New destination vectors facilitate Modular Cloning for Chlamydomonas

Niemeyer, Justus; Schroda, Michael

doi:10.1007/s00294-022-01239-x

Cited by 9 publications

(7 citation statements)

References 25 publications

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“…The level 1 ble modules were assembled into destination vector pICH47732 [ 31 ] by combining the following parts: A1-B2_pCM0-016 ( PSAD promoter+5′-UTR), A1-B2_pMBS96 ( AR promoter+5′-UTR), A1-B2 AβSAP(i) promoter+5′-UTR(i) [ 2 ], A1-B2_pMBS978-pMBS983, B3-B5_pCM0-077 ( ble(i) CDS) and B6-C1_pCM0-119 ( RPL23 3′-UTR ). Level 2 devices for mCherry and NanoLUC reporters were generated by assembling the same parts as well as B3-B4 pCM0-063 ( NanoLUC CDS), B3-B4_pCM0-067 ( mCherry(i) CDS), and B5_pCM0-100 ( 3xHA CDS) into destination vector pMBS807 [ 32 ] conferring resistance to spectinomycin after transformation into Chlamydomonas .…”

Section: Methodsmentioning

confidence: 99%

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Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas

Niemeyer¹,

Fischer²,

Aylward

et al. 2023

Genes

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Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, Chlamydomonas reinhardtii has shown great potential as a host for biotechnological exploitation. However, low expression of nuclear transgenes is still a problem and needs to be optimized. In many model organisms, viral promoters are used to drive transgene expression at high levels. However, no viruses are known to infect Chlamydomonas, and known viral promoters are not functional. Recently, two different lineages of giant viruses were identified in the genomes of Chlamydomonas reinhardtii field isolates. In this work, we tested six potentially strong promoters from these viral genomes for their ability to drive transgene expression in Chlamydomonas. We used ble, NanoLUC, and mCherry as reporter genes, and three native benchmark promoters as controls. None of the viral promoters drove expression of any reporter gene beyond background. During our study, we found that mCherry variants are produced by alternative in-frame translational start sites in Chlamydomonas. We show that this problem can be overcome by mutating the responsible methionine codons to codons for leucine and by using the 5′-UTR of βTUB2 instead of the 5′-UTRs of PSAD or RBCS2. Apparently, the βTUB2 5′-UTR promotes the use of the first start codon. This could be mediated by the formation of a stem-loop between sequences of the βTUB2 5′-UTR and sequences downstream of the first AUG in the mCherry reporter, potentially increasing the dwell time of the scanning 40S subunit on the first AUG and thus decreasing the probability of leaky scanning.

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Section: Methodsmentioning

confidence: 99%

“…Whole-cell protein extraction, SDS-PAGE, semi-dry blotting and immunodetections were performed as described previously [ 32 ]. Total cell proteins corresponding to 1 µg chlorophyll were used [ 33 ].…”

Section: Methodsmentioning

confidence: 99%

Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas

Niemeyer¹,

Fischer²,

Aylward

et al. 2023

Genes

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“…A standardized library of synthetic biology parts and workflows has now been established to build a Chlamydomonas Modular Cloning (MoClo) toolkit (Crozet et al, 2018). This kit contains constitutive, inducible, and repressible promoters, N‐ and C‐terminal epitopes, fluorescent and bioluminescent reporters, and selectable marker genes and terminators, among other building blocks (Niemeyer & Schroda, 2022; Schroda & Remacle, 2022).…”

Section: Moclo Toolkitmentioning

confidence: 99%

Chlamydomonas: Fast tracking from genomics

Findinier

Grossman

2023

Journal of Phycology

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Elucidating biological processes has relied on the establishment of model organisms, many of which offer advantageous features such as rapid axenic growth, extensive knowledge of their physiological features and gene content, and the ease with which they can be genetically manipulated. The unicellular green alga Chlamydomonas reinhardtii has been an exemplary model that has enabled many scientific breakthroughs over the decades, especially in the fields of photosynthesis, cilia function and biogenesis, and the acclimation of photosynthetic organisms to their environment. Here, we discuss recent molecular/technological advances that have been applied to C. reinhardtii and how they have further fostered its development as a “flagship” algal system. We also explore the future promise of this alga in leveraging advances in the fields of genomics, proteomics, imaging, and synthetic biology for addressing critical future biological issues.

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“…The level 1 modules were then combined with pCM1-01 (level 1 module with the aadA gene conferring resistance to spectinomycin flanked by the PSAD promoter and terminator) from the Chlamydomonas MoClo kit, with plasmid pICH41744 containing the proper end-linker, and with destination vector pAGM4673 (Weber et al, 2011), digested with BbsI, and ligated to yield seven of the eight level 2 devices displayed in Supplemental Table 2. For the VPL2 construct pMBS970, level 0 parts were directly assembled into level 2 destination vector pMBS807 already containing the aadA resistance cassette (Niemeyer and Schroda, 2022). All newly generated level 0 and level 2 plasmids can be ordered from the Chlamydomonas Research Center (https://www.chlamycollection.org/).…”

Section: Cloning Of Coding Sequences For Baits Apex2 Bioid and Turboidmentioning

confidence: 99%

TurboID reveals the proxiomes of CGE1, VIPP1, and VIPP2 inChlamydomonas reinhardtii

Kreis¹,

König²,

Sommer³

et al. 2022

Preprint

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In Chlamydomonas reinhardtii, VIPP1 and VIPP2 play a role in the sensing and coping with membrane stress and in thylakoid membrane biogenesis. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CGE1 and stromal HSP70B as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in significant biotinylation in vivo. TurboID-mediated PL with VIPP1/2 as baits under ambient and H2O2 stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B and CDJ2. Novel proteins in the VIPP1/2 interaction network can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport. A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL1-11) and confirmed the proximity of VIPP1 and VPL2 in a reciprocal experiment. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs in thylakoid biogenesis and stress responses.

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New destination vectors facilitate Modular Cloning for Chlamydomonas

Cited by 9 publications

References 25 publications

Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas

Analysis of Viral Promoters for Transgene Expression and of the Effect of 5′-UTRs on Alternative Translational Start Sites in Chlamydomonas

Chlamydomonas: Fast tracking from genomics

TurboID reveals the proxiomes of CGE1, VIPP1, and VIPP2 inChlamydomonas reinhardtii

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