Introduction

The construction of our transgenic vector for transformation into C. reinhardtii involved the use of 2A “self-cleaving” peptide sequences, the vast majority of which have not been characterized for use in C. reinhardtii. Our team decided to test five different 2A peptide sequences to determine which one had the highest cleaving efficiency and would therefore be best to use in a multicistronic expression vector.

Design: Initial Design of Multicistronic Vector

The genes we planned to insert into the C. reinhardtii nuclear genome were genes encoding for phytochelatin synthase (PCS), arsenate reductase (ACR2p), and paromomycin resistance (ParoR).

Several strategies were undertaken to increase the nuclear transgene expression of the PCS and ACR2p genes to be integrated into our construct. Altering factors such as promoters, introns, codon usage, or terminators has been shown to have the potential to improve nuclear transgene expression in C. reinhardtii ^[1]. The PCS and ACR2p genes to be used were codon optimized in accordance with the codon bias for C. reinhardtii. This codon optimization step is crucial for high transgene expression as transcripts with improper codon usage are not only less translated, but also, they are more likely to be degraded ^[1]. Additionally, introns were inserted into the codon-optimized PCS and ACR2p genes in order to further increase transgene expression. This increase in transgene expression results from enhancer sequences in intron sequences as well as the increase in transcript levels via intron mediated enhancement ^[1]. The ParoR gene was a component taken from the Chlamydomonas MoClo Toolkit and thus was already codon optimized for use in C. reinhardtii and contained introns.

To the PCS, ACR2p, and ParoR genes which were optimized for nuclear transgene expression in C. reinhardtii, further elements were added which would aid in cloning and analysis of the transformants. FLAG and HA tags added to PCS and ACR2p respectively served to enable protein production to be detected via western blotting. In addition, Golden Gate restriction enzyme sites containing overhangs compatible with the Chlamydomonas MoClo Toolkit were added to PCS and ACR2p in order to aid plasmid construction. Restriction enzyme sites were not added to ParoR as it was included in the Chlamydomonas MoClo Toolkit and already possessed these.

In our project, we not only planned to design a construct containing our genes of interest, but also, we aimed to expand the number of 2A peptides available for genetically engineering in C. reinhardtii by testing them via Förster resonance energy transfer (FRET). To do this, we designed a construct containing mCerulean, mVenus, and ParoR from the Chlamydomonas MoClo Toolkit. In this construct, mCerulean and mVenus would be separated by an experimental 2A peptide not well characterized in C. reinhardtii, and mVenus and ParoR would be separated by FMDV, a 2A peptide demonstrated to be functional in C. reinhardtii and included in the C. reinhardtii MoClo Toolkit.

Figure 1. The 2A peptide mechanism produces independent polypeptides under the control of a single promoter.

The multiple different iterations of plasmids used in our FRET testing all possessed the same structure aside from the experimental 2A peptide which was varied between each plasmid. In order to avoid building the same construct multiple times, we created a modular plasmid containing all of the parts which would remain consistent throughout each plasmid as well as a mScarlet reporter in the place of the experimental 2A peptide. This mScarlet reporter, a visual selection marker which caused successful E. coli colonies to fluoresce red, not only enabled us to detect the successful assembly of this modular plasmid, but it also allowed us to easily switch it out with different experimental 2A peptides, as it was designed to be flanked by Golden Gate restriction enzyme sites. All of the 2A peptides which were used in this project were designed to be flanked with compatible Golden Gate restriction enzyme sites. A similar modular design was employed in the plasmid containing our genes of interest to enable us to easily insert a new experimental 2A peptide into our construct after determining the most effective 2A peptide via FRET testing.

Build: Golden Gate Assembly

Prior to constructing the final constructs for our genes of interest as well as our 2A peptide FRET testing, the modular constructs containing the mScarlet reporter were constructed for each. To build the modular construct for the genes of interest, a five-fragment Golden Gate assembly was performed with PCS, the mScarlet reporter, ACR2p, FMDV, and ParoR. The modular construct for FRET testing was constructed by assembling the following parts: mCerulean, the mScarlet reporter, mVenus, FMDV, and ParoR.

After the modular constructs for our genes of interest and our 2A peptide FRET testing were constructed, various 2A peptides were inserted via one-fragment Golden Gate assemblies. The TaV 2A peptide was inserted into the genes of interest modular construct. The TaV, TME-GD7, DrosC, HuRV, and IFV 2A peptides were inserted into the FRET testing modular construct respectively.

Test: FRET Assay:

Once C. reinhardtii transformants which successfully integrated the plasmids containing the fluorophore FRET pair as well as experimental 2A peptides were obtained, they were subjected to FRET analysis in order to determine the cleaving behavior of the 2A peptides contained therein. The successful transformants for each of the five FRET plasmids as well as wild type C. reinhardtii were screened in 96 well plates in a plate reader. Autofluorescence of C. reinhardtii was obtained using untransformed, wild type C. reinhardtii. The values of autofluorescence were used to correct the readings obtained from the FRET plasmid transformants. After performing a Z-test and scoring the data accordingly, it was concluded that at least one representative transformant from each of the 2A peptides tested exhibited a lack of FRET activity, indicating cleavage activity of the 2A peptide. While this result is promising, further FRET testing trials are needed in order to determine the precise cleaving efficiencies of each of the 2A peptides under study.

Figure 2. Successful cleavage of proteins with a 2A peptide results in no detectable FRET.

Learn: Multicistronic Vector 2.0

While this initial FRET assay provided evidence of successful cleavage activity exhibited in all five of the tested 2A peptides, we plan to conduct further improved FRET experiments which will enable us to parse the precise cleaving efficiencies of each of the 2A peptides under investigation. With this data, we hope to not only improve the number and characterization of 2A peptides available for use in C. reinhardtii engineering, but we also hope to improve the construct containing our genes of interest used in our own project. After obtaining the relative cleaving efficiencies of each of the 2A peptides under study, we plan to integrate the most effective 2A peptide into our modular construct, thus producing a construct containing 2A peptides with the highest cleavage efficiencies in C. reinhardtii. Higher cleaving efficiency means that this construct will allow for an increased production of our proteins of interest. Thus, we expect it to result in a measurable improvement in C. reinhardtii’s arsenic sequestration rate.

References