Genetic engineering tools
Over the years, various selectable markers for transformation of the nuclear or chloroplast genome of Chlamydomonas have been developed in the Purton group and made available to the research community. Plasmids carrying these markers can be obtained through the Chlamydomonas Center.
This marker for nuclear transformation was developed by Saul Purton whilst a post-doc in the lab of Jean-David Rochaix, University of Geneva. ARG7 encodes argininosuccinate lyase, the last enzyme of the arginine biosynthesis pathway, and the cloned gene can be used to rescue arg7 mutants to arginine prototrophy. The gene was cloned into the low copy number vector pBR329 to give pARG7.8 [1,2]. We subsequently, inserted a ‘molecular flag’ into one of the introns to allow discrimination between the introduced ARG7 DNA and the endogenous arg7 copy. The flag was a 392 bp HaeII fragment of phiX174 and the resulting plasmid named pARG7.8phi3 .
ARG7 has proved popular as a marker and an insertional mutagen because of the ease of selection and the high transformation rates [e.g. 4].
1. Debuchy R, Purton S, Rochaix JD (1989) The
argininosuccinate lyase gene of Chlamydomonas
reinhardtii: an important tool for nuclear transformation and for
correlating the genetic and molecular maps of the ARG7 locus.
EMBO J. 8:
2. Purton, S. & Rochaix,
Characterisation of the ARG7 gene of Chlamydomonas reinhardtii
application to nuclear transformation. European
Journal of Phycology 30: 141-148.
Gumpel NJ, Rochaix JD, Purton S (1994) Studies
on homologous recombination in the green alga Chlamydomonas
Genetics 26: 438-442.
4. Lumbreras V,
Purton S (1998) Recent advances in Chlamydomonas transgenics. Protist 149: 23-27.
Genbank accession number for ARG7 is X16619.
Download Word files for pARG7.8 and pARG7.8phi3 maps and sequences.
This dominant selectable
marker for nuclear transformation
confers resistance to the bleomycin family of DNA-damaging antibiotics.
The original version of the marker is
described in ref 5, with subsequent improvements in ref 6.
Download further details of the latest version (plasmid pSP124S).
5. Stevens DR, Rochaix JD, Purton S
bacterial phleomycin resistance gene ble
as a dominant selectable marker in Chlamydomonas.
Gen Genet. 251: 23-30.
6. Lumbreras V,
Stevens DR, Purton S
(1998) Efficient foreign gene expression in Chlamydomonas
reinhardtii mediated by an endogenous intron. Plant J 14: 441–447.
The plasmid pDBle is a modified version of the nuclear expression vector pGenD described by Fischer and Rochaix (2001). We have modified the vector by inserting the BLE marker downstream of the expression cassette. Download a Word file for further details and DNA sequence of pDBle.
The marker was developed for chloroplast transformation and confers resistance to kanamycin and amikacin, and is described in ref 7. Download further details of the plasmid (pSK.KmR) carrying the aphA6 cassette.
7. Bateman JM,
Purton S (2000) Tools for
chloroplast transformation in Chlamydomonas:
expression vectors and a new dominant selectable marker. Mol
Gen Genet. 263: 404-410.
This conditional negative selectable marker for use in the Chlamydomonas reinhardtii chloroplast is described in ref 8. It is a modified version of the E. coli cytosine deaminase (codA) gene. The CrCD enzyme confers sensitivity to 5-fluorocytosine by converting it to toxic 5-fluorouracil.
Download further details of the plasmid pRY127d, which contains the gene encoding HA-tagged CrCD and is available from the Chlamydomonas Center.
8. Young R, Purton S (2014) Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression. Plant J 80:915-925.
Codon Usage Optimizer
CUO is a GUI-based multiplatform software written in Java. It is open source and built with a user/developer-friendly structure. The main function of the software at the current development stage is to optimize genes to be transformed into the Chlamydomonas reinhardtii chloroplast genome although it can be used to optimize genes into other hosts as well. The main tool in CUO, Moptimizer, introduces a semi-automatic way of gene optimization which provides more flexibility and accuracy during the optimization process. The future plan for CUO is to be developed into a multipurpose bioinformatics software where data, findings, planning and learning in biology labs can be created and shared. CUO can be downloaded using the link below.
Please report feedback to khai.kong.09[at]ucl.ac.uk.
Page last modified on 22 apr 16 09:48