Synthesis of L0 and L1 Constructs
By using Golden Gate cloning we designed Level 0, Level 1 and Level 2 vectors. We created several Level 0 parts which can be fused into the Level 0 backbone. We used our basic parts to assemble them into Level 1 backbones to create composite parts as transcription units. 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 |
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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-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 Paromomycine 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 |
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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 restiction site | ✓ | ✓ | ✓ |
L1c | BBa_K2984002 | Level 1 backbone with ampicillin resistance and RFP flanked by GoldenGate restiction site | ✓ | ✓ | ✓ |
L2 | BBa_K29863 | Level 2 backbone with paromycin resistance flanked by GoldenGate restiction site | - | - | - |
Examples for Successful Results of L1 Composite Parts
Successful Transformation into E.coli | Successful Sequencing | Successful Transformation into C. reinhardtii |
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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 | ✓ | ✓ | ✓ | ✓ |
bleR+scp | BBa_K2984045 | Bleomycin resistance fused to self cleaving peptide | ✓ | ✓ | ✓ | ✓ |
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 | ✓ | ✓ | ✓ | ✓ |
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 (photo systems, 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 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.
Secretion of Enzymes
There were three level 1 constructs which we wanted to check, compare and characterize for the secretion efficiency:
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, in context with two different promoters. The first and second could be compared to see how the SP20 glycomodule ramped the ARS secretion. 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 construct worked, we prepared a supernatant measurement. 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 new 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 left over chlamys and an absorbance scan to scan for the YFP absorption. Additionally we measured the fluorescence with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fluorescence intensity, plotted with 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 debree in the medium. There is no clear indication of mVenus presence in the medium.
The activity of the Arylsulfatase secretion signal could not be observed in interaction with our Level 1 constructs containing the YFP mVenus. Nevertheless, we could verify that the signal works in principle during the screening of one of our CRIPRR-Cas 9 transformations, where secretion is used to color positive clone blue (Fig. 5).
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 control of contamination and competition in cultivation. Transforming the phosphite decarboxylase (ptxD) into C. reinhardtii enables the alga 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, transforming such an enzyme into a C. reinhardtii strain, these transformants gain a selection advantage in comparison to the wild type. Trough coupling PtxD to PETase or MHETase we could avoid the problem that C. reinhardtii cells, which lost PETase/MHETase activity by expulsion of the plasmid prevailed.
On the basis of these advantages we want to transform the ptxD gene into C. reinhardtii. To determine the impact of phosphate and phosphite on C. reinhardtii, we tested different strains under several media conditions. As a reference, we used the TAP media containing phosphate. We further tested the growth in a media without phosphate (TA media) and in TAP media supplemented with phosphite (TAPi).
The first graph shows the growth curve of the wildtype SAG11-32b strain. The cells growing in TAP media show growth up to OD 2.3 [R.U.]. Cultures growing on phosphorus deficient media (TA) grow almost similar. This is 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. The reason is that phosphonates act as potent enzyme inhibitors which compete with their 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 media for 9 days and were then used to inoculate the cultures on TAPi media.
The graph shows three replicates of UVM4 cultures that were cultivated in phosphate-depleted media (blue, red, green). These cells were not able to grow at all. Cells that grew in TAP media as a control did grew normally. When cells were rescued in TAP media, they were able to grow again. This test show that C. reinhardtii cannot grow at phosphate starvation when the storage of a phosphate source were previously depleted.
CRISPR-Cas 9 Directed Transgene Insertion
As described in the Design page we hoped to ramp up expression, by inserting our DNA construct into certain regions of the C.reinhardti genome where we expected a higher expression of transgene constructs. Here we targeted three different loci.
To see if our Cas9 assays work in general we targeted the SNF-related serine/threonine-protein kinase (SNRK) locus. The targetside within this locus inhibits expression of the arylsulfatase. If a successful cleavage appears, expression of the arysulfatase is ramped up. When the enzyme is secreted, a supplemented color is turned blue by arylsulfatase activity (Fig. 1). This relations allows efficient screening of algae colonies were Cas9 restriction appeared.
The first construct we transformed into C. reinhardtii while targeting the SNRK locus was the level 1 construct L1c-Psad-YFP-Rbcs2. This experiment delivered us our first positive yfp clone, which exhibits a robust expression of mVenus. Subsequently, we performed a Colony PCR with SNRK locus specific primers, to amplify the insert. The SNRK specific amplification without an insert would lead to bands with a length of approximately 200 bp. If the insert was successfully inserted into the SNRK gene, we would have expected a band of approximately 2000 bp. The PCR results showed that the insertion of the YFP construct into the SNRK locus was not successful (Fig. 2). we can see bands at a length of 200 bp and also one bands at around 5000 bp. The YFP clone is labeled on Fig. 2 as A4. We sent the bands of a length of 5000 bp to sequencing but obtained unclear results. It seems that the construct was inserted into another locus, regardless of the CRISPR-Cas9 restriction directed to the SNRK gene.
As a second approach we designed guide RNAs for CRISPR-Cas9 for two different loci where we expected boosted expression. These loci are loci of highly expressed genes in C. reinhardtii. By inserting our construct into these loci we expected a boosted expression of our YFP mVenus. The guide RNAs were designed to guide CRISPR-Cas9 between two genes of the PsaD protein and the RubisCo enzyme (RbcS2) respectively. After the transformation of our algae with CRISPR-Cas9 we did colony PCRs as a screening method to examine if the insertion into the loci was successful. Hereby we designed primers that bind to the PsaD and RubisCo genes to investigate if the insertion worked as planned.
The PCR results, seen on Fig. 3, show no clear bands for the RubisCo (Rbcs2) and PsaD probes. A clear band at the expected length can be seen for the wild type (WT) probe of RbcS2. The same can not be said for the PsaD wild type, where no band can be observed. This indicates that there might have been a problem with the PCR parameters and fragments were not amplified correctly. On the other hand it is also possible that the directed insertion of our construct by CRISPR-Cas9 was unsuccessful. 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 answer the question of high expression loci in C. reinhardtii.
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 had 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 Gonzáles-Ballester et al. (2005) and is based on the amplification of the transgene from the genome using primers that bind on recurrent restriction sites in the genome (Gonzáles-Ballester et al. 2005) . For the amplification of the transgene, a primer is used that binds on the insert, and another primer that bind on 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.
González-Ballester, D., de Montaigu, A., Galván, A., & Fernández, E. (2005). Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA. Analytical biochemistry, 340(2), 330-335.
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 model of PET degradation in 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 took into account enzyme expression, secretion and kinetics and also the cultivation density of the algae as decisive factors for the PET degradation rate. The model 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 transgene expression and the flat panel cultivation method to achieve higher cultivation densities. For more information please visit our model page here.
PET degradation by C. reinhardtii
Expressing and secreting PETase 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. For more constructs you can visit our composite parts page.
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 a HA-tag to detect and isolate the enzyme. In Fig. 2, the successful amplification of the PETase enzyme out of C. reinhardtii via colony PCR can be seen. The expected length of the fragment was 885 bp or approximately 900 bp. The probes 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. 4). Because of these problems with the MHETase screening we were not able to assess in time if one of our transformed clones was positive.
Growth Experiments: Toxicity of Microplastic and it's 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 degrate PET, we also checked if the presence of substantial amounts of microplastic in the media affects the growth of C. reinhardtii .
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