Team:Humboldt Berlin/Results

1. Establishing Chlamy in the iGEM competition

Synthesis of L0 and L1 Constructs

We created a basic set of genetic parts for the use in C. reinhardtii. We chose to use the MoClo assembly standard for two reasons: First, C. reinhardtii is a eukaryotic organism with full transcription units that comprise more elements than those of bacterial systems (promoter, 5'UTR, signal peptides, CDS, 3'UTR, terminator). To assemble multiple units through the BioBrick RFC[10] assembly is time consuming, while other assembly standards like the Golden Gate based MoClo syntax allow the cloning of multiple parts in a one-step reaction. Second, C. reinhardtii has only been rarely used as chassis in iGEM so far, so there is a need to add more parts to the iGEM Registry. For us this was also the chance to start over with a more advanced cloning standard like MoClo.

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We created a set of parts, (our Chlamy-HUB Collection), including basic regulatory sequences, reporter genes and epitope-tags. Most importantly, we created new MoClo backbones to comply with the iGEM standard (by exchanging the lacZ cloning reporter by an RFP insert). Furthermore, we designed L1 and L2 backbones that include a C. reinhardtii selection cassette. This means L1-constructs can directly be used for transformations of C. reinhardtii.

All parts were synthesized or amplified by PCR. Successful amplifications were verified by an agarose gel showing a band at the expected size and were ligated into the backbones using Golden Gate cloning. Vectors were then transformed into E. coli. Those containing the L0 part formed white colonies. Transformants containing empty backbones grew red since the backbones contain RFP as a selection marker. Correct transformants were confirmed by DNA sequencing. L1 plasmids that were successfully transformed into C. reinhardtiii were monitored by Colony PCR. Successful amplification-, transformation- and sequencing procedures are marked by a tick.

Examples for successful Results of L0 parts

Successful Amplification	Successful Transformation into E. coli	Successful Sequencing

Cloning Results of L0 parts

Part Name	Registry Name	Description	Fusion Sites	Successful Amplification	Successful Transformation into E. coli	Successful Sequencing
L0 - Backbone	BBa_K2984010	L0 backbone (plasmid with RFP insert)	-	✓	✓	✓
L0-AR	BBa_K2984009	L0 backbone with AR promoter	A1-A3	✓	✓	✓
L0-AR	BBa_K2984025	L0 backbone with AR promoter	A1-B1	✓	✓	✓
L0-PsaD	BBa_K2984022	L0 backbone with PsaD promoter	A1-A3	✓	✓	✓
L0-PsaD	BBa_K2984008	L0 backbone with PsaD promoter	A1-B1	✓	✓	✓
L0-bleR+scp	BBa_K2984045	Bleomycin resistance fused to self cleaving peptide	B1-B1	✓	✓	✓
L0-ARS1	BBa_K2984000	L0 backbone with 3'UTR Arylsulfatase1 secretion signal	B2-B2	✓	✓	✓
L0-GLE	BBa_K2984001	L0 backbone with 3'UTR gametolysin secretion signal	B2-B2	✓	✓	✓
L0-PETase	BBa_K2984011	L0 backbone with PETase CDS	B3-B4	✓	✓	✓
L0-Hyg	BBa_K2984012	L0 backbone with Hygromycin B resistance CDS	B3-B4	✓	✓	✓
L0-Paro	BBa_K2984006	L0 backbone with Paromomycin resistance CDS	B3-B4	✓	✓	✓
L0-ptxD	BBa_K2984026	L0 backbone with phosphite oxidoreductase CDS	B3-B3	✓	✓	✓
L0-ptxD	BBa_K2984013	L0 backbone with phosphite oxidoreductase CDS	B3-B4	✓	✓	✓
L0-YFP	BBa_K2984017	L0 backbone with yellow fluorescent protein CDS	B3-B4	✓	✓	✓
L0-YFP	BBa_K2984024	L0 backbone with yellow fluorescent protein CDS	B4-B4	✓	✓	✓
L0-YFP	BBa_K2984020	L0 backbone with yellow fluorescent protein CDS	B5-B5	✓	✓	✓
L0-6xHis tag	BBa_K2984014	L0 backbone with 6x histidine tag	B5-B5	✓	✓	✓
L0-3xHis tag	BBa_K2984015	L0 backbone with 3x histidine tag	B5-B5	✓	✓	✓
L0-SP₂₀	BBa_K2984016	L0 backbone with glycomodule for secretion enhancement	B5-B5	✓	✓	✓
L0-RbcS2	BBa_K2984018	L0 backbone with RuBisCo terminator	B5-C1	✓	✓	✓
L0-RbcS2	BBa_K2984021	L0 backbone with RuBisCo terminator	B6-C1	✓	✓	✓
L0-Linker	BBa_K2984034	L0 backbone with Linker sequence	B2-B2	✓	✓	✓
L0-Ble	BBa_K2984040	L0 backbone with Bleomycin resistance	B1-B1	✓	✓	✓
L0-scp	BBa_K2984044	L0 backbone with Bleomycin resistance	B1-B1	✓	✓	✓
L0-PETase	BBa_K2984049	L0 backbone with PETase	B3-B3	✓	✓	✓
L0-PsaDIntron	BBa_K2984046	L0 backbone with PsaDIntron	A1-A3	-	-	-
L0-RFP	BBa_K2984004	L0 backbone with RFP	-	✓	✓	✓
L0-amp	BBa_K2984007	L0 backbone with ampilicin resistance	-	✓	✓	✓
L0-amp	BBa_K29858	Linker L1c -> L0	-	✓	✓	✓
L0 Linker	BBa_K29860	Linker L0 -> L1a	-	✓	✓	✓
L0 Linker	BBa_K29861	Linker L0 -> L1b	-	✓	✓	✓
L2 Linker	BBa_K29862	Linker L2 -> L1b	-	✓	✓	✓
Linker sequence	BBa_K29834	Linker sequence	B2-B2	✓	✓	✓

Examples for Successful Results of L1 and L2 Backbones

Successful Amplification	Successful Transformation into E. coli	Successful Sequencing

Cloning Results of Backbones

Part Name	Registry Name	Description	Successful Amplification	Successful Transformation into E. coli	Successful Sequencing
L1a	BBa_K2984003	Level 1 backbone with ampicillin resistance and RFP flanked by GoldenGate restiction site	✓	✓	-
L1b	BBa_K2984005	Level 1 backbone with ampicillin resistance and RFP flanked by GoldenGate restriction sites	✓	✓	✓
L1c	BBa_K2984002	Level 1 backbone with ampicillin resistance and RFP flanked by GoldenGate restriction sites	✓	✓	✓
L2	BBa_K29863	Level 2 backbone with paromycin resistance flanked by GoldenGate restriction sites	-	-	-

Examples for Successful Results of L1 Composite Parts

Successful Transformation into E. coli	Successful Sequencing	Successful Transformation into C. reinhardtii

Cloning Results of L1 Composite Parts

Part Name	Registry Name	Description	Successful Amplification	Successful Transformation into E. coli	Successful Sequencing	Successful Transformation into C. reinhardtii
L1c-PsaD-YFP-RbcS2	BBa_K2984019	Reference for fluorescence measurements	✓	✓	✓	✓
L1c-PsaD-ARS-YFP-Rbsc2	BBa_K2984027	Secretion of YFP	✓	✓	✓	-
L1-PsaD-ARS-PETase-YFP-SP₂₀-RbcS2	BBa_K2984028	Secretion of PETase and expression measurement via YFP fluorescence	✓	✓	✓	✓
L1-PsaD-GLE-PETase-YFP-SP₂₀-RbcS2	BBa_K2984029	Secretion of PETase and expression measurement via YFP fluorescence	✓	✓	✓	✓
L1c-PsaD-ARS-YFP-SP₂₀-Rbcs2	BBa_K2984030	Secretion of YFP to test the ARS secretion signal and SP₂₀ efficiency	✓	✓	✓	✓
L1-PsaD-ARS-PETase-YFP-RbcS2	BBa_K2984031	Secretion of PETase and expression measurement via YFP fluorescence	✓	✓	✓	-
L1-PsaD-ARS-PETase-3xHA-RbcS2	BBa_K2984032	Secretion of PETase and purification via HA-tag	✓	✓	✓	-
L1c-AR-ARS-YFP-Rbcs2	BBa_K2984033	Secretion of YFP	✓	✓	✓	✓
L1c-PsaD-PETase-YFP-His-RbcS2	BBa_K2984035	Expression of PETase and expression measurement via YFP fluorescence to be purified via His-Tag	✓	✓	✓	-
L1c-PsaD-PETase-YFP-Rbcs2	BBa_K2984036	Secretion of PETase and expression measurement via YFP fluorescence	✓	✓	✓	-
L1c-PsaD-PtxD-Rbsc2	BBa_K2984037	Evaluation of C. reinhardtii growth on phosphite-containing media	✓	✓	✓	✓
L1c-Psad-PETase-ptxD-Rbcs2	BBa_K2984038	Expression of PETase and evaluation ofC. reinhardtiigrowth on phosphite-containing media	✓	✓	-	-
L1c-PsaD-PETase-3xHA-RBCS2	BBa_K2984039	Expression of PETase and purification via HA-Tag	✓	✓	✓	-
L1c-PsaD-ARS-PETase-3xHA-SP₂₀-RbcS2	BBa_K2984041	Enhanced Secretion of PETase and SP₂₀ efficiency and purification via HA-Tag	✓	✓	✓	✓
L1c-PsaD-MHETase-YFP-RbcS2	BBa_K2984042	Expression of MHETase and expression measurement via YFP fluorescence	✓	✓	✓	-
L1c-PsaD-MHETase-3xHA-RbcS2	BBa_K2984043	Expression of MHETase and purification via HA-Tag	✓	✓	✓	-
L1c-AR-PETase-YFP-RbcS2	BBa_K2984047	Expression of PETase and expression measurements via YFP fluorescence	✓	✓	-	-
L1c-PsaD-bleRscp-ARS-PETase-SP₂₀-RbcS2	BBa_K2984048	Enhanced Secretion of PETase and SP₂₀ efficiency and bleomycin resistance to C. reinhardtii	✓	✓	-	-
L1-PsaD-Paro-RbcS2	BBa_K2984055	Construct lending paromomycin resistance to C. reinhardtii	✓	✓	✓	✓
L1-PsaD-Hyg-RbcS2	BBa_K2984056	Construct lending hygromycin resistance to C. reinhardtii	✓	✓	✓	✓

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Expression of YFP-containing constructs

The yellow fluorescent protein YFP was used as a fluorescent tag in some of our constructs. The goal of using YFP as a tag was to be able to measure enzyme expression and secretion and to screen for successful mutants using YFP as a marker. Additionally, we wanted to use a YFP-expressing C. reinhardtii to analyse possible locus effects on expression. We were able to successfully transform a YFP-expressing C. reinhardtii with a construct of our own design.

But measurements of YFP in C. reinhardtii turned out to be a great challenge, because of the strong interaction of the algae with light (by their photosystems, pigments, chlorophyll and light antennae). More information on our process of measuring YFP can be found on our Measurement page.

We were able to successfully measure YFP fluorescence intensity and fluorescence spectra of YFP-expressing C. reinhardtii clones in comparison to the wild type (WT). The results showed that our clone exhibited a higher fluorescence intensity at 528 nm than the WT (YFP emission peak) and the fluorescence spectrum of YFP confirmed the presence of the yellow fluorescent protein. This YFP-expressing clone also allowed us to characterize the light induction of the PsaD promoter by doing a time-resolved measurement of the fluorescence intensity.

During this measurement we exposed C. reinhardtii cultures which contained our YFP construct to different growth conditions. One was exposed only to the dark, the other to synchronized growth lighting conditions (10 hours dark, 14 hours illuminated). Then, we started a time-resolved fluorescence intensity measurement in the dark, with a WT control. After approximately four hours, we activated a light source and exposed the cultures to light, thus activating the light inducible PsaD promoter. Our results showed that for the dark and synchronized cultures containing the YFP construct a peak in fluorescence intensity could clearly be seen after the light induction. This proved the light induction of the PsaD promoter. If you are interested in this measurement, please visit the page of our YFP mVenus construct in the iGEM registry here.

fluorescence difference spectrum — **Fig. 2 - YFP emission spectrum of a *C. reinhardtii* clone with YFP with an emission maximum at approximately 530 nm. Difference spectrum of WT and YFP spectra.**

fluorescence intensity time resolved — **Fig. 3 - Time-resolved measurement of YFP fluorescence intensity.** Light induction of the PsaD promoter (white area). It can clearly be seen that at the time of illumination the fluorescence intensity of YFP increases, indicating a light induced expression of the protein.

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Secretion of Enzymes

Secretion of enzymes is of high importance to solve complex problems with the help of cells like C. reinhardtii. For PET degradation specifically, the secretion is fundamental, so that the enzyme PETase can reach its substrate PET. We used several different secretion signal peptides and designed constructs specifically to test and quantify the secretion out of the cell. There were three level 1 contructs we designed to test secretion:

1. L1c-Psad-ARS-YFP-RbcS2
2. L1c-Psad-ARS-YFP-SP₂₀-RbcS2
3. L1c-AR-ARS-YFP-RbcS2

The first and third construct were designed for measuring the influence of arylsulfatase secretion signal (ARS) on mVenus secretion, expressed by two different promoters. The first and second constructs could be compared to see how the SP₂₀ glycomodule increased the secretion driven by the ARS peptide. The level 1 constructs were transformed into C. reinhardtii via electroporation. After two weeks of growth on TAP-agar plates containing paromomycin, the present clones were picked for colony PCR or sequencing.

To see if and how our different constructs worked, we conducted supernatant measurements. For each positive clone 50 ml flasks were prepared 4 days in advance. Of each flask 150 µl were transferred to a black 96-well plate and sequentially diluted up to 1:128. These flasks were then centrifuged for 10 min with a speed of 2000 rpm at 4°C. The supernatant was separated from the pellet in a fresh falcon tube for measurement. Before starting another centrifugation process, 12 x 150 µl of each supernatant were transferred to a black 96-well plate. This step was repeated after the second centrifugation, creating a third well plate. All three well plates were then analyzed under the plate reader to measure for fluorescence emission.

Unfortunately, we were unable to measure fluorescence in any of the supernatants. For every well in each plate, absorbance was measured at a wavelength of 680 nm to check the supernatants for possible cell debris or leftover Chlamys and absorbance scans were performed to scan for the absorption by YFP. Additionally, we measured the fluorescence with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fluorescence intensity, plotted against the absorbance of the medium, can be seen in Fig. 4. Here we see that the fluorescence intensity correlates with the optical density of the medium, which explains the fluctuations in fluorescence intensity due to the presence of residual cells or debris in the medium. It was not possible to assess with certainty if there was YFP present in the medium.

Nevertheless, we were able to prove that YFP was present in vesicles near the membrane area of one of our YFP mVenus secretion clones (BBa_K2984030). This was done by imaging C. reinhardtii cells using confocal microscopy (Fig. 4.1). Although we were not able to measure YFP in the medium surrounding the algae, which is difficult, as can be read in our measurements page, it is possible to assume that secretion did occur if we observe the confocal micrsocopy images. The presence of the vesicle strongly suggests secretion of YFP mVenus.

Additionally, we can verify that the ARS secretion signal works in principle during the screening of one of our CRISPR/Cas9 transformations, where secretion is used to color positive clone blue (Fig. 5). By knocking out the SNRK gene, which regulates the expression of the arylsulfatase, uncontrolled secretion of this enzyme takes place. By adding a special pigment to the colonies, the knockout colonies become blue, demonstrating the function of the ARS secretion signal.

secretion measurements — **Fig. 4 - Fluorescence intensity of the medium probes in correlation with the measured optical density of the medium.** There is no clear indication that mVenus is present in the medium.

confocal microscopy - YFP — **Fig. 4.1 - Presence of YFP mVenus in *C. reinhardtii*.** A vesicle carrying a high concentration of YFP mVenus can be seen for construct BBa_K2984030.

blue screening — **Fig. 5 - Blue colony screening of cell colonies using the SNRK knockout.** The blue color of the colonies demonstrates that the secretion signal ARS works.

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Phosphite decarboxylase Controlled Growth: PtxD as a selection advantage?

Utilizing an alga that can compete against other organisms in culture by using another phosphorus source, e.g. phosphite, can have great advantages in controlling contamination and competition in a cultivation. The phosphite decarboxylase (ptxD) enables C. reinhardtii to outcompete against other algae as Loreza-Quezada et.al. (2016) have shown. Thus, the combination of PtxD expression coupled with expression of PETase and MHETase would enable C. reinhardtii to degrade microplastic within a closed or open cultivation setup.

Furthermore, b< transforming such an enzyme into a C. reinhardtii strain these transformants gain a selection advantage in comparison to the wild type. Trough coupling of PtxD to PETase or MHETase we could avoid the problem of C. reinhardtii cells, which lost PETase/MHETase activity by expulsion of the plasmid prevailing.

Based on these advantages we want to transform the PtxD gene into C. reinhardtii. To determine the impact of phosphate and phosphite on growth of C. reinhardtii, we tested different strains under several media conditions. As a reference we used the TAP medium containing phosphate. We further tested growth in a medium without phosphate (TA medium) and in TAP medium supplemented with phosphite (TAPi).

The first graph shows the growth curve of the wildtype strain SAG11-32b. The cells growing on TAP media show growth up to OD 2.3 [R.U.]. Cultures growing on phosphorus-deficient media (TA) grow similarly. This is probably due to the phosphate storage of C. reinhardtii in the cytosol (Vered Irihimovitch et al., 2008).

In comparison, the cultures growing on phosphite (TAPi) media show strongly reduced growth. This is explained by the fact that phosphonate acts as a potent enzyme inhibitor which competes with its structural analogues for binding the enzyme's active sites (White & Metcalf, 2007). In conclusion, the metabolism of phosphate is inhibited by phosphite in C. reinhardtii wild type cells.

To show how the C. reinhardtii mutant strain UVM4 (mutant without cell wall) can handle phosphate starvation, cultures were grown in TA medium for 9 days and were then used to inoculate the cultures on TAPi medium. The graph shows three replicates of UVM4 cultures that were cultivated in phosphate-depleted medium (blue, red, green). These cells were not able to grow at all but if these were rescued in TAP medium they were able to grow again. Cells that grew on TAP medium as a control displayed normal growth. This experiment shows that C. reinhardtii cannot grow under phosphate starvation if the storages of phosphate were previously depleted.

Sag11 growth TA and TAPi — **Fig. 6 - Mean (MW) of three replicates of growth of UVM4 for each culture media TAP(Tris-Acetat-Phosphate), TA (no Phosphate KH₂PO₄) and TAPi (Phosphite KH₂PO₃).** The graph shows relative absorption (OD) at 680 nm and 720 nm at 150 µE light and 30°C.

UVM4 growth TA and TAPi — **Fig. 7 - UVM4 was starved for 9 days on TA medium (no Phosphate KH₂PO₄), 1 ml was used to inoculate in the respective media.** Relative absorption (OD) was measured at 680 nm at 150 µE light and 30°C.

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CRISPR/Cas9 Directed Transgene Insertion

Achieving high expression rates of recombinant proteins is often difficult in C. reinhardtii. Transgenes are randomly integrated into the genome and consequently their expression is influenced by the genetic surrounding of the insertion site. We hoped to increase expression, by inserting our DNA construct into a defined region of the C. reinhardtii genome. With CRISPR/Cas9, we targeted three different loci to insert our transgenic constructs.

To see if our Cas9 assays work in general, we first used the easy screenable SNF-related serine/threonine-protein kinase (SNRK2.2) locus. Inactivation of the SNRK2.2 gene leads to the constitutive expression of arylsulfatase, which gets secreted into the medium. The presence of arylsulfatase can easily monitored with a color reaction. By cleaving the sulfate group from the colourless substrate 5-bromo-4-chloro-3-indolyl-sulfate (X-SO₄) the dye indole blue is formed (Fig. 8). This blue-green screen poses as an efficient method of finding CRISPR/Cas9-directed insertion mutants.

The first construct we inserted into the SNRK2.2 locus was the level 1 construct L1c-Psad-YFP-Rbcs2. This experiment delivered us our first positive YFP-expressing clone exhibiting a robust expression of mVenus. Then, we performed a colony PCR with SNRK2.2 locus-specific primers to amplify the insert. The SNRK2.2 specific amplification without an insert would lead to bands with a length of approximately 200 bp. If the insert was successfully inserted into the SNRK2.2 gene, we expected a band of approximately 2000 bp. The PCR results showed that the insertion of the YFP construct into the SNRK2.2 locus was only partly successful (Fig. 9). We can see a bigger band at around 5000 bp. The YFP-expressing clone is labeled on Fig. 9 as A4. We sent the bands of a length of 5000 bp to sequencing but obtained only overlayed signals from a mix of PCR products. So the SNRK2.2 target site was cut by CRISPR/Cas9 but we have no certainty if the YFP construct is inserted along with other sequences, inserted twice or not inserted at all.

After confirming that we can insert our YFP construct at targeted regions into the genome we now thought of loci that would boost the expression of our transgene. We chose two of the most highly expressed genes in C. reinhardtii, PSAD and RBCS2 and designed guide RNAs for CRISPR/Cas9 to insert our YFP-constructs into the 3'UTR region of these genes. By inserting our construct into these loci we hoped for an increased expression of our YFP mVenus.

After the transformation of our algae with CRISPR/Cas9 ribonucleoproteins we performed colony PCRs as a screening method to examine if the insertion into the loci was successful. For this, we designed primers that bind to the PSAD and RBCS2 genes to investigate if the insertion worked as planned.

The PCR result, seen on Fig. 10, shows the analysis of 96 clones that were transformed to target a YFP construct into PSAD and RBCS2. Only for the wild type (WT) probe of RbcS2 a clear PCR band can be seen at the expected length. No clear bands can be observed in the other samples. This indicates that there was a problem with the PCR parameters and fragments were not amplified correctly. Due to the fact that we did this experiment at the end of our project we did not have enough time to do further screening or transformations to identify high expression loci in C. reinhardtii.

Crispr PCR — Fig. 10 - PCR of the PSAD and RBCS2 loci to investigate a successful insertions into these loci. The PCR seems to not have worked well because of the smeared bands.

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RESDA PCR for Identification of Transgene Insertion Locus

The locus where a transgene is inserted is of great importance for the expression rate of the gene. Many conventional methods of transformation lead to random insertion of the transgene, so that there is no influence on the insertion locus. When a transformation is successful and a transgene is abundantly expressed, it might be of interest to know in which locus the gene was inserted. The RESDA-PCR method is a method that allows localization of the insertion locus.

We cultivated a C. reinhardtii clone which was successfully transformed with our YFP mVenus construct and showed abundant expression of the protein. We wanted to investigate in which locus our transgene was inserted, so we planned a RESDA PCR.

The RESDA PCR method is a PCR method designed to identify the insertion locus of transgenes. The method was developed by González-Ballester and his colleagues and is based on the amplification of a transgene from the genome using primers that bind on recurrent restriction sites in the genome (González-Ballester et al. 2005). To amplify the transgene, one primer that binds to the insert is used and another primer that binds to one of the recurrent enzymes in the genome, in hope that the insert is close enough to a restriction site to be amplified. The amplification is done through two different rounds where the second round uses nested primers to narrow down the specificity of the amplification. After the amplification the fragment can be sequenced to determine its locus.

Our goal was to determine the locus of insertion of our YFP mVenus construct, to see if we could use this locus for future directed insertion of transgenes with CRISPR/Cas9. Additionally, we also performed the RESDA-PCR with a luciferase-expressing clone kindly provided by the research group of Prof. Dr. Hegemann of the Humboldt University of Berlin.

The PCR was made with primers for four different restriction sites. These are AluI, PstI, SacII and TaqI. We performed the PCR with a temperature gradient and with different primer concentrations to check for optimal conditions of amplification. Unfortunately, we were not able to observe any bands on the agarose gel after our RESDA-PCR (Fig. 1). We repeated the PCR twice, varying the conditions each time to optimize the formation of bands. Still, we were not able to reproduce the results described by González-Ballester et al. (2005). This means that we were unfortunately unsuccessful to determine the locus of insertion of our YFP expressing clone.

**Fig. 11 - Agarose gel of a RESDA-PCR; there are no clear bands to be observed, only primer clouds at the bottom of the probes.** Labels: TM05 and F7: luciferase expressing clone, A4: YFP expressing clone, WT: wild type.

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2. PET-degradation

PET-degradation in-silico

The viability of PET degradation by C. reinhardtii at a larger scale is yet unknown. Models of biological systems allow us to design experiments in silico that are difficult to reproduce in vivo and give us special insights into the role that parameters might play in the given biological system. Therefore, to assess the efficiency of PET degradation by C. reinhardtii, a mathematical model of PET degradation in a continuous culture of C. reinhardtii was designed.

The overall goal of the model is to determine the time needed to degrade 1 mg of PET. The model takes into account enzyme expression, secretion and kinetics and also the cultivation density of the algae as decisive factors for the PET degradation rate. It predicted that for a cultivation density of 1:10, a 40 g PET bottle would be degraded in approximately 10 years. For a cultivation density of 1:100, the predicted time to degrade a bottle was 100 years. Additionally, an improvement of the PETase enzyme by a factor 1000 was made. For a cultivation density of 1:10 and the improved enzyme, the time needed to degrade a bottle was 119 days. For a cultivation density of 1:100 and the improved enzyme, the time needed to degrade a bottle was 3,3 years. The results of the model led us to take several decisions regarding the improvement of the PET degradation. We chose to use the light-inducible PsaD promoter for our constructs, the secretion enhancing glycomodule SP₂₀, the specialized C. reinhardtii strain UVM4 for increased transgene expression and the flat panel cultivation method to achieve higher cultivation densities. For more information please visit our model page.

PET 1 to 10 — Fig. 12 - Results of the PET degradation simulation for the cultivation density 1:10.

PET 1 to 100 — Fig. 13 - Results of the PET degradation simulation for the cultivation density 1:100.

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PET degradation by C. reinhardtii

Expressing and secreting PETase and MHETase in C. reinhardtii was one of the main goals of our project. To be able to express the PETase, we designed a wide variety of constructs with functional parts from our Chlamy-HUB Collection.

These constructs were systematically designed to experimentally test the expression and secretion of PETase. Additionally, we designed the constructs with different markers for detection or measurement of the enzyme, like the yellow fluorescent protein mVenus, HA-tags and His-tags. Construct BBa_K2984028 was for example designed for enhanced secretion of PETase, marked with the YFP mVenus. Or construct BBa_K2984039, designed for expression of the enzyme, marked with a HA-tag for detection, isolation and purification. To see more constructs you can visit our composite parts page.

PETase Gel — **Fig. 15 - Amplification of the PETase transgene of *C. reinhardtii* through a colony PCR shows successful transformation of the construct.** Expected band length: 885 bp.

Fig. 16 - Crystal structure of the MHETase enzyme.

Fig. 17 - Amplification of the MHETase transgene out of C. reinhardtii through a colony PCR. Due to problems with the primer we had issues with unspecific bands in the gel. The successful transformation of the construct could not be confirmed.

The constructs were all assembled using the MoClo standard and then transformed into C. reinhardtii. After the transformation, the clones had to be screened for positive transgene insertion. The screening was done through colony PCRs, where the inserted fragments were fully or partially amplified to verify transgene insertion. Unfortunately, our initial PETase level 0 part was missing one nucleotide, which led to a frameshift of all subsequent parts in the construct. Although we had done many transformations of PETase constructs it was not until two months into our transformation workflow that we discovered this mistake. This made our PETase impossible to screen, because all markers for detection were located after the PETase sequence and had the aforementioned frameshift.

After successfully correcting the mistake in the PETase sequence, we were able to successfully transform a PETase construct into C. reinhardtii. We transformed construct BBa_K2984032 into C. reinhardtii, which contains the secretion signal ARS and an HA-tag to detect and isolate the enzyme. In Fig. 15, the successful amplification of the PETase enzyme of C. reinhardtii via colony PCR can be seen. The expected length of the fragment was 885 bp or approximately 900 bp. The samples were sent to sequencing and we could verify that the sequence of the PETase was intact after the insertion into C. reinhardtii. Unfortunately we did not have enough time to do further tests and experiments with the transformed clone.

Additionally to the PETase enzyme, we wanted to transform the MHETase enzyme into C. reinhardtii. For this enzyme we also designed constructs systematically, as can be seen in our composite parts overview. The MHETase we used for these constructs was kindly given to us by the TU Kaiserslautern 2019 iGEM team. Nevertheless, after various attempts to transform the MHETase into C. reinhardtii, we were unable to find a successful clone in our screening. We had problems amplifying the MHETase through a colony PCR because we had difficulties with the primer specificity. The primers we designed first showed well defined bands at the wrong length in the gels. We sent our probes to sequencing and it turned out that our primers were amplifying parts of chromosome 16 of C. reinhardtii. After ordering new primers, we encountered another specificity issue: several unspecific bands were appearing in our gel (Fig. 17). Because of these problems with the MHETase screening we were not able to assess in time if one of our transformed clones was positive.

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Growth Experiments: Toxicity of microplastic and its degradation products

When it comes to cultivating our transgenic alga in the bioreactor it is not only important to know about the toxicity of the PET degradation products. Since the alga should degrade PET, we also checked if the presence of substantial amounts of microplastic in the media affected the growth of C. reinhardtii. To determine the range of possible applications for C. reinhardtii for plastic degradation we performed toxicity tests on different strains. We assessed whether the degradation products of MHET, terephthalic acid (TPA) and ethylene glycol (EG) are toxic for C. reinhardtii and showed that TPA is not toxic for the alga whereas EG is toxic in concentrations only above 7%. As shown by our model, even the most optimistic catalytic rates would not yield such high EG concentrations. Therefore, we can be confident that our algae can survive degrading PET in a bioreactor setup.

Toxicity test setup

We performed our toxicity tests in a Multi-cultivator “MC1000”. This cultivator enables measurement of growth in different media creating growth curves.

The growth of C. reinhardtii was observed by measuring the optical density at OD 680 nm. At this wavelength, photosystem II has its absorption maximum, so measuring at this wavelength is common when cultivating phototrophic organisms. To exclude a possible contamination the cultivator also measured the OD at 720 nm where every other organism absorbs. The ratio of both measurements shows the reproduction of either C. reinhardtii or a contamination (e.g. bacteria).

Toxicity test for Terephthalic acid (TPA)

To determine the toxicity of TPA growth experiments were performed with the wild type C. reinhardtii strain CC-125. The solubility of TPA is fairly low with ~15 µg/l. In these measurements different dilutions of TPA in the standard TAP medium were tested inside the Multi-cultivator 1000. Figure 19 shows the growth curve of C. reinhardtii determined at 680 nm (shown in green) by measuring the OD. TPA was dissolved in TAP medium. Even concentrations of more than 15 µg/l (where TPA precipitates) did not have an influence on the reproduction of C. reinhardtii. The red curve illustrates the OD at 720nm. Since these curves do not show an abnormal increase, the cultivator was not contaminated. This shows that growth of C. reinhardtii is not influenced by TPA.

Toxicity test for ethylene glycol

Ethylene glycol, Ethan-1,2-diol, (EG) is a colourless organic compound with the formula (CH₂OH)2. It has a LD₅₀ in rats (orally) 4700 mg·kg⁻¹. The toxic effect relies on its byproducts glycolaldehyde, glyoxal and glyoxylic acid. To determine the toxic effect of EG on C. reinhardtii the growth of several strains at concentrations of 1 % to 10 % EG was tested.

Figure 20 shows that C. reinhardtii wild type CC125 is able to tolerate EG concentrations up to 5 % without showing growth limitation. A concentration of 4 % decreases the growth speed but nevertheless growth maximum is reached. Concentrations starting at 7 % are lethal to the alga. But due to the turnover rate of the enzymes PETase and MHETase the concentration of EG will never reach a concentration more than 1-2 %. This fact makes it possible for C. reinhardtii to survive in an environment where these products arise. Thus, Chlamydomonas represents a good model organism to tolerate the process of PET degradation. We further did the same measurement using the C. reinhardtii mutant strain UVM4 that lacks a cell wall. The graph in figure 21 also demonstrates that already concentrations of 6 % inhibit the growth of this mutant strain.

Toxicity test on the effect of microplastic

When it comes to cultivating our transgenic alga in the bioreactor it is not only important to know about the toxicity of the PET degradation products. Since the alga should degrade PET, we also checked if the presence of substantial amounts of microplastic in the media affects the growth of C. reinhardtii. From our visit at the wastewater treatment facility we learned that the cleaned water that leaves the facility has an average microplastic concentration of 15 mg/L (A. Matzinger, 2018). We set up cultivation experiments with varying concentrations of microplastic particles (<40 µm).

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3. C. reinhardtii for industrial use:

C. reinhardtii in our use-case scenario

One of the main goals targeted by projects like ours is the development of a use-case scenario. We therefore visited a wastewater treatment facility in Berlin to test growth of C. reinhardtii under natural and industrial conditions. Our idea: The C. reinhardtii mutant strain UVM4 should degrade microplastic inside a wastewater treatment facility.

As UVM4 has no flagella it sediments. Together with the sinking terephthalic acid (TPA) both can be pumped out. This could be included into the existing cleaning process: Inside the secondary clarifier the sewage sludge settles on the ground of the pond and gets pumped out. The clean top layer of this water flows into the river network of Berlin. To test this if such a system would be feasible we took some water samples from the different cleaning stages of the wastewater treatment facility into our lab and performed growth tests with C. reinhardtii in it. We took samples from the secondary clarifier as well as water that had been cleaned by UV light in the facility.

Unfortunately Chlamy did not grow in any taken wastewater samples. We realized that cultivating Chlamy in wastewater requires many further examinations, experiments and modifications. Thus, different water conditions tolerable by C. reinhardtii must be examined.

Secondary_clarifier — **Fig. 23 - Water received from wastewater facility in Berlin.**

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Filtration of C. reinhardtii for selection inside the bioreactor

When using a bioreactor to degrade PET, Chlamy will mix with PET, MHET, EG and TPA. We wanted to find a way to separate C. reinhardtii from the other components and to extract the cells out of our bioreactor. Therefore we tested several microfilters with different pore widths. For this test we used the C. reinhardtii cell-wall-deficient UVM4 mutant strain. By using vacuum filtration, UVM4 cells were filtered through different filters.

We tested three different pore sizes: 7 µM, 5 µM, 0.8 µM for filtration success. To determine the efficiency of the filtration process we measured the optical density of the culture and the weight of the filters. The table shows that with decreasing pore size the filter gains weight and the optical density of the filtrate decreases. By the second filtration through a 0.8 µM filter less cells stick to the filter.

filter pore size	filter's weight increase	culture's decrease of OD
7 µM	0.0007 g	0.0007
5 µM	0.0043 g	0.159
0.8 µM (first filtration step)	0.0061 g	0.277
0.8 µM (secondary filtration step)	0.0041 g	0.224

The pictures taken of the filters after the filtration process are shown in Figure 25. The first filtration through a 0.8 µM filter yielded the greenest color and therefore shows the highest net filtration. By this measurement we could show that only a pore size of 0.8 µM can filter C. reinhardtii cells sufficiently well. Since C. reinhardtii cells have a size of 10 µM this result is surprising. An explanation for the small required pore size could be the missing cell wall, as we used the cell-wall-deficient mutant strain. With this knowledge we can build filter systems inside our bioreactor to sufficiently filter C. reinhardtii out of the bioreactor and to separate it from other components.

vacuum filtration setup with different filter pads. — **Fig. 25 - Vacuum filtration set-up with different filter pads.**

Different pore sizes and their cut-off rate — **Fig. 26 - *C. reinhardtii* cell-wall-deficient UVM4 mutant strain filtered through microfilters with different pore sizes.** Cells stick to the filter with 0.8 µM sized pores.

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Learn more...

Plant Synthetic Biology

Curious about our synthetic biology approaches in algae? Read about our Chlamy-HUB Collection and more here.

Achievements

Find out about our achievements and the goals we reached during our iGEM project.

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