB. The Troygenics presenting L-Proline on their surfaces will bind and inactivate PrnB on the Surface
of A. niger and will be actively internalized by the target cell because it will sense the reduced functionality of the transporter and will
aim to replace the putative inactive transporter to replace them with functional ones.
CeDIS
To express the CeDIS in the target cell we constructed a DNA sequence containing two expression sites, separated by a spacer sequence. The first expression site is responsible for the production of Cas13a. Here we were looking for two different types of promoters; one to test our system and one which will replace the test promoter in the final construct.
The guide RNAs belonging to Cas13a are created within the second expression site. For their successful expression, a special kind of promoter, which includes a lot of restriction enzyme recognition sites, is needed. Since CRISPR/Cas systems have been established in many organisms, we chose to use the most commonly used combination of promoter and terminator in the target organism for the expression of our single guide RNAs.
Both expression sites contain a promoter and a terminator. We decided to use regulatory elements, which originate from the target cell and show as few similarities in their bp-sequence to other species as possible, to add another layer of specificity to the CeDIS.
CeDIS non target cell. Cas13a doesn´t work because there are no matching sequences
CeDIS target cell. Cas13a works and leads to RNA cleavage because of matching sequences
As the spacer sequence, we chose the 234bp lacZα fragment, a part of the β-Galactosidase gene. This could be used for controlling transformation success with plasmids carrying this system.
To test our CeDIS, we wanted to use a strong, inducible promoter which can be repressed completely. That way, the results we were going to obtain concerning the efficiency of our cell death inducing system wouldn't be affected by the transformation efficiency.
For the promoter which will be used to express the Cas13a in the final CeDIS, we were looking for a strong, constitutive promoter to obtain the highest possible efficiency in the target cell, without the need of induction.
Since the terminator, downstream of the expressed gene, is important for its expression level as well as it‘s promoter, we chose the terminator which leads to the highest expression levels in combination with our promoter of choice.
To verify the specificity of a promoter or terminator sequence, we performed a blast search of all possible candidates using the NCBI database (downloaded in may 2019). The results where annotated and the S. cerevisiae hits among them were excluded using python scripts. In each case, we decided to use the promoter or terminator which has led to the most promising results in previous studies and shows the least number of similarities to sequences of other species.
Lab application
As our main aim of the Troygenics is to transform eukaryotic cells, we could not only focus on damaging them with our CeDIS. For this we
parallel designed a lab application. This lab application contains a sfGFP as gene of interest, instead of the CRISPS/Cas-System from the CeDIS.
When the target organism has taken up the Troygenic, the gene of interest is integrated in the target organism’s DNA. In the case of the lab
application among other genes sfGFP is integrated into the genome. This will cause the expression of GFP in the target organism and let it fluoresce green.
This application can be very beneficial for different experiments, as you can e. g. select organisms by their color. It is also possible to
integrate other genes than sfGFP into the plasmid to transform eukaryotic cells many possible ways depending on the planned experiment. The lab
application also demonstrates that our Troygenics are a platform system which is customizable and useful in many cases.
Biosafety
Biosafety is an important aspect of genetic engineering, especially when a project like Troygenics has a possible application that requires the output
beyond the lab. Therefore, we have designed several biosafety mechanisms to control that our Troygenics cannot affect other organisms than the target organism.
First, we designed the Troygenics on two plasmids. None of both plasmids is able to build a Troygenic or something similar on their own, but both
plasmids in combination are needed for the Troygenic assembly.
The next thing is, that we bound a specific endocytosis ligand to the Troygenics. So, only the target organism and closely related species can take up the
Troygenics. If they are not taken up, they do not operate in their determined way.
We also constructed our CeDIS with seven guide RNAs which are specific to the target organism and only to this. They are related to essential genes
and with seven gRNAs, the target organism cannot mutate from all of these.
All in all, our Troygenics are very safe and we demonstrated this with many experiments, too.
References
Adhikari, H., Caccamise, L. M., Pande, T., & Cullen, P. J. (2015). Comparative Analysis of Transmembrane Regulators of the Filamentous Growth Mitogen-Activated Protein Kinase Pathway Uncovers Functional and Regulatory Differences. Eukaryotic Cell, 14(9), 868–883. Link
Bardwell, L. (2004). A walk-through of the yeast mating pheromone response pathway. Peptides, 25(9), 1465–1476. Link
Braunsdorf, C., Mailänder‐Sánchez, D., & Schaller, M. (2016). Fungal sensing of host environment. Cellular Microbiology, 18(9), 1188–1200. Link
Douglas, L. M., Li, L., Yang, Y., & Dranginis, A. M. (2007). Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion. Eukaryotic Cell, 6(12), 2214–2221. Link
Fujisawa, R., & Masuda, M. (2007). Ecotropic murine leukemia virus envelope protein affects interaction of cationic amino acid transporter 1 with clathrin adaptor protein complexes, leading to receptor downregulation. Virology, 368(2), 342–350. Link
Gianvito, P. D., Tesnière, C., Suzzi, G., Blondin, B., & Tofalo, R. (2017). FLO 5 gene controls flocculation phenotype and adhesive properties in a Saccharomyces cerevisiae sparkling wine strain. Scientific Reports, 7(1), 1–12. Link
Goossens, K. V. Y., & Willaert, R. G. (2012). The N-terminal domain of the Flo11 protein from Saccharomyces cerevisiae is an adhesin without mannose-binding activity. FEMS Yeast Research, 12(1), 78–87. Link
Gournas, C., Evangelidis, T., Athanasopoulos, A., Mikros, E., & Sophianopoulou, V. (2015). The Aspergillus nidulans proline permease as a model for understanding the factors determining substrate binding and specificity of fungal amino acid transporters. Journal of Biological Chemistry, jbc.M114.612069. Link
Karunanithi, S., Vadaie, N., Chavel, C. A., Birkaya, B., Joshi, J., Grell, L., & Cullen, P. J. (2010). Shedding of the Mucin-like Flocculin Flo11p Reveals a New Aspect of Fungal Adhesion Regulation. Current Biology : CB, 20(15), 1389–1395. Link
Kotta-Loizou, I., & Coutts, R. H. A. (2017). Mycoviruses in Aspergilli: A Comprehensive Review. Frontiers in Microbiology, 8. Link
Kurjan, J., & Herskowitz, I. (1982). Structure of a yeast pheromone gene (MFα): A putative α-factor precursor contains four tandem copies of mature α-factor. Cell, 30(3), 933–943. Link
Liu, S., Xie, J., Cheng, J., Li, B., Chen, T., Fu, Y., … Jiang, D. (2016). Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector. Proceedings of the National Academy of Sciences, 113(45), 12803–12808. Link
Lo, W., & Dranginis, A. (1997). FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin. Journal of Bacteriology, 178, 7144–7151. Link
Lo, W., & Dranginis, A. (1998). The Cell Surface Flocculin Flo11 Is Required for Pseudohyphae Formation and Invasion by Saccharomyces cerevisiae. Molecular Biology of the Cell, 9, 161–171. Link
Manfredi, J. P., Klein, C., Herrero, J. J., Byrd, D. R., Trueheart, J., Wiesler, W. T., … Broach, J. R. (1996). Yeast alpha mating factor structure-activity relationship derived from genetically selected peptide agonists and antagonists of Ste2p. Molecular and Cellular Biology, 16(9), 4700–4709.
Meem, M. H., & Cullen, P. J. (2012). The Impact of Protein Glycosylation on Flo11-Dependent Adherence in Saccharomyces cerevisiae. FEMS Yeast Research, 12(7), 809–818. Link
Moreno-García, J., Martín-García, F. J., Ogawa, M., García-Martínez, T., Moreno, J., Mauricio, J. C., & Bisson, L. F. (2018). FLO1, FLO5 and FLO11 Flocculation Gene Expression Impacts Saccharomyces cerevisiae Attachment to Penicillium chrysogenum in a Co-immobilization Technique. Frontiers in Microbiology, 9. Link
Novodvorska, M., Hayer, K., Pullan, S., Wilson, R., Blythe, M., Stam, H., … Archer, D. (2013). Trancriptional landscape of Aspergillus niger at breaking of conidial dormancy revealed by RNA-sequencing. BMC Genomics, 14, 246. Link
Olah, Z., Lehel, C., Anderson, W. B., Eiden, M. V., & Wilson, C. A. (1994). The cellular receptor for gibbon ape leukemia virus is a novel high affinity sodium-dependent phosphate transporter. Journal of Biological Chemistry, 269(41), 25426–25431.
Siliciano, P. G., & Tatchell, K. (1984). Transcription and regulatory signals at the mating type locus in yeast. Cell, 37(3), 969–978. Link
Sophianopoulou, V., & Scazzocchio, C. (1989). The proline transport protein of Aspergillus nidulans is very similar to amino acid transporters of Saccharomyces cerevisiae. Molecular Microbiology, 3(6), 705–714. Link
Swart, K., Debets, A., Bos, C., Slakhorst, M., Holub, E., & Hoekstra, R. F. (2001). Genetic analysis in the asexual fungus Aspergillus Niger. Acta Biologica Hungarica, 52, 335–343. Link
Tailor, C. S., Willett, B. J., & Kabat, D. (1999). A Putative Cell Surface Receptor for Anemia-Inducing Feline Leukemia Virus Subgroup C Is a Member of a Transporter Superfamily. Journal of Virology, 73(8), 6500–6505.
Tatebayashi, K., Yamamoto, K., Nagoya, M., Takayama, T., Nishimura, A., Sakurai, M., … Saito, H. (2015). Osmosensing and scaffolding functions of the oligomeric four-transmembrane domain osmosensor Sho1. Nature Communications, 6, 6975. Link
Thorley, J., Mckeating, J., & Rappoport, J. (2010). Mechanisms of viral entry: Sneaking in the front door. Protoplasma, 244, 15–24. Link
Van, A. D., Debets, A. J., & Hoekstra, R. F. (2006). Dynamics of dsRNA mycoviruses in black Aspergillus populations. Fungal Genetics and Biology : FG & B, 43(6), 446–452. Link
Wingfield, P. T. (2017). N-Terminal Methionine Processing. Current Protocols in Protein Science, 88(1), 6.14.1-6.14.3. Link
Yamamoto, K., Tatebayashi, K., & Saito, H. (2016). Binding of the Extracellular Eight-Cysteine Motif of Opy2 to the Putative Osmosensor Msb2 Is Essential for Activation of the Yeast High-Osmolarity Glycerol Pathway. Molecular and Cellular Biology, 36(3), 475–487. Link
