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High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii
Published on: December 5, 2016
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Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant
Ashley E Sproles1,2, Anthony Berndt1,2, Francis J Fields1,2
1The California Center for Algae Biotechnology, University of California, San Diego, La Jolla, CA, USA.
Applied Microbiology and Biotechnology
|February 7, 2022
Summary
We developed a new vector and screening method for Chlamydomonas reinhardtii to efficiently produce recombinant proteins. This dual antibiotic selection and fluorescence-activated cell sorting (FACS) process rapidly identifies high-expressing algae, improving production efficiency.
Area of Science:
- Biotechnology
- Algal Biotechnology
- Molecular Biology
Background:
- Chlamydomonas reinhardtii is a model organism for algal genetic engineering.
- Producing high-value recombinant proteins in algae is challenging due to low expression frequencies from random transgene integration.
Purpose of the Study:
- To design an optimized expression vector for enhanced recombinant protein production in Chlamydomonas.
- To develop a high-throughput screening (HTS) method for rapid isolation of high-expressing algal transformants.
Main Methods:
- Designed a novel expression vector featuring dual antibiotic resistance genes.
- Employed a dual antibiotic selection strategy to enrich for transformants.
- Utilized fluorescence-activated cell sorting (FACS) for efficient screening of recombinant protein expression.
Main Results:
- The new vector and HTS process enabled rapid identification of high-expressing transformants within 3 weeks.
- Achieved 60-100% of cells producing the target recombinant protein, a significant improvement over existing methods (<1%).
- Demonstrated an order of magnitude improvement in screening efficiency for recombinant protein production.
Conclusions:
- The developed vector and screening protocol significantly enhance the efficiency of identifying high-expressing recombinant protein producers in Chlamydomonas.
- This approach accelerates the development of algal platforms for producing valuable bioproducts.
- The method is adaptable for other algal strains and recombinant proteins, broadening its applicability.
