Journal of Biotechnology 195 (2015) 60–66

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A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii Ivo Bertalan ∗ , Matthias C. Munder, Caroline Weiß, Judith Kopf, Dirk Fischer, Udo Johanningmeier Martin-Luther-Universität Halle-Wittenberg, Institut für Pflanzenphysiologie, Weinbergweg 10, 06120 Halle, Germany

a r t i c l e

i n f o

Article history: Received 18 September 2014 Received in revised form 16 December 2014 Accepted 19 December 2014 Available online 30 December 2014 Keywords: Algal biotechnology Chlamydomonas reinhardtii Fud7 Transgenic chloroplast Rekombinant protein expression

a b s t r a c t In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts. © 2015 Published by Elsevier B.V.

1. Introduction Microalgae represent a group of photosynthetic organisms, which efficiently produce valuable biomass in inexpensive media with the help of solar energy. They are used as a source for proteins, lipids, vitamins and pigments, play a role in animal and human nutrition and are promising for sustainable production of fuels (Pulz and Gross, 2004; Spolaore et al., 2006; Walker et al., 2005; Johanningmeier and Fischer, 2010; Wijffels et al., 2013; Corchero et al., 2013). With recent advances in genetic engineering techniques it becomes possible to manipulate some of those organisms in order to boost the production of specific intrinsic biomolecules or express new compounds or pathways not originating in the organism (Radakovits et al., 2010; Rosales-Mendoza et al., 2012; Wijffels et al., 2013). The model alga for which the most comprehensive toolbox is available and which has been studied most intensively is

∗ Corresponding author. Tel.: +49 3455526256. E-mail address: ivo.bertalan@pflanzenphys.uni-halle.de (I. Bertalan). http://dx.doi.org/10.1016/j.jbiotec.2014.12.017 0168-1656/© 2015 Published by Elsevier B.V.

the eukaryotic green alga Chlamydomonas reinhardtii (Harris, 2001; Rochaix, 2002; Purton, 2007). Specifically, its chloroplast has been exploited as a powerful prokaryotic-like expression platform for the production of recombinant therapeutic and diagnostic proteins (Maliga and Bock, 2011; Rasala and Mayfield, 2014) using biolistic transformation methods. As opposed to nuclear transformation, chloroplast transformation has distinct advantageous features like homologous recombination facilitating gene replacement, high transgene copy numbers, absence of RNAi and gene silencing mechanisms, or sequestration of the recombinant protein within the organelle containment. Although much effort has been invested in the last years to improve selection methods and transgene expression in Chlamydomonas chloroplasts, it is still a laborious procedure generally involving several cloning steps. Efficient transgene expression further requires the development of proper combinations of promoter, 5 and 3 UTR sequences as well as codon adjustment of the foreign gene (Franklin et al., 2002; Weiß et al., 2012). Recent progress in synthetic biology permits the modular and seamless assembly of such genetic elements in a single tube by exploiting type IIs

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restriction endonucleases like BsaI, hence reducing cloning steps and simplifying cloning strategies (Weber et al., 2011; Engler and Marillonnet, 2013). Based on this so called Golden Gate shuffling technology, we present here a fast, versatile and reliable method for integrating foreign genes into the intergenic region between the psbA gene and 5S rRNA gene on the chloroplast genome. The specifically designed transformation vector pMM2 contains BsaI restriction sites for insertion of the gene of interest together with the allotted regulatory sequences, the complete psbA gene for selection and flanking sequences for homologous recombination in the recipient strain Fud7 (Bennoun et al., 1986). This genetically stable Chlamydomonas mutant has a large deletion on the chloroplast genome between the psbA gene and 5S rRNA gene encompassing part of the psbA gene, which results in photosynthetic incompetence due to the absence of the psbA-encoded D1 subunit of photosystem II. Biolistic transformation of vector pMM2 with or without insertions into Fud7 gives rise to photosynthetically competent colonies when selected on media without a carbon source. In this study we choose to express MPT64, a protein released from Mycobacterium tuberculosis and relevant for diagnostic purposes (Bekmurzayeva et al., 2013). In order to demonstrate the versatility of our approach, the mpt64 gene was assembled with different promoter/UTR sequences already known to drive expression to various levels.

2. Materials and methods

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Fig. 1. Map of transformation vector pMM2. The plastomic DNA region is flanked by EcoRI and BamHI sites and includes the intronless psbA gene, a multiple cloning site (MCS), the 5S gene and a part of the 23S gene (dark gray line). The light gray line represents vector sequences with the ampR gene indicated. The BsaI recognition sequences highlighted in gray are positioned in inverse orientation within the MCS. BsaI enzymes cleave outside (arrows) their recognition sites leaving non-compatible 5 -overhangs (underlined sequences) and permit any DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly. There is a unique NheI site (GCTAGC) located between the two BsaI sites (not indicated).

2.1. Algal and bacterial strains The C. reinhardtii psbA deletion mutant CC-4147 Fud7 (mt+) was obtained from the Culture Collection of Algae (Chlamydomonas Resource Center) and grown heterotrophically in Tris-acetatephosphate medium (TAP; Harris, 1989). Reference strain IL (Johanningmeier and Heiss, 1993) with an intronless psbA gene and transformants were grown in TAP or high salt medium (HS; Harris, 1989) at 23 ◦ C under continuous light (40 ␮E m−2 s−1 ). For growth on plates, 1.5 % (w/v) agar was added to the medium. Escherichia coli strain Top 10 was grown at 37 ◦ C in LB medium (Sambrook et al., 1998) and was used for Golden Gate shuffling (Engler et al., 2009). Bacterial clones were selected on LB plates containing 100 ␮g/ml ampicillin.

2.2. Construction of vector pMM2 Starting point for pMM2 construction was the plasmid pSH4-IL, which contains a 4.5 kb EcoRI–BamHI cpDNA fragment with the psbA cDNA, and plasmid pSHc6 (Johanningmeier and Heiss, 1993), which served as template for a PCR fragment containing 2 kb DNA sequences around the 5S rRNA gene. A multiple cloning site (MCS) was prepared by using a forward primer containing the MCS sequence (underlined) 5 -GAAGATCTTGAGGTAGAGACCAAAGCTAGCAAAGGTCTCAGCTTGACCCGGGGAAGGGGAAGGGGACGTCCTAAACGGAGCAT-3 and the reverse primer 5 -CACTTTATGCTTCCGGCTCGTATGTTGT-3 (located in the plasmid backbone of pSHc6) and inserting the fragment digested with BglII and NotI into pSH4-IL cut with BamHI and NotI. In the ampR gene of the resulting vector pMM1 an additional BsaI site (GGTCTC) was removed by site-directed mutagenesis without changing the amino acid sequence of the gene product. The complete vector was amplified with 5 -phosphorylated primers 5 -GTGAGCGT-GGGTCCCGCGGTATCATTG-3 and 5 CGGCTCCAGATTTATCAGC-AATAAACC-3 and subsequently self-circularized. The new plasmid was named pMM2 (Fig. 1), its correct sequence verified and used for Golden Gate shuffling.

2.3. Construction of transformation vectors A set of eleven PCR fragments composed of sequences for promoter, 5 UTR, gene of interest, tags and 3 UTR were generated for Golden Gate shuffling of four individual expression cassettes (Figs. 2 and 4). These fragments were generated with primers containing a BsaI restriction site at their 5 -ends. Care was taken to ensure that no additional BsaI restriction sites were present in the fragments. The template for promoter and UTR fragments was genomic DNA from mutant strain IL. The coding sequence for mpt64 was optimized accounting for the codon usage of C. reinhardtii chloroplasts (Fig. S1) and synthesized by Eurofins MWG Operon. This sequence has a codon adaptation index (CAI) = 0.757 (Sharp and Li, 1987) and an effective number of codons (Nc) = 29.4 (Puigbò et al., 2008). Tag sequences for His and HA were generated by primer extension and primer dimerization, respectively. All primers and the length of resulting PCR fragments are listed in Table 1. Up to five PCR fragments together with pMM2 were combined (Table 2) in one step by Golden Gate shuffling. A pipetting scheme and thermo cycler program for this restriction ligation reaction is exemplarily shown in Table S1. 10 ␮l of the reaction mixture was used to transform competent E. coli cells. Plasmids were sequenced to confirm the correct assembly and insertion of the various expression cassettes into pMM2. Supplementary Fig. S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2014.12.017. Supplementary Table S1 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec. 2014.12.017. 2.4. Chloroplast transformation Plastids were transformed by particle bombardment (Klein et al., 1987). Recipient strain Fud7 was grown in TAP medium to late exponential growth phase. 1.5 × 107 cells were concentrated onto Polyamide filters (0.45 ␮m; 47 mm diameter; Whatman, Dassel,

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Fig. 2. Illustration of the cloning and transformation strategy using vector pMM2. Vector pMM2 contains the truncated plastome region as described in the text and compared with the wildtype region outlined at the top of the figure. The gene of interest (GOI) with its hemagglutinin-tag (HA) and regulatory regions (promoter P, 5 and 3 UTR) is inserted in a “one pot – one step” cloning procedure, giving rise to transformation vector pTrans with its large flanking regions homologous to those existing in the Fud7 recipient strain.

Germany) and transformed with tungsten particles coated with the transformation vector. To select for primary transformants, the bombarded Fud7 cells were transferred from TAP to HS agar plates and placed under continuous light as described by Dauvillee et al. (2004). Individually transformed alga colonies were grown for several generations in liquid HS medium until homoplasmic. Homoplasmicity was checked by Southern blot analysis. Total cellular DNA of Fud7 and transformant strains was prepared according to Newman et al. (1990), digested with HindIII, separated

by agarose gel electrophoresis, blotted onto positively charged nylon membrane (Roche® ) and hybridized to a 5S rDNA probe. The probe was digoxigenin (DIG) labeled using the PCR-DIG-labeling mix (Roche® ) with primers (5 -CCCCTTGCGGGTAACTATCG-3 and 5 -CGAAGGGGACGTCCTTCGGAGT-3 ) and pMM2 as template. After washing and blocking the membrane was incubated with AntiDIG fab fragments (Roche® ), then washed, equilibrated and finally incubated with CSPD solution (ready-to-use, Roche® ). Chemiluminescence was detected with HyperfilmTM (GE Healthcare).

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Table 1 Oligonucleotides and resulting PCR-fragments used for Golden Gate shuffling. No.

PCR-fragment (size in bp)

Primer

Sequence (5 → 3 )a

1

P 16S (239)

2

atpA 5 UTR HisX (433)

3

psbA P + 5 UTR (280)

4

psbD P + 5 UTR (540)

5

atpA P + 5 UTR (560)

6

psbA/atpA MPT (711)

7

psbD MPT (715)

8

HisX MPT (731)

9

HA tag (53)

10

rbcL 3 UTR (480)

11

psbA 3 UTR (146)

for rev for rev for rev for rev for rev for rev for rev for rev for rev for rev for rev

TATA GGTCTCAAGGTCAGGCAACAAATTTATTTATTGTCCCGTAAGGGG TATA GGTCTCAGATACTCTTTAAAGTTTAAATTTTGTCGGGATTTTAAACCC TATA GGTCTCATATCTTTACCTTTTTTTTAATTTGCATGATTTTAATGCTTATGC TATA GGTCTCAGATGGTGGTGATGCATAAAAAAGAAAAAATAAATAAAAGATTAAAAAAG TATA GGTCTCAAGGTCGTCCTATTTTAATACTCCGAAGGAGGCAG TATA GGTCTCTCATATGTTAATTTTTTTAAAGTTTTAATTTCTCCGTAAAATATTG TATA GGTCTCAAGGTCCAGGCAATTGTCACTGGCGTC TATA GGTCTCATGCGTGTATCTCCAAAATAAAAAAACAACTCATCG TATA GGTCTCAAGGTCAAAAGTCATTTTTATAACTCGTCTCAA TATA GGTCTCCCATAAAAAAGAAAAAATAAATAAAAGATTAAAAAAG TATA GGTCTCATATGCGTATCAAAATCTTCATGCTTGTTACTGCTG TATA GGTCTCAGGTAAGCAAGCATTGAGTCGATAGCAG TATA GGTCTCTCGCAATGCGTATCAAAATCTTCATGCTTG TATA GGTCTCAGGTAAGCAAGCATTGAGTCGATAGCAG TATA GGTCTCACATCATCATCACCATCACCACATGCGTATCAAAATCTTCATGCTTGTTA TATA GGTCTCAGGTAAGCAAGCATTGAGTCGATAGCAG TATA GGTCTCATACCCTTACGATGTTCCTGATTAC TATA GGTCTCAATTAAGCGTAATCAGGAACATCGTAAGGG TATA GGTCTCATAATTTTTATTTTTCATGATGTTTATGTGAATAGCATAAACATCG TATA GGTCTCAAAGCAACACATAACTCCACGTAAGCGCA TATA GGTCTCATAATTTTTTTTTAAACTAAAATAAATCTGGTTAACCATACCTGG TATA GGTCTCAAAGCGGGACGTCCTGCCAACTGCC

a

Recognition site of BsaI restriction enzyme is shown in gray and the resulting overhang is underlined.

2.5. RNA analysis Total nucleic acids were isolated from algae cells grown to late log phase in 50 ml TAP medium under continuous illumination (40 ␮E m−2 s−1 ). 15 ml of the cell culture were centrifuged and resuspended in 1 ml denaturing solution (38% saturated phenol, 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate pH 5.0, 5% glycerol) and incubated at 55 ◦ C for 5 min to lyse the cells. 200 ml chloroform were added, incubated for 5 min and centrifuged. The aqueous phase was precipitated by addition of 500 ml isopropanol. The RNA pellet was washed in 75% ethanol and finally resuspended in 40 ml of RNase-free water. RNA yield was quantified spectrophotometrically. Northern blot analysis was performed according to standard procedures (Sambrook et al., 1998). Blots were hybridized with mpt64 specific probe that was DIG-labeled by PCR using the forward primer 5 -ATGCGTATCAAAATCTTCATGCTTG-3 and reverse primer 5 - CGTAATCAGGAACATCGTAAGG-3 .

2.6. Protein analysis For total protein preparation 15 ml of cell culture was harvested by centrifugation (3.000 × g, 5 min). The pellet was washed once in 1 ml of solution A (0.1% Na2 CO3 ) and resuspended in 300 ␮l solution A. 200 ␮l of solution B (5% SDS, 30% sucrose) and 25 ␮l ß-mercaptoethanol were added and agitated for 25 min. Cell debris was removed by centrifugation (16.000 × g, 5 min) and the chlorophyll concentration of the supernatant (total cellular protein) was determined as described by Arnon (1949). Proteins were separated by SDS PAGE (Schägger and von Jagow, 1987), transferred onto nitrocellulose membranes (BA-85, Schleicher and Schüll, Dassel, Germany) as described by Towbin et al. (1992) and

immunodecorated with horseradish peroxidase (HRP)-coupled anti-HA-HRP antibody (Mitenyi Biotec GmbH, Germany). Detection was carried out using the ECL-AdvancedTM Western blotting detection kit (GE Healthcare) following manufacturer’s instructions. 3. Results 3.1. Design of transformation vector pMM2 The vector pMM2 (Fig. 1) contains sequences for homologous recombination with the plastome of recipient strain Fud7, a functional psbA gene to restore the ability for photoautotrophic growth and a MCS that allows to utilize the type IIs restriction enzyme based cloning system described by Engler et al. (2008, 2009). For efficient recombination this vector encompasses large 5 - and 3 -regions (1.654 bp and 2.021 bp, respectively) homologous to those flanking the deletion breakpoint in strain Fud7 (Fig. 2). This breakpoint has been determined by sequencing a PCR fragment obtained with primers located within the 5 UTR of the psbA and the 5S rRNA genes, indicating that an 8.225 bp fragment was deleted in this mutant (Fig. S2). In order to reduce the size of the transformation vector, an intronless psbA gene (Johanningmeier and Heiss, 1993) was introduced and a 1161 bp intergenic sequence between the psbA and 5S rRNA genes was replaced by a short MCS (42 bp) resulting in vector pMM2. With a length of about 9 kb pMM2 is suitable for integrating transgenes by Golden Gate shuffling. Upon biolistic introduction of this vector into Fud7 and selection for photoautotrophic growth transformation rates between 1 and 10 transformants per ␮g plasmid DNA are achieved. Supplementary Fig. S2 related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jbiotec.2014.12.017.

Table 2 List of the individual PCR-fragments, which are assembled into specific expression cassettes. Expression cassettes

Fused PCR-fragments into the MCS of pMM2 by golden gate shuffling

Size (bp)

16S MPT psbA MPT psbD MPT atpA MPT

(P16S) + (atpA 5 UTR HisX) + (HisX MPT) + (HA tag) + (rbcL 3 UTR) (psbA P + 5 UTR) + (psbA/atpA MPT) + (HA tag) + (psbA 3 UTR) (psbD P + 5 UTR) + (psbD MPT) + (HA tag) + (psbA 3 UTR) (atpA P + 5 UTR) + (psbA/atpA MPT) + (HA tag) + (rbcL 3 UTR)

1.814 1.079 1.342 1.711

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by particle bombardment into the Fud7 strain. Transformants were selected for photoautotrophic growth on minimal media and colonies were detected after about ten days. Three transformants of each construct were selected and grown in liquid minimal (HS) medium until homoplasmic. PCR analysis was performed to test the correct integration of the expression cassette (data not shown). One representative transformant of each construct was further checked for homoplasmicity by PCR (not shown) and Southern blot analysis (Fig. 5). In all four strains only the specific fragment for the inserted transgene fragment with the correct size was detected, indicating that all plastome copies were identical. Sequence analyses confirmed that the expression cassettes contained no undesired mutations (data not shown). Fig. 3. PCR fragments used for Golden Gate shuffling contain BsaI restriction sites at their 5 and 3 end. M, 100 bp DNA Ladder; lane 1, 16S promoter (16S); lane 2, atpA 5 UTR with a partial sequence for an N-terminal His-tag (atpA 5 UTR HisX); lane 3, psbA promoter and 5 UTR (psbA P + 5 UTR), lane 4, psbD promoter and 5 UTR (psbD P + 5 UTR); lane 5, atpA promoter and 5 UTR (atpA P + 5 UTR); lane 6, MPT64 coding sequence suitable for the psbA and atpA P + 5 UTR fragment (psbA/atpA MPT); lane 7, MPT64 coding sequence suitable for the psbD P + 5 UTR fragment (psbD MPT); lane 8, MPT64 coding sequence with a partial sequence for an N-terminal His-tag suitable for fragment atpA 5 UTR HisX (HisX MPT); lane 9, sequence for a C-terminal HA-tag suitable for all MPT and 3 UTR fragments (HA tag); lane 10, rbcL 3 UTR (rbcL 3 UTR); lane 11, psbA 3 UTR (psbA 3 UTR).

3.2. Construction of mpt64 gene cassettes and transformation MPT64 as a foreign protein was chosen to test the recombinant protein expression system in combination with various UTR/promoter regions, which have been shown to provide high transcription and/or translation rates (Barnes et al., 2005; Rasala et al., 2011). A codon optimized mpt64 gene sequence adjusted to the Chlamydomonas chloroplast codon usage was generated (Fig. S1) and furnished with a C-terminal HA-tag for protein detection with an HA-specific antibody. Individual PCR-fragments (Fig. 3) consisting of HA-tag sequences, promoter sequences of 16S, psbA, psbD, and atpA genes and UTR sequences of these genes were generated with primers containing BsaI restriction sites as 5 extensions (Table 1), thus forming suitable overhangs for correct assembly into the multiple cloning site of pMM2 during Golden Gate shuffling (Fig. 2). Individual restriction-ligation reactions were transformed into E. coli giving rise to four vectors containing gene expression cassettes as outlined in Fig. 4. These vectors were transformed

Fig. 4. Gene expression cassettes were assembled and inserted into the MCS of pMM2 by Golden Gate cloning and used subsequently for the expression of MPT64 in C. reinhardtii chloroplasts. Resulting algae strains were named by the individual used promoter sequences (A) 16S MPT, (B) psbA MPT, (C) psbD MPT, (D) atpA MPT.

3.3. Mutant analysis In order to determine the expression levels of each construct, Northern and Western blot analyses were performed on total cellular RNA and protein, respectively. As can be seen from Fig. 6a, transcript levels vary drastically, being highly abundant in construct 16S MPT and very low in strain psbA MPT (only detectable upon longer exposure; not shown). A similar result is observed for MPT64 protein levels in strains 16S MPT and psbA MPT (Fig. 6b). However, although transcript levels for strains psbD MPT and atpA MPT are similar their protein levels are quite different. 4. Discussion In order to generally improve the chloroplast of the green alga Chlamydomonas reinhardtii as a host system for heterologous protein expression, this study sought to establish an efficient and versatile procedure for the generation of stable recombinant algal strains. The key elements of this approach is the newly generated transformation vector pMM2, which is able to transform mutant strain Fud7 (Bennoun et al., 1986) and allows selection for photoautotrophic growth. Fud7 has been used as recipient for chloroplast transformation in many instances (Mayfield et al., 1994; Minagawa and Crofts, 1994; Michelet et al., 2011). In order to significantly reduce the size of the vector for better handling, we introduced the intronless psbA gene, thus removing 5.5 kbp of intron sequences without affecting cellular metabolism (Johanningmeier and Heiss, 1993; Minagawa and Crofts, 1994). Furthermore, a 1.1 kbp intergenic sequence between the psbA and 5S rRNA genes was substituted by a short MCS, which allows shuffling of genes due to BsaI restriction sites. To demonstrate the versatility of this arrangement, we chose mpt64 as a heterologous test gene and combined it with various promoter/UTR regions using the strategy outlined in Fig. 2. Analyses of the transformants (Fig. 6) reveal that strain 16S MPT displays the highest expression at both the mRNA and protein levels. In strain psbA MPT both mRNA and MPT64 are hardly detectable. One possible explanation for this result could be a modified structure of the mRNA due to the heterologous sequence, which could affect ribosome binding, translation factor binding and/or RNA stability. These observations are basically in accordance with data obtained by Barnes et al. (2005) and Rasala et al. (2011). Thus, using vector pMM2 we succeeded in assembling up to five DNA fragments in a “one pot and one step” cloning procedure (Engler et al., 2008, 2009; Weber et al., 2011) without leaving short DNA sequences often remaining by using standard cloning procedures. Furthermore, the approach described here depends on restoration of photoautotrophic growth for selecting transformants. Although this strategy has been used before

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Fig. 5. Test of homoplasmicity performed by restriction fragment length polymorphism (RFLP)-analysis (Southern blot analysis). (a) Restriction map of the relevant plastom region for the four transformation strains and recipient strain Fud7. Indicated are the used 5S rDNA probe and the HindIII restriction sites that produce strain specific DNAFragments. (b) Result of the Southern blot analysis. Total cellular DNA from recipient strain Fud7 and transformant strains (A) 16S MPT, (B) psbA MPT, (C) psbD MPT, (D) atpA MPT was digested with HindIII. Detection was realized by a 5S rDNA specific probe.

Fig. 6. Northern and Western blot analyses of transgenic strains 16S MPT (A), psbA MPT (B), psbD MPT (C), atpA MPT (D) and control strain IL-MCS (IL) containing the multiple cloning site without insert. (a) Detection of mpt64 mRNA. 5 ␮g of total RNA samples were separated and analyzed with an mpt64 specific probe. As loading control the ethidium bromide stained 28S rRNA band is shown. (b) Western blot analysis of MPT64 accumulation. Total cellular protein samples equivalent to 1 ␮g chlorophyll were analyzed with an anti-HA antibody.

(Boynton et al., 1988; Michelet et al., 2011), most published procedures rely on cotransformation with genes like aadA conferring antibiotic resistance (Goldschmidt-Clermont et al., 2008; Dreesen et al., 2010; Wu et al., 2011; Noor-Mohammadi et al., 2012; Demurtas et al., 2013), putting additional strain on the expression machinery of the organism already stressed by foreign gene production. In addition, the presence of antibiotic resistance genes could be an unwanted property if the whole organism is to be used e.g. for feeding purposes. Thus, the combination of a fast and efficient cloning method with a marker-free selection procedure will ameliorate the expression of foreign proteins in the algal chloroplast. 5. Conclusion We established an efficient and fast method to generate transplastomic C. reinhardtii strains. One of the advantages of our approach is the generation of new transformation vectors with different promoter/UTR combinations, tags, etc. in only one step and without undesired sequences. In combination with a marker-free selection procedure using psbA deletion mutant Fud7 we consider this an important advancement in expanding the toolbox for biotechnological modifications of the green alga C. reinhardtii. Acknowledgments This work was supported by the Land Sachsen-Anhalt and COST (European Cooperation in Science and Technology, TD1102).

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A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii.

In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Partic...
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