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-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

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

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	✓	✓	✓	✓

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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.

Fig. - Fluorescence intensity of C. reinhardtii WT and a YFP-carrying clone, in decreasing optical density of the cell culture. Excitation at 490 nm and emission measurement at 528 nm. The results clearly show that the fluorescence of the YFP-expressing clone is higher than the autofluorescence of the WT algae. Fig. - 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 Fig. - Time-resolved measurement of the 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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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.

Fig. 1 - Agarose 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.

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.

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.

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