Genetic engineering tools
Over the years, various selectable markers and cell lines 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 and cell lines can be obtained through the Chlamydomonas Center.
- ARG7 nuclear selectable marker
- BLE nuclear selectable marker
- C. reinhardtii TN72 (CC-5168)
- Chloroplast expression vectors pSRSapI and pASapI
- Stop-codon readthrough using trnWUCA
- aphA6 chloroplast selectable marker
- crCD chloroplast negative marker
- Codon Usage Optimizer
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.
BLE nuclear selectable marker
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.
C. reinhardtii TN72 (CC-5168)
TN72 (see ref 7) serves as a recipient strain for chloroplast transformation using the pASapI, pSRSapI and pWUCA2 expression vectors developed in the Purton lab.
This mutant is a chloroplast transformant in which the aadA (SpcR) cassette has been inserted between the BstXI site in the middle of the psbH coding sequence and the MluI site in the psbH-trnE2 intergenic region, thereby deleting a 0.36 kb genomic region including a large part of psbH. The aadA gene is in the same orientation as psbH. TN72 is PSII-minus (i.e. acetate-requiring) due to the loss of PsbH, and spectinomycin resistant due to the presence of AadA. TN72 is a cw15 (cell wall mutant) strain, allowing simple transformation using glass beads without the need for autolysin.
7. Wannathong T, Waterhouse JC, Young REB, Economou CK, Purton S (2016). New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Applied Microbiology & Biotechnology 100: 5467-5477
Chloroplast expression vectors pSRSapI and pASapI
pSRSapI is a vector for expressing transgenes in the C. reinhardtii chloroplast (see ref 7 above). The transgene should be inserted into the expression site using SapI and SphI restriction enzymes (or their isoschizomers LguI and PaeI, respectively). It will then be flanked by the C. reinhardtii psaA exon 1 promoter and 5′ UTR (perfect scarless fusion at the ATG start codon) and the C. reinhardtii rbcL 3′ UTR. The external flanking regions in the construct include an intact psbH gene and target the expression cassette downstream of psbH by homologous recombination.
Plasmid pASapI has an atpA promoter and 5'UTR instead of the psaA exon 1 element; pSRSapI generally gives stronger transgene expression.
Use minimal medium for the restoration of psbH in C. reinhardtii, selecting for phototrophic growth. The recipient cell line must therefore be a psbH mutant such as TN72.
Stop-codon readthrough using trnWUCA
The pWUCA2 plasmid for transgene expression in the C. reinhardtii chloroplast is used for biocontainment and to aid the cloning of genes whose products are toxic to E. coli (ref 8).
pWUCA2 can be used to introduce the trnWUCA gene downstream of psbH in the C. reinhardtii chloroplast genome. A psbH mutant recipient line, e.g. TN72, must be used to allow selection on minimal medium. TrnWUCA is a synthetic tRNA that allows the readthrough of internal UGA codons. This plasmid also contains an empty expression cassette with a psaA exon 1 promoter and 5' UTR, where a gene of interest can be inserted (e.g. one with internal TGA codons).
An alternative plasmid, pWUCA1, can be used to introduce the trnWUCA gene downstream of psaA exon 3 in the C. reinhardtii chloroplast genome using spectinomycin selection.
8. Young REB, Purton S (2016). Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnology Journal 14: 1251-1260
aphA6 chloroplast selectable marker
The marker was developed for chloroplast transformation and confers resistance to kanamycin and amikacin, and is described in ref 9. Download further details of the plasmid (pSK.KmR) carrying the aphA6 cassette.
9. 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.
crCD chloroplast negative marker
This conditional negative selectable marker for use in the Chlamydomonas reinhardtii chloroplast is described in ref 10. 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.
10. 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.