Introduction

The eukaryotic microalga Chlamydomonas reinhardtii serves as a model system for studying flagellar function, photosynthesis, and organelle biogenesis mainly due to its well-defined genetics and the ease of nuclear and plastid transformation (1). Whereas the introduction of foreign DNA into the nuclear genome of Chlamydomonas is achieved very efficiently owing to random non-homologous recombination (2), the actual expression of transgenes has been complicated by several difficulties.

As a main problem, promoter sequences tested for the expression of homologous or heterologous genes yielded in reasonable transgene expression as long as their gene product led to a selectable benefit (e.g., resistance against antibiotics or complementation of a mutant phenotype) (3,4). If a non-selectable gene of interest (GOI), [e.g., a reporter gene or a modified gene from Chlamydomonas itself was to be (over) expressed], the same promoters mostly resulted in low cotransformation rates and a great variability in the level of transgene expression (5,6), although in some cases rather high expression levels have been reported (7,8). As in cotransformation experiments, both expression cassettes (i.e., the GOI and the selectable marker), are usually delivered on two separate plasmids, their integration into the nuclear genome occurs independently from each other, usually leading to rates of co-expression of only about 10% (9). For convenience, both genes are arranged within one vector, since the integration and expression of the marker more frequently includes expression of the GOI, resulting in co-expression rates of up to 70% (9,10). Although such a placement of two expression cassettes within one transformation vector has proven to be a good method to optimize transformation results, practically, the molecular construction of large plasmids containing more than one transgene often fails due to the lack or unfavorable presence of restriction sites. The Cre/lox-system of bacteriophage P1 for site-specific recombination offers a possibility to circumvent such problems, as two arbitrary plasmids containing a specific recognition sequence (so called lox-site) are efficiently fused by the activity of the enzyme Cre-recombinase in vivo and in vitro (11). Therefore, Cre/lox-mediated recombination has been employed in numerous ways for the construction of plasmids and the manipulation of genomes (12). In this study we used the Cre/lox-system to create large module vectors carrying tandem expression cassettes for Chlamydomonas without the need for restriction enzymes in the final cloning steps. As an example, a luciferase gene was expressed from different commonly used promoter sequences (wild-type and artificial arrangements), and the expression cassettes were integrated together with a selection marker on tandem vectors or delivered as separate plasmids.

Materials and Methods

Construction of Plasmids

A part of the backbone of plasmid pBlue BAC4.3-E/Uni-CAT (Invitrogen, Carlsbad, CA, USA) carrying a loxP-site, a kanamycin selection marker, and an R6Kγ origin of replication for maintenance in Escherichia coli PIRI cells (Invitrogen) was used for the construction of all pProm-Chlamy vectors. After digestion of pBlueBAC4.3-E/Uni-CAT with SalI, the 2157-bp fragment was purified and religated, forming pUni-soloII.

Plasmid pRbcRL(Hsp196) contains a C. reinhardtii codon-adapted luciferase gene (crluc) from Renilla reniformis (13) under the chimeric HSP70A/RBCS2 promoter and the 3′-untranslated region (3′-UTR) of the Chlamydomonas RBCS2 gene (14). This plasmid was incubated with SacI and KpnI, and the complete expression cassette for luciferase was subcloned into pUC18 cut with the same enzymes to get a new set of restriction sites. From the resulting plasmid the expression cassette was isolated with SacI and SmaI and transferred to pUnisoloII digested with SacI and HincII, leading to pProm-Chlamy vector pHsp70A/RbcS2-Chlamy. To create additional pProm-Chlamy vectors, the HSP70A/RBCS2 promoter was excised with SacI/XhoI or XbaI/XhoI and replaced by the promoter sequences of the PSAD, LHCB1, or HSP70A gene. Therefore, promoter sequences were provided with a 5′-SacI or 5′-XbaI and a 3′-XhoI restriction site (underlined) by recombinant PCR using pGenD-ble (15) and oligonucleotides psa1(5′-TAGAGCTCCACACACCTGCCCGTCTG-3′) and psa2(5′-CATCTCGAGCATTTTGGCTTGTTGTGAGTAGCAG-3′) for the PSAD promoter and pMS188 (16) and HspXba(5′-AATCTAGAGCCATATCGCCGCCGCTTTG-3′) and HspXho(5′-ATCTCGAGCATGTTTACTGACTCTTAAGCGAGTTGGTGGTTATGTATAGCGGC-3′) for the HSP70A promoter. PCR products were directly incubated with the corresponding restriction enzymes and ligated into the promoterless pProm-Chlamy vector fragment generating pPsaD-Chlamy and pHsp70A-Chlamy. The LHCB1 promoter was obtained from plasmid pLhcRL (14).upon digestion with SacI and XhoI, finally resulting in pLhcb1-Chlamy.