Genetic engineering tools

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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.

ARG7

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 [3].

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].

References:

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: 2803-2809.

2. Purton, S. & Rochaix, J.-D. (1995). Characterisation of the ARG7 gene of Chlamydomonas reinhardtii and its application to nuclear transformation. European Journal of Phycology 30: 141-148.

3. Gumpel NJ, Rochaix JD, Purton S (1994) Studies on homologous recombination in the green alga Chlamydomonas reinhardtii. Current Genetics 26: 438-442.

4. Lumbreras V, Purton S (1998) Recent advances in Chlamydomonas transgenics. Protist 149: 23-27.

The Genbank accession number for ARG7 is X16619.

Download Word files for pARG7.8 and pARG7.8phi3 maps and sequences.

BLE

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 (1996) The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol 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.

aphA6

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.

Codon Usage Optimizer programme

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 at finger tips.

Download CUO and optimize away!

Please report feedback to khai.kong.09[at]ucl.ac.uk.

Page last modified on 20 apr 12 14:24

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