Type IIS restriction enzymes cut outside their recognition sites, making them useful in this cloning method for consecutive assembly of fragments. Through the restriction, overhangs are formed which allow the fusion of said genetic fragments to complementary overhangs of the syntax and thereby determine the order of each in a transcriptional unit (Figure 1). These fusion sites allow for the assembly of several fragments in the right order in just one cloning reaction. The used MoClo-kit offers ten different options for the positioning inside a L1 plasmid which are defined by the parts’ functions.

Within the MoClo syntax, there are three different cloning vectors, level 0, 1 and 2 (referred to as “L0”, “L1” and “L2”, respectively). L0 vectors carry one basic genetic fragment or part, L1 vectors are assembled fragments creating a transcriptional unit and L2 are multigenic constructs. Construction of an L0-part is done by flanking a gene of interest with the specific fusion site and the recognition site of BpiI by a PCR reaction. Upon digestion by BpiI it can be inserted into a previously digested L0-backbone. To then clone it into a L1-backbone, it is digested by BsaI, revealing the fusion sites for its assembly in a transcription unit. Lastly, a fusion of several transcription units (L1) into a L2 multigenic device is possible with the MoClo syntax.

As part of our contribution to a toolkit usable by future iGEM teams we design and construct not only the parts we intend to use on our goal of PET-degradation but several more, a L0-backbone and L1-backbone.

To ensure that all parts were designed correctly we cloned the PCR fragments into a L0 vector. To this end, we used a self-modified version of a L0 backbone containing RFP. After ligation, we transformed the L0 plasmids containing the parts into Escherichia coli and checked for white transformants. Only genes with the correct fusion and restriction sites could be inserted into the L0 backbone resulting in growth of white colonies, since the RFP gene was interrupted. For further verification, we checked the parts by DNA sequencing. We used the same control mechanisms for L1 assembly constructs.

Pierre Crozet and his team created a MoClo toolkit adapted for the model organism C. reinhardtii with 119 openly distributed genetic parts (Crozet et al., 2018) including resistance cassettes. However, these resistance cassettes are not located on any level 1(L1) plasmid backbone. Consequently, an additional cloning step is required to assemble a L2 device, since a single L1 construct cannot be transformed without a resistance cassette. To simplify this cloning process, we design a resistance cassette specifically for C. reinhardtii to serve as a plasmid component for the transformation into Chlamydomonas reinhardtii.

To build a selection cassette using the Golden Gate cloning standard, each part required for a selection cassette (promoter, linker, selection marker and terminator) must be cloned into the L0 backbone. The “selection marker” can be any antibiotic resistance for C. reinhardtii. Golden Gate cloning using BsaI can assemble the parts into a L1 backbone which results in an antibiotic resistance transcriptional unit. Using primers amplifying the selection cassette on a L1 MoClo backbone attaching the restriction sites compared to restriction sites on the “new” L1a,b,c vectors. Attaching BamHI and XhoI sites gives rise to a PCR product which is linkable with the new constructed L1 plasmids when digested by BamHI and XhoI. The resulting plasmids can then represent level 1 Golden Gate cloning vectors specified for C. reinhardtii. The picture shows an example to produce a selection cassette containing a hygromycin resistance linkable to a new L1c plasmid.

Once constructed, the designed plasmids are transformed into the SAG32-11b and UVM4 strains of C. reinhardtii and tested for expression. As a transformation method we chose electroporation, during which two different electric fields are applied to the algal cell. The first, a high voltage pulse applied for relatively short time causes the membrane to form pores and the second, of a low voltage for relatively long time transfers the DNA into the cell. We co-transform each of our constructs containing the transcriptional unit (L1 constructs) with a plasmid conferring antibiotic resistance (paromomycin or hygromycin, in our case).

After transformation, the algae has to be tested for successful uptake of the plasmids. If the clones are able to grow on TAP-agar plates containing antibiotics, the resistance plasmid was transformed and is proven to work accordingly. Subsequently, the clones have to be screened for the presence of the genetic constructs. A colony PCR is used to amplify the constructs. If the respective bands show up, the clones are continuously cultivated under thriving expression.

We take native signals from arylsulphatase 1 (ARS1) and a putative signal sequence of gametolysin (GLE) (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012) and build L0 modules with them. As the yield of heterologous expressed proteins fused to these secretion signals was previously described as relatively low, we additionally designed a 5’UTR glycomodule (SP₂₀) as a L0 part which also enhances the stability of the attached protein when secreted (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012).

In order to test these secretion signals, we tagged them with a YFP protein, which can be observed under a fluorescence microscope (excitation peak at 480 nm and emission peak at 528 nm). If secreted, the medium outside the cells is expected to emit fluorescence as opposed to the medium surrounding wild type algae.

The phosphite oxidoreductase from Pseudomonas stutzeri WM88 (PtxD) oxidizes phosphite to phosphate using NAD⁺ as a co-substrate (Loera-Quezada et al., 2016; White & Metcalf, 2007). We provide our C. reinhardtii strains with the PtxD enzyme to increase its competitiveness against other microorganisms in culture. This way, we enable it to degrade phosphite, presumably as only organism in our culture, avoiding contamination.

We observe the growth of our engineered C. reinhardtii in media containing phosphite, phosphate or phosphorus to prove the enzyme activity. In the phosphite-supplied media, we expect to observe growth of the strains expressing PtxD and inhibition of wild type algae due to a lack of phosphorus.

To test if they behave as expected, we designed constructs making it possible to directly compare them by expressing YFP as fluorescent marker protein under the regulation of both promoters. Using a plate reader and fluorescence microscope made comparing numerous AR- and PsaD-constructs very easily. We also planned to compare expression rates of constructs containing the PsaD promoter with and without introns. PsaD with introns was designed taking the AR promoter as a model. The AR promoter is composed of the HSP70 promoter fused to the RbcS2 intron. We followed a similar approach by fusing the RbcS2 intron to the end of the PsaD promoter, hoping to achieve a higher expression yield. Unfortunately, we were not able to compare expression driven by these three variants of promoters in the wet lab.

Target recognition inside the nuclear genome of C. reinhardtii relies on the presence of protospacers and protospacer-adjacent motifs (PAM) on the target DNA. A gRNA, which is designed to match specific regions on the genome and bind to them (Mali et al., 2013) is used to direct the Cas9 endonuclease. Upon binding to the target region, a cut of Cas9 creates a double-stranded break (DSB) with blunt ends. Into the DSB artificial DNA could be inserted through DNA repair-mechanisms. We wanted to find out if there were regions in the genome of C. reinhardtii that displayed an increased protein expression. To this end, we designed sgRNA complementary to three different genome regions.

The first locus is the SNRK locus. Since Kelterborn et al. had implemented a straight-forward screening process, successful cleavages could easily be identified. We want to use these loci mainly as a comparison to different locus insertions, since we do not expect high expression. As we are using the Promoter of the Photosystem Subunit II, we want to check if we are able to increase the expression by inserting our DNA Fragments at the end of the PsaD gene. Strenkert et al. (2019) have shown that a light dependent increase of PsaD expression occurs. When grown under synchronized light conditions a higher protein yield may be achieved.

Ribulose-1,5-bisphosphat-carboxylase (RuBisCO) is often referred to as the most abundant soluble protein. Carrying the function of carbon dioxide fixation it is present in all plants and phototrophic organisms. Due to this frequent appearance, we want to see if insertions inside the RuBisCO locus lead to higher expression. By targeting this locus for the insertion of our constructs, we hope to increase the protein yield, while characterizing the expression pattern of C. reinhardtii to ease the further use for the SynBio community.

During the process of electroporation transgenic DNA is inserted randomly into the genome of C. reinhardtii. This opens up the possibility that a high expression locus might be found by coincidence during our screening process. This locus could then be considered for further transformations to yield a high protein expression, useful in all genetic engineering projects.

To identify such a locus we try to amplify this genomic region with a restriction enzyme site-directed amplification PCR (RESDA-PCR). This polymerase chain reaction uses primers that are designed in a way that they anneal at restriction enzyme recognition sites of the enzymes AluI, SacII, PstI or TaqI. These sites are widely spread inside the genome. This increases the probability that the transgene DNA is inserted next to one of these sites, so that a PCR-product containing the genome sequences might be amplified and sequenced.

We use the syntax defined in the Constraint Based Reconstruction Analysis (COBRA) Toolbox for Python (Ebrahim et al., 2013), already existing metabolic reconstructions of Chlamy (Imam et al., 2015, Kliphuis et al., 2011), the fully sequenced genome (Merchant et al., 2007 ) and a recent -omics dataset (Strenkert et al., 2019) to define the components of the model. In a second step we combine these components in such a way that we are able to assess how they work together to give rise to growth of a Chlamy culture. The model should also provide the synthetic biologist with information useful for performing specific tasks in C. reinhardtii.

Unfortunately, we could not finish the model because there was not enough time. We still were able to use knowledge gathered along the way, though.