An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas
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An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas. / Specht, Elizabeth A; Nour-Eldin, Hussam Hassan; Hoang, Kevin T D; Mayfield, Stephen P.
In: Biotechnology Journal, Vol. 10, No. 3, 2015, p. 473-479.Research output: Contribution to journal › Journal article › Research › peer-review
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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
Y1 - 2015
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.
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.
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
VL - 10
SP - 473
EP - 479
JO - Biotechnology Journal
JF - Biotechnology Journal
SN - 1860-6768
IS - 3
ER -
ID: 155939207