Synthesis of L0 and L1 Constructs
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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 (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
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PtxD Controlled Growth
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CRISPR-Cas 9 Directed Transgene Insertion
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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 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. XXX, 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. XXX). 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
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