An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas

TY - JOUR

T1 - An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas

AU - Specht, Elizabeth A

AU - Nour-Eldin, Hussam Hassan

AU - Hoang, Kevin T D

AU - Mayfield, Stephen P

N1 - Special Issue: Protein Stabilization

PY - 2015/3/1

Y1 - 2015/3/1

N2 - The model alga Chlamydomonas reinhardtii has been used to pioneer genetic engineering techniques for high-value protein and biofuel production from algae. To date, most studies of transgenic Chlamydomonas have utilized the chloroplast genome due to its ease of engineering, with a sizeable suite of reporters and well-characterized expression constructs. The advanced manipulation of algal nuclear genomes has been hampered by limited strong expression cassettes, and a lack of high-throughput reporters. We have improved upon an endogenous reporter gene - the ARS2 gene encoding an arylsulfatase enzyme - that was first cloned and characterized decades ago but has not been used extensively. The new construct, derived from ARS2 cDNA, expresses significantly higher levels of reporter protein and transforms more efficiently, allowing qualitative and quantitative screening using a rapid, inexpensive 96-well assay. The improved arylsulfatase expression cassette was used to screen a new transgene promoter from the ARG7 gene, and found that the ARG7 promoter can express the ARS2 reporter as strongly as the HSP70-RBCS2 chimeric promoter that currently ranks as the best available promoter, thus adding to the list of useful nuclear promoters. This enhanced arylsulfatase reporter construct improves the efficiency and ease of genetic engineering within the Chlamydomonas nuclear genome, with potential application to other algal strains. Manipulation of algal nuclear genomes has been hampered by a lack of adequate molecular tools. In this study, the authors construct a new arylsulfatase reporter (ARS2) which is developed for transforming Chlamydomonas reinhardtii more efficiently, and can produce thousands of colonies per transformation that can be grown in deep 96-well plates for downstream analysis. Furthermore, the improved reporter construct produces much higher expression, enabling rapid visual analysis using an inexpensive, high-throughput chromogenic assay. The availability of this ARS2-derived nuclear reporter improves the tractability of algal nuclear genome engineering.

AB - The model alga Chlamydomonas reinhardtii has been used to pioneer genetic engineering techniques for high-value protein and biofuel production from algae. To date, most studies of transgenic Chlamydomonas have utilized the chloroplast genome due to its ease of engineering, with a sizeable suite of reporters and well-characterized expression constructs. The advanced manipulation of algal nuclear genomes has been hampered by limited strong expression cassettes, and a lack of high-throughput reporters. We have improved upon an endogenous reporter gene - the ARS2 gene encoding an arylsulfatase enzyme - that was first cloned and characterized decades ago but has not been used extensively. The new construct, derived from ARS2 cDNA, expresses significantly higher levels of reporter protein and transforms more efficiently, allowing qualitative and quantitative screening using a rapid, inexpensive 96-well assay. The improved arylsulfatase expression cassette was used to screen a new transgene promoter from the ARG7 gene, and found that the ARG7 promoter can express the ARS2 reporter as strongly as the HSP70-RBCS2 chimeric promoter that currently ranks as the best available promoter, thus adding to the list of useful nuclear promoters. This enhanced arylsulfatase reporter construct improves the efficiency and ease of genetic engineering within the Chlamydomonas nuclear genome, with potential application to other algal strains. Manipulation of algal nuclear genomes has been hampered by a lack of adequate molecular tools. In this study, the authors construct a new arylsulfatase reporter (ARS2) which is developed for transforming Chlamydomonas reinhardtii more efficiently, and can produce thousands of colonies per transformation that can be grown in deep 96-well plates for downstream analysis. Furthermore, the improved reporter construct produces much higher expression, enabling rapid visual analysis using an inexpensive, high-throughput chromogenic assay. The availability of this ARS2-derived nuclear reporter improves the tractability of algal nuclear genome engineering.

KW - Algal Proteins

KW - Arylsulfatases

KW - Cell Nucleus

KW - Chlamydomonas reinhardtii

KW - Gene Expression

KW - Genes, Reporter

KW - Genetic Engineering

KW - Promoter Regions, Genetic

KW - Transgenes

U2 - 10.1002/biot.201400172

DO - 10.1002/biot.201400172

M3 - Journal article

C2 - 25224580

SN - 1860-6768

VL - 10

SP - 473

EP - 479

JO - Biotechnology Journal

JF - Biotechnology Journal

IS - 3

ER -