» Articles » PMID: 35129657

Improved High-throughput Screening Technique to Rapidly Isolate Chlamydomonas Transformants Expressing Recombinant Proteins

Overview
Authors
Affiliations
Soon will be listed here.
Abstract

The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas, that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products. KEY POINTS: • A protein expression vector using double-antibiotic resistance genes was designed • Double antibiotic selection causes fewer colonies with more positive for phenotype • Coupling the new vector with FACS improves microalgal screening efficiency > 60.

Citing Articles

An automated high-throughput lighting system for screening photosynthetic microorganisms in plate-based formats.

Noonan A, Cameron P, Dofher K, Sukkasam N, Liu T, Ronn L Commun Biol. 2025; 8(1):438.

PMID: 40087381 DOI: 10.1038/s42003-025-07853-y.


Bioinformatic Prediction and High Throughput In Vivo Screening to Identify Cis-Regulatory Elements for the Development of Algal Synthetic Promoters.

Torres-Tiji Y, Sethuram H, Gupta A, McCauley J, Dutra-Molino J, Pathania R ACS Synth Biol. 2024; 13(7):2150-2165.

PMID: 38986010 PMC: 11264317. DOI: 10.1021/acssynbio.4c00199.


Horizon scanning of potential environmental applications of terrestrial animals, fish, algae and microorganisms produced by genetic modification, including the use of new genomic techniques.

Miklau M, Burn S, Eckerstorfer M, Dolezel M, Greiter A, Heissenberger A Front Genome Ed. 2024; 6:1376927.

PMID: 38938511 PMC: 11208717. DOI: 10.3389/fgeed.2024.1376927.


The Clinical Promise of Microalgae in Rheumatoid Arthritis: From Natural Compounds to Recombinant Therapeutics.

Cutolo E, Caferri R, Campitiello R, Cutolo M Mar Drugs. 2023; 21(12).

PMID: 38132951 PMC: 10745133. DOI: 10.3390/md21120630.

References
1.
Aharoni A, Amitai G, Bernath K, Magdassi S, Tawfik D . High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. Chem Biol. 2005; 12(12):1281-9. DOI: 10.1016/j.chembiol.2005.09.012. View

2.
Berthold P, Schmitt R, Mages W . An engineered Streptomyces hygroscopicus aph 7" gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist. 2003; 153(4):401-12. DOI: 10.1078/14344610260450136. View

3.
Chen W, Zhang C, Song L, Sommerfeld M, Hu Q . A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J Microbiol Methods. 2009; 77(1):41-7. DOI: 10.1016/j.mimet.2009.01.001. View

4.
Chew K, Yap J, Show P, Suan N, Juan J, Ling T . Microalgae biorefinery: High value products perspectives. Bioresour Technol. 2017; 229:53-62. DOI: 10.1016/j.biortech.2017.01.006. View

5.
Daugherty P . Protein engineering with bacterial display. Curr Opin Struct Biol. 2007; 17(4):474-80. DOI: 10.1016/j.sbi.2007.07.004. View