DE102008013304B4 - Method and expression strain for heterologous gene expression - Google Patents

Abstract

A method of expressing heterologous genes, the method comprising the steps of: - preparing at least one expression vector; Cloning at least one heterologous target gene into the expression vector; Preparing a Rhodobacter capsulatus expression strain, wherein the expression strain is prepared by integration of a T7 RNA polymerase gene under the control of the Rhodobacter-specific promoter Pfru into the recA gene of the Rhodobacter capsulatus chromosome; Introducing the recombinant expression vector into the cells of the expression strain; and expression of the heterologous target gene in the cells of the expression strain.

Description

The invention relates to a method and an expression strain for the expression of heterologous genes.

The use of proteins with defined catalytic and structural properties is indispensable for pharmaceutical and biotechnological development and production processes. An immediate consequence of this development is the exponentially increasing demand for biological and technical processes for producing such biocatalysts.

Heterologous expression of genes is by far the simplest and most cost-effective way of producing large quantities of a particular protein for scientific and commercial purposes. Over the past three decades, a broad spectrum of molecular genetic techniques has been developed that allow the efficiency of protein synthesis to be empirically optimized in some manageable bacterial and eukaryotic microorganisms. The production of alien proteins in organisms such as the bacterium Escherichia coli and the baker's yeast Saccharomyces cerevisiae is now a standard method used worldwide.

In fact, some recombinant proteins can be produced very easily by expressing the heterologous genes at high rates in a fast-growing organism such as E. coli. Unfortunately, this is rarely the case. The normal case is an insufficient protein yield, for which there are complex causes that lie at different levels of the very complex process of protein biosynthesis and can vary from host to host. Frequently, inefficient transcription, instability of the transcript or inadequate translation is the cause. However, there may also be numerous problems with the maturation of the protein, such as. As an incorrect protein folding or the absence of essential cofactors occur.

In heterologous expression, the synthesis of any target protein is not carried out in the original organism, but is instead carried out by genetic engineering in an undemanding and genetically and technically manageable microorganism or cell culture. For this purpose, a variety of established expression systems exist today, based on microorganisms such as bacteria and fungi or on higher organisms such as insects, plants and animals.

Of particular importance is the expression in prokaryotic and eukaryotic microorganisms, as they can be easily manipulated using a variety of genetic methods and allow by their short generation times and their easy and inexpensive cultivation of a very economical production of large quantities of the recombinant gene product. The yields of the heterologously expressed protein can be up to 50% of the total protein (Alldread et al., 1992). However, not every expression host is equally suitable for the expression of any heterologous protein.

The most commonly used host for expression of heterologous proteins is the Gram-negative enterobacterium Escherichia coli (Hannig & Makrides, 1998, Jana & Deb, 2005, Sørensen & Mortensen, 2005). In countless studies, both proteins of prokaryotic and eukaryotic origin have been successfully expressed in this organism. Nevertheless, the heterologous expression of proteins in E. coli is sometimes problematic.

For example, it is known that different organisms often prefer different alternative codons to encode a particular amino acid (Gutman & Hatfield, 1989). This preference for particular codons is referred to as "codon bias" (Carbone et al., 2003). The preference for certain codons depends mainly on the type of organism, but may also vary within one and the same cell, e.g. Between strong and weakly expressed genes or between genes that are active under different growth conditions (Saier, 1995, Makrides, 1996). A related problem arises because some organisms do not use the "universal" genetic code but an alternative form (Santos et al., 2004).

In functional heterologous expression, problems can easily arise if the parent organism has a different codon usage than the heterologous expression host. The preference for particular codons is usually associated with the fact that the specific tRNA molecules for these codons in the tRNA pool of the cell are significantly more represented than the tRNA molecules that recognize rare codons. If a gene now contains many such rare codons, the supply of cognitive tRNA species for these codons is rapidly depleted (Smith, 1996, Gustafsson et al., 2004). The consequence of this may be delays in the elongation of the peptide chain during translation (Zahn, 1996), frame shifts (McNulty et al al., 2003), translational "jumps" (Kane et al., 1992), or defective incorporation of other amino acids (Calderone et al., 1996; Forman et al., 1998, McNulty et al., 2003).

Another problem with heterologous expression is toxic proteins. Toxic effect of the recombinant proteins can lead to instability of the expression plasmids or even death of the cells, thereby greatly reducing the yields of the heterologous protein. Sometimes expression vectors containing genes for highly toxic gene products can not even be established in the cells if the promoter controlling their transcription also has a basal expression level uninduced (Doherty et al., 1993).

In some cases, it does not even have to be a toxic protein per se in order to sustainably damage the cell. For example, overexpression of non-toxic proteins may lead to overloading of the metabolism and thus to decreased growth rates (Andersson et al., 1996) and even death of the cells (Dong et al., 1995). This may be related to the fact that the expression of large amounts of recombinant protein costs the cell important resources and energy through the maintenance and replication of the expression plasmids (Corchero & Villaverde, 1998), as well as through the expression of these genes (Glick, 1995). This in turn can not only affect the expression of the heterologous protein, but also the functionality of vital cell processes (Glick, 1995, Hoffmann & Rinas, 2001).

Another problematic consequence of heterologous overexpression may be the induction of a stress response of the cell. Among other things, heat shock genes and genes for DNA repair are up-regulated (Rinas, 1996, Gill et al., 2000, Lesley et al., 2002). One of the consequences is increased proteolytic activity of the cells due to increased expression of proteases (Harcum & Bentley, 1999, Ramirez & Bentley, 1999), which can lower the yield of the recombinant protein.

The folding of the heterologous protein can be another problem in heterologous expression (Baneyx & Mujacic, 2004). Some proteins rely on specific folding aids, such as foldases and chaperones, which are partially unexpressed by E. coli (Schlieker et al., 2002). Incorrect folding of a protein favors its degradation by host proteases (Goff & Goldberg, 1985, Makrides 1996) or can lead to aggregation of the protein and thus formation of insoluble inclusion bodies (Wall & Plückthun, 1995, Baneyx & Mujacic, 2004).

The formation of inclusion bodies is a common problem in heterologous expression (Rinas & Bailey, 1993, Villaverde & Carrió, 2003). It is influenced by a variety of factors, including the expression level (Strandberg & Enfors, 1991) and the structural properties of the recombinant protein (Murby et al., 1995).

Some proteins rely on additional components to be expressed in soluble and active form. For example, some membrane proteins require the presence of specific associated lipids (Opekarová & Tanner, 2003) that are not synthesized in E. coli. Other proteins undergo a variety of modifications before they reach their active conformation (Wenzel & Müller, 2005). Still other proteins require cofactors or prosthetic groups to be activated.

All of these and other problems can result in a recombinant protein not being expressed in E. coli, or only in low yield, or only an insoluble and / or inactive form of the protein being synthesized.

In the past 30 years, numerous strategies have been developed to overcome the problems mentioned in heterologous expression. In principle, two different paths can be taken. On the one hand, it is possible to optimize a standard expression strain using molecular genetic methods specifically for the expression of a problematic target protein. On the other hand, however, the expression of problematic proteins can also take place in alternative host organisms, which, because of their physiological, genetic or structural properties, are better suited for efficient synthesis of the proteins.

There are two basic methods for overcoming a different codon preference. One possibility is the coexpression of genes encoding the cognitive tRNAs of the rare codons (Forman et al., 1998, McNulty et al., 2003, Sørensen et al., 2003). However, the tRNA in question must be overexpressed for this purpose, thus subtracting part of the total synthesis capacity of the cell for its own expression. In addition, additional expression plasmids with the genes of these tRNAs be established and maintained in the organism. This limits the choice of expression plasmids on the one hand, since incompatibilities must be avoided, and on the other hand can mean additional metabolic stress for the cell.

In the second approach to overcoming codon bias, rare codons of the target gene are transformed by targeted mutations into other codons that code for the same amino acid but are more commonly used by the expressionist (Calderone et al., 1996; Deng, 1997; Chang et al., 2006). In the event that a great deal of change has to be made, this method may extend to complete resynthesis of the gene of interest (Pan et al., 1999, Feng et al., 2000, Patterson et al., 2005). However, this codon optimization is a very time-consuming and costly process.

The most effective method of expressing a toxic protein in E. coli is the use of strictly regulated, inducible promoters so that the cells can first generate sufficient biomass before commencing expression (Studier, 1991, Rossi & Blau, 1998; Wycuff & Matthews, 2000; Jonasson et al., 2002). Another method is the secretion of the recombinant proteins to minimize their cytoplasmic concentration (Hockney, 1994, Gumpert & Hoischen, 1998, Jana & Deb, 2005). However, this method requires that the recombinant gene be fused with a signal sequence specific for secretion. In other cases, one makes use of the formation of inclusion bodies, in which the recombinant protein is included and therefore can not develop a toxic effect (Baneyx & Mujacic, 2004). However, the problem arises again that the proteins must be recovered from the inclusion bodies laboriously.

The folding of proteins can be improved, for example, by the coexpression of folding modulators (Wall & Plückthun, 1995, Schlieker et al., 2002, Baneyx & Mujacic, 2004), although similar problems arise as in the co-expression of tRNAs. The coexpression of the molecular chaperones DnaK, DnaJ and GrpE (Nishihara et al., 1998), GroEL and GroES (Amrein et al., 1995, Yasukawa et al., 1995, Nishihara et al., 1998) has been successful in recent studies ) as well as FkpA (Arié et al., 2001). Optimization of growth conditions can also increase the yield of correctly folded protein (Georgiou & Valax, 1996).

The proteolytic degradation of recombinant proteins is often avoided by the use of protease-deficient expression strains (Nishihara et al., 1998, Chen et al., 2004, Sørensen et al., 2005). Also, the fusion of the recombinant protein with stabilizing partner proteins (Martinez et al., 1995, Corchero et al., 1996, Skosyrev et al., 2003) or the coexpression of stabilizing factors (LeThanh et al., 2005) have been successful in some cases ,

Sometimes, when a protein is to be expressed in active or soluble form, additional specific components such as cofactors or complexing partners, this problem can be overcome by the co-expression of the necessary components (Weickert et al., 1996).

Often, however, optimizing a single factor does not lead to the goal or even adversely affects heterologous expression, so additional factors need to be modified.

The proposed solution strategies for the elimination of expression problems in many cases allowed the synthesis of problematic proteins in E. coli. However, they are associated with a high expenditure of time and money and often still do not lead to the desired success. In addition, an individual optimization of the expression host must be carried out for each "problem protein". Therefore, expression in an alternative organism is often the better solution. Thus, other organisms may have a more effective folding or secretion machinery or their codon usage is similar to that of the original producer of the heterologous protein.

An example of an alternative expression organism is the phototrophic purple bacterium Rhodobacter capsulatus.

R. capsulatus belongs to the group of phototrophic non-sulfur purple bacteria from the group of α-proteobacteria and is therefore a member of the Rhodospirillaceae family. The habitat of this rod-shaped Gram-negative bacterium mainly comprises the light-penetrated layers of stagnant or slow-flowing waters and lakes (Weaver et al., 1975). It is characterized among other things by its red pigmentation, which under phototrophic growth conditions by the pigments of the Photosynthesis apparatus comes about. The proteins of the photosynthetic complex are incorporated in a specialized photosynthetic membrane, the so-called intracytoplasmic membrane (ICM).

R. capsulatus is able to use several different metabolic pathways to provide energy and cell building blocks. Thus it is capable of both aerobic and anaerobic respiration (Zannoni, 1995) and can also exploit light energy for anoxygenic photosynthesis (Gregor & Klug, 1999, Bauer et al., 2003). In anoxygenic photosynthesis, light energy is converted into a proton gradient, which in turn serves to synthesize ATP. In contrast to photosynthesis in plants, neither oxygen nor redox equivalents are produced. Photosynthesis is used by R. capsulatus exclusively as an energy source under anaerobic conditions.

Under nitrogen deficiency, R. capsulatus is able to use atmospheric nitrogen as an N-source and thus grow diazotrophically. For this purpose it has two different nitrogenase complexes (Masepohl et al., 2002, Drepper et al., 2003). Other metabolic activities in R. capsulatus include hydrogen metabolism (Vignais et al., 2000) and CO ₂ fixation (Tabita, 1995).

This organism is one of the best studied phototrophic bacteria. The R. capsulatus genome has been completely sequenced (Haselkorn et al., 2001), providing the basis for detailed analysis of global processes using advanced analysis techniques (eg, microarray technology).

Over the past 30 years, numerous bacterial expression vectors have been constructed, based on different promoters depending on the application. Such expression vectors can usually only be replicated in a limited group of closely related bacterial species (eg, representatives of the enterobacteria group) and are referred to as narrow range vectors (vectors with a narrow host spectrum). In addition, there are currently few vectors that can be established in a larger group of bacteria. In this case one speaks of broad host range vectors. Frequently, overexpression expression vectors with strong, inducible promoters are preferred, which allow to separate the growth and the expression phase. The induction may be depending on the promoter and expression host z. For example, by the addition of metabolites or other compounds, the withdrawal of nutrients or a change in temperature.

Examples of inducible promoters in E. coli include the lac promoter (Fuller, 1982), the tac promoter generated by recombination of the promoters P _lac and P _trp (de Boer et al., 1983) and the araC-P _BAD - Promoter based on a fusion of the arabinose-dependent promoter araBAD and the regulator araC (Guzman et al., 1995, Newman & Fuqua, 1999).

In R. capsulatus, promoters from E. coli are poorly or not recognized at all. Instead, the nifH promoter was used in this organism for expression, which normally controls the transcription of the R. capsulatus genes for nitrogen fixation (Pollock et al., 1988). Furthermore, fructose- and DMSO-inducible promoters were used in R. capsulatus (Duport et al., 1994, Kappler & McEwan 2002).

The T7 system is of particular importance among bacterial expression systems: in the case of the T7 expression system, the recombinant target genes are under the transcriptional control of a bacteriophage T7 promoter (Tabor & Richardson, 1985, Studier & Moffatt, 1986). This 23 bp sequence is recognized exclusively by the specific RNA polymerase from the same phage (Kochetkov et al., 1998). The intrinsic bacterial RNA polymerase does not recognize these promoters and therefore has no influence on the expression of the target genes. T7 RNA polymerase is one of the simplest enzymes to catalyze RNA synthesis: it has only a single subunit and does not require additional protein factors. Their mechanism has been well studied (Kochetkov et al., 1998).

The T7 RNA polymerase has a very high processivity compared to bacterial RNA polymerases. Therefore, very high protein yields can be achieved with T7 RNA polymerase-based expression systems (Studier & Moffatt, 1986). In principle, two components are required for T7 polymerase-dependent expression of a heterologous target gene: On the one hand, expression plasmids are needed in which the target genes can be placed under the control of the T7 promoter (Rosenberg et al., 1987). There are now more than 40 commercial T7 expression vectors for use in E. coli (eg pET plasmids from Novagen). On the other hand, one needs the T7 RNA polymerase gene, which must be under the control of an (inducible) bacterial promoter.

In E. coli, the T7 expression system is one of the most widely used expression systems today. Mostly, strain BL21 (DE3) is used (Studier & Moffatt, 1986), which integrates a prophage into the chromosome that expresses the T7 RNA polymerase under the control of the inducible promoter lacUV5. Meanwhile, however, T7 expression systems for other bacteria, such. Pseudomonas aeruginosa (Brunschwig & Darzins, 1992), Pseudomonas putida (Herrero et al., 1993), Salmonella enterica (McKinney et al., 2002; Santiago-Machuca et al., 2002) or Ralstonia eutropha (Barnard et al ., 2004).

Drepper et al. (2005) describe, for example, an expression system in R. capsulatus which comprises a nifH gene which is under the control of the T ₇ promoter. For hierologic expression of this gene, a plasmid is additionally introduced, which carries the gene for the T ₇ polymerase, so that after induction with IPTG both the polymerase and the NifH protein can be expressed.

From the US 2007/0212782 A1 Furthermore, bacterial cells are known which contain a T ₇ expression plasmid on which a target gene is under the control of a T ₇ promoter. The bacterial cells may contain DE3 derivatives for gene expression. E. coli and R. capsulatus are proposed as bacterial cells, gene expression with DE3 derivatives being shown only for E. coli.

Furthermore, Elsen et al. (2003) the expression of mutant genes under the control of the fructose inducible fru promoter in R. capsulatus.

The gene data obtained from (meta) genome research represents an unprecedented reservoir of new biomolecules with enormous application potential for the chemical and pharmaceutical industries. However, for the use of this reservoir and thus for the identification and provision of new enzymes as well as biotechnological generated special and fine chemicals efficient expression and screening systems are the key enabling technologies.

It is the object of the invention to provide an efficient method for heterologous gene expression as well as an efficient expression and screening system.

• – Herstellen mindestens eines Expressionsvektors;
• – Klonieren mindestens eines heterologen Zielgens in den Expressionsvektor;
• – Herstellen eines Rhodobacter capsulatus Expressionsstamms, wobei der Expressionsstamm durch Integration eines T7-RNA-Polymerasegens, das unter der Kontrolle des Rhodobacter-spezifischen Promotors P_fru steht, in das recA-Gen des Rhodobacter capsulatus – Chromosoms hergestellt wird;
• – Einbringen des rekombinanten Expressionsvektors in die Zellen des Expressionsstamms; und
• – Expression des heterologen Zielgens in den Zellen des Expressionsstamms.

The object is achieved by a method for expressing heterologous genes, which comprises the following steps:

• - producing at least one expression vector;
• Cloning at least one heterologous target gene into the expression vector;
• - producing a Rhodobacter capsulatus expression strain, whereby the expression strain by incorporating a T7 RNA polymerase gene is under the control of Rhodobacter-specific promoter P _fru, in the recA gene of Rhodobacter capsulatus - chromosome is produced;
• Introducing the recombinant expression vector into the cells of the expression strain; and
• - Expression of the heterologous target gene in the cells of the expression strain.

The object is further achieved by an expression strain of the cells of the bacterium Rhodobacter capsulatus comprises obtained by integration of a T7 RNA polymerase gene is under the control of Rhodobacter-specific promoter P _fru, in the recA gene of Rhodobacter capsulatus - chromosome are modified. The expression strain thus has two special properties which are of crucial importance for heterologous expression. First, integration of the T7 RNA polymerase gene into the chromosome produces a genetically stable expression host that allows highly reproducible and controllable expression of the phage polymerase. Second, by integrating the T7 RNA polymerase gene into the recA gene, the expression strain has a recA mutation, thereby completely preventing homologous recombination events between expression vectors and host chromosome.

The invention thus relates to a method and the provision of an expression system, each based on the Gram-negative, phototrophic bacterium Rhodobacter capsulatus. R. capsulatus is due to its special physiology compared to other microbial expression hosts such. B. E. coli particularly suitable to produce membrane-localized proteins in large quantities: Under phototrophic growth conditions R. capsulatus forms an enormously enlarged vesicular membrane system, which has a high intrinsic capacity for membrane proteins. Due to the high protein concentration in the membrane vesicles formed, both efficient translocation systems and suitable chaperones are provided in this organism.

In addition, R. capsulatus provides advantages in the functional expression of redox enzymes and proteins that exhibit O ₂ sensitivity or are toxic to the host in the presence of oxygen. Under phototrophic conditions, this bacterium is able to synthesize almost all of the described redox cofactors in large quantities and to incorporate them into corresponding (heterologous) proteins. In addition, due to its special physiology, the organism is optimally adapted to protein production under anaerobic conditions.

The expression system described here is composed of various R. capsulatus expression strains as well as a set of new tailor-made (over) expression vectors. It enables both the identification of new enzymes in high-throughput screening and the overexpression of heterologous problem proteins in R. capsulatus under aerobic and anaerobic conditions.

R. capsulatus has a high GC content of 68%. It is therefore more suitable for the heterogeneous expression of GC-rich genes than E. coli (51% GC, Blattner et al., 1997).

By forming an intracytoplasmic membrane system, R. capsulatus undergoes a tremendous increase in membrane surface area under phototrophic growth conditions. This property can be exploited for the effective overexpression of membrane proteins, which can be embedded in large numbers in this membrane, without causing toxic effects.

Since R. capsulatus preferentially grows under anaerobic conditions and achieves comparatively high cell division rates due to photosynthesis under these conditions, it is particularly suitable for the heterologous synthesis of oxygen-sensitive proteins or enzymes which have a toxic effect in the presence of O ₂ .

In E. coli, many cofactors and prosthetic groups are only synthesized in small amounts or not at all. In contrast, R. capsulatus synthesizes a number of these components under phototrophic and diazotrophic conditions, for example, heme groups (Lang et al., 1996; de Smet et al., 2001a, Smart et al., 2004), molybdenum cofactors (Wang et al , 1993, Solomon et al., 1999), and in particular various iron-sulfur clusters (Jeong & Jouanneau, 2000, Chevallet et al., 2003) in large quantities. This bacterium is therefore well suited for the expression of heterologous proteins carrying such cofactors. Thus, several redox proteins that are not expressible in E. coli have been successfully overexpressed in R. capsulatus. These include, for example, the sulfite: cytochrome c oxidoreductase (SorAB) from Starkeya novella (Kappler & McEwan, 2002), and the flavocytochrome c from purple bacteria (de Smet et al., 2001b). These could not be synthesized in E. coli.

According to the invention, the vector pBBR22b is modified to produce the expression vector. When the vector pBBR22b is modified by deletion of the lacI gene, LacI-independent expression of the target genes is made possible. Additionally or alternatively, the vector pBBR22b may be modified by insertion of the aphII gene of the Tn5 transposon under the control of the Km promoter. Expression of this gene confers selectable resistance to the recipient strain on the antibiotic kanamycin.

In a particularly advantageous embodiment of the invention, the heterologous gene is cloned in the reading direction of a T7 promoter in the expression vector. In this embodiment, when the aphII gene is transcribed in an opposite orientation to the T7 promoter, the expression of a heterologous target gene is merely the control of the T7 promoter. It may therefore also be advantageous to clone the heterologous gene into the expression vector opposite to the reading direction of the Km promoter of the aphII gene.

In a further advantageous embodiment of the invention, the heterologous gene is cloned in the reading direction of the Km promoter of the aphII gene in the expression vector. For example, if the aphII DNA fragment contains the natural promoter of the gene, but not the transcription terminator, all target genes that are cloned into the polylinker of the new expression vector are constitutively expressed by the aphII promoter.

The invention further comprises a kit for the heterologous expression of at least one gene which comprises at least one modified pBBR22b vector with a polylinker downstream of a T7 promoter and additionally an expression strain according to the invention.

The invention also includes a cell culture comprising cells of the expression strain of the invention.

A particular advantage of the method and expression system according to the invention is that it is suitable or can be used in particular for the effective overexpression of the target gene both under aerobic and under anaerobic conditions.

The invention will be explained in more detail by way of example with reference to the following figures and exemplary embodiments.

: Features of the new Rhodobacter expression vectors. Black: the promoters for expression (in pRhok: Km promoter, in pRhot: T7 promoter); Dark gray: antibiotic resistance (AB); Light gray: origin of replication to mobilize the vectors (MOB); White: broad host range ori (REP).

: Functional DNA elements of the cloning and expression regions of the pRho expression vectors. Black arrow: The T7 promoter (the Km promoter in pRhok lies outside the region shown upstream of the T7 promoter); Gray: Interfaces of the polylinker (the bracketed interfaces are not singular); Black box: tag sequences; White Box: Ribosome Binding Site (RBS)

: The Rhodobacter expression vectors pRhokHi-2 and pRhotHi-2. Black: the promoters for expression (in pRhok: Km promoter, in pRhot: T7 promoter); Dark gray: chloramphenicol resistance gene (Cm); Light gray: origin of replication to mobilize the vectors (MOB); White: broad host range ori (REP)

: The Rhodobacter expression vectors pRhokHi-6 and pRhotHi-6. Black: the promoters for expression (in pRhok: Km promoter, in pRhot: T7 promoter); Dark gray: Spectinomycin resistance gene (Sp); Light gray: origin of replication to mobilize the vectors (MOB); White: broad host range ori (REP)

: The Rhodobacter expression vectors pRhokS-2 and pRhotS-2. Black: the promoters for expression (in pRhok: Km promoter, in pRhot: T7 promoter); Dark gray: chloramphenicol resistance gene (Cm); Light gray: origin of replication to mobilize the vectors (MOB); White: broad host range ori (REP)

: Plasmid map of the mutagenesis construct pRc-T7 to produce the R. capsulatus T7 expression strain B10-T7. White: the antibiotic resistance genes; Dark gray: R. capsulatus recA gene; Light gray: fructose-inducible R. capsulatus promoter; Black: T7 RNA polymerase gene.

: Scheme for the production of the R. capsulatus expression strain B10S-T7. (A) Since there are two recA homologous regions on the plasmid pRc-T7, homologous recombination can in principle occur at two different sites of the chromosome. Thus, a total of three different recombination results can occur: In a simple recombination event (B), the entire plasmid, including both plasmid-encoded resistance genes, is integrated into the genome. Since there may theoretically be excision from the chromosome by recombining the duplicated homologous regions together, these plasmid integrations are not stable. Conversely, a double recombination event (C) results in insertion of the interposon cassette ( _Pfru > T7-Pol Gm ^R ), while the remainder of the plasmid (including Km ^R ) is lost. Since there is no duplication of homologous DNA regions after this recombination event, no further recombination can occur. (Gm ^R = gentamycin resistance gene, Km ^R = kanamycin resistance gene, Km ^S = kanamycin sensitive, P _fru = fructose-dependent promoter, T7pol = T7 RNA polymerase gene).

: Immunological detection of expression of T7 RNA polymerase and YFP in R. capsulatus (A) Detection of T7 RNA polymerase. (B) Evidence of YFP. (-F / + F = not induced / induced; -O ₂ / + O ₂ = anaerobic / aerobic; + = positive control for T7 RNA polymerase and YFP; P _Km yfp = R. capsulatus B10S (pRhok-yfp); P _T7 yfp = R. capsulatus B10S-T7 (pRhot-yfp)

: Characterization of the new R. capsulatus expression system by heterologous expression of the yellow fluorescent protein YFP. The relative fluorescence was measured at an excitation wavelength of 529 nm and related to a cell density of 1 (OD ₆₆₀ ) and a sample volume of 1 ml. Further explanations are in the text.

: Synthesis and Localization of Bacterio-opsin in R. capsulatus. Bacterio-opsin was detected immunologically using an anti-His ₆ antiserum in cell-free protein extracts of the R. capsulatus strain B10S-T7 with pRhotHi2-bop. By adding the inducer fructose (8 mM), bop expression was induced. By fractionation of the protein extracts were the cell debris and Inclusion body separated both from membranes and membrane components as well as soluble cell components and also immunologically analyzed.

: Total protein distribution in sucrose density gradient. Cell membranes and membrane fractions were separated by ultracentrifugation in sucrose density gradient and analyzed by Bradford analysis for relative total protein content.

: Accumulation of bacterio-opsin in sucrose density gradient. The individual fractions of the sucrose density gradient were analyzed immunologically with respect to bacterio-opsin using an anti-His ₆ antiserum. Bop could only be detected in fraction 29 of the sucrose density gradient.

: In vivo fluorescence labeling of E. coli using the pRhokHi-2-FbFP vectors. The individual FbFP fluorescent markers (BsFbFP, EcFbFP and PpFbFP) were expressed in E. coli DH5α. Subsequently, the in vivo fluorescence was determined (FbFP excitation wavelength: 450 nm, fluorescence detection: 495 nm). The unmodified BsFbFP wild type protein YtvA, which fluoresces only weakly, served as a control.

: In vivo fluorescence labeling of E. coli using the pRhotHi-2-FbFP vectors. The individual FbFP fluorescent markers (BsFbFP, EcFbFP and PpFbFP) were expressed in E. coli BL21 (DE3). Subsequently, the in vivo fluorescence was determined (FbFP excitation wavelength: 450 nm, fluorescence detection: 495 nm). The unmodified BsFbFP wild type protein YtvA, which fluoresces only weakly, served as a control.

: The Rhodobacter expression vectors pRhokHi-2 and pRhotHi-2. Sequences see SEQ ID NO: 1 and 2.

: The Rhodobacter expression vectors pRhokS-2 and pRhotS-2. Sequences see SEQ ID NO: 3 and 4.

: The Rhodobacter expression vectors pRhokHi-6 and pRhotHi-6. Sequences see SEQ ID NO: 5 and 6.

: The Rhodobacter expression vector pRhokHi-2-YFP. Sequence see SEQ ID NO: 7.

: The Rhodobacter expression vector pRhotHi-2-YFP. Sequence see SEQ ID NO: 8.

: The Rhodobacter expression vector pRhokHi-2-bop. Sequence see SEQ ID NO: 10.

: The Rhodobacter expression vector pRhotHi-2-bop. Sequence see SEQ ID NO: 11.

: The Rhodobacter expression vectors pRhokHi-BsFbFP and pRhotHi-BsFbFP. Sequences see SEQ ID NO: 13 and 14.

: The Rhodobacter expression vectors pRhokHi-EcFbFP and pRhotHi-EcFbFP. Sequences see SEQ ID NO: 15 and 16.

: The Rhodobacter expression vectors pRhokHi-2-PpFbFP and pRhotHi-2-PpFbFP. Sequences see SEQ ID NO: 17 and 18.

In order to be able to use the physiological advantages of R. capsulatus for the heterologous expression of any target genes, various broad host range expression vectors were first constructed. In addition, a R. capsulatus T7 expression strain was generated which carries a chromosomal insertion of the T7 RNA polymerase gene, which is under the control of a Rhodobacter-specific promoter.

The functional properties of R. capsulatus expression vectors

To allow expression of heterologous genes in the phototrophic bacterium R. capsulatus, a comprehensive set of expression vectors (pRho series) was constructed. The general properties of these vectors are in shown.

For the heterologous expression of the recombinant target genes, two different promoters are alternatively present on the expression vectors: (1) The P _Km promoter (promoter of the kanamycin resistance gene aphII) allows moderate expression of the target gene (pRhok). Since expression of the kanamycin resistance gene is constitutively successful, no induction of P _Km- dependent expression is necessary. The pRhok vectors are thus ideal for high throughput screening of enzyme or metagenomic banks. (2) The T7 promoter enables inducible overexpression depending on the T7 RNA polymerase (pRhot, suitable for biotechnological production processes).

The vectors were designed so that the respective target genes via the NdeI restriction site and another interface of the polylinker (see clone in areas with gray background). The heterologous gene products can also be fused at the C-terminal end to a His ₆ tag (eg pRhotHi) or StrepII tag (eg pRhotS), which allows easy detection and affinity chromatographic purification (Schmidt & Skerra 2007 Lichty et al., 2005).

The broad host range ori replication origin also allows for direct comparison of protein yields, protein localization, and activity after expression in various Gram-negative bacteria, such as. E. coli. In order to ensure the most flexible use of pRho expression vectors in different expression hosts, the plasmids carry different antibiotic resistance genes. For the His ₆ -day expression vectors, variants with a chloramphenicol or spectinomycin resistance gene are available. The StrepII tag vectors exist so far only as chloramphenicol variants. Table 1 lists the various vectors with the eponymous characteristics and the corresponding vector designation. Tab. 1: The pRho expression vectors Km promoter T7 promoter has Cm resistance Sp resistance Cm resistance Sp resistance His _6-day Strep-tag His _6-day Strep-tag His _6-day Strep-tag His _6-day Strep-tag pRhokHi-2 pRhokS-2 pRhokHi-6 - pRhotHi-2 pRhotS-2 pRhotHi-6 -

Construction of the R. capsulatus expression vectors

In the following section the construction of the R. capsulatus expression vectors is explained in detail.

Construction of the R. capsulatus expression vectors pRhokHi and pRhotHi

For the construction of the pRho vectors, the expression vector pBBR22b (Rosenau and Jaeger, 2004) was used as the backbone. The pBBR22b vector is a hybrid vector consisting of parts of the broad host range vector pBBR1MCS (Kovach et al., 1994) and the E. coli T7 expression vector pET22b (Novagen). This vector was modified for use in R. capsulatus as follows: (i) The vector-localized lacI gene encoding the Lac repressor and responsible for the regulation of the T7 promoter in E. coli was deleted. This should allow LacI-independent expression of the target genes in R. capsulatus. (ii) Since the chloramphenicol resistance gene of pBBR1MCS is not expressed in R. capsulatus, the aphII gene of the Tn5 transposon was inserted into the vector. The expression of this gene confers on the R. capsulatus recipient strain selectable resistance to the antibiotic kanamycin.

In order to carry out the abovementioned modifications of pBBR22b, initially a 1.2 kb EcoRI fragment which completely carries the aphII gene was excised from the vector pBSL15 (Alexeyev et al., 1995). Restriction of the pBBR22b plasmid with Bg / II and ApaI removed most of the lacI gene from this vector. The resulting in the restriction overhanging ends of both DNA fragments were then made up by blending with the T4 DNA polymerase to blunt ends and then ligated together. Due to the unforced cloning, the aphII resistance gene was cloned into the pBBR22b derivative in two different orientations shortly above the T7 promoter: in the first orientation, the reading direction of the aphII gene corresponds to that of the T7 promoter. Although the aphII DNA fragment contains the natural promoter of the gene, but not the transcription terminator, all target genes that are cloned into the polylinker of the new expression vector are constitutively expressed by the aphII promoter. The new 6678bp Rhodobacter expression vector was named pRhokHi 2 denotes. In the second orientation, the aphII gene is transcribed in opposite orientation to the T7 promoter. Therefore, in this case, the expression of a heterologous target gene is only subject to the control of the T7 promoter. The resulting expression vector is named pRhotHi-2. The two recombinant vectors are in shown. For the construction of the spectinomycin resistant derivatives, a 2 kb ΩSp cassette was excised by HindIII restriction from plasmid pHP45Ω (Prentki & Krisch, 1984). The starting plasmids pRhokHi-2 and pRhotHi-2 were then opened with the restriction enzyme SphI, whose recognition sequence is located in the Cm resistance gene. The ligation was carried out here after the Auffüllreaktion the overhanging DNA ends. The two 8.7 kb, Sp-resistant plasmid variants pRhokHi-6 and pRhotHi-6 are in shown.

Construction of the R. capsulatus expression vectors pRhotS-2 and pRhokS-2

Since the purification of enzymes with metal cofactors over a His ₆ affinity day is often problematic, two further pRho vectors were generated which code for a StreptavidinII tag instead of the His tag. The StrepII tag is an eight amino acid peptide (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys), which has a very high intrinsic affinity for streptavidin (Schmidt and Skerra 2007).

To replace the His ₆ tag coding region of the pRhotHi-2 and pRhokHi-2 vectors with a StrepII tag coding sequence, a rapid-change PCR was performed (Wang & Malcom, 1999). The primers were designed so that the homologous primer regions can bind upstream and downstream of the His ₆ coding sequence in the original vectors. In addition, both primers have a region which has the complete sequence for the StrepII tag as well as a recognition sequence for the restriction enzyme PstI (Table 2). The StrepII tag coding sequence was optimally adapted for this purpose to the codon usage of the host organism R. capsulatus.

Tab. 2: Characteristics of the primers for generating the strep-tag vectors pRhotS-2 and pRhokS-2

By means of a polymerase chain reaction, the respective StrepII tag-encoding vector variants pRhotS-2 and pRhokS-2 could then be completely amplified. For the rapid change PCR, the following approaches and PCR programs were used: (1) PCR approaches: component ad 20 μl Final conc. template 0.4 μl Primer 100 pM each 2 μl 0.5 μM Buffer 5 × 4 μl 1 × dNTP mix 10 mM 0.4 μl 200 μM Phusion Pol. 0.2 μl 0.02 U / μl dH ₂ O 10.4 μl

(2) PCR program (PCR cycler: EP gradient from Eppendorf)

The chosen primers and PCR methods are suitable for the generation of both StrepII tag expression vectors, since the crucial parameters, such as the homologous primer sequences, are identical in both cases. The product of the PCR reaction is the respective linearized pRho expression vector, which has a sequence for the streptavidin tag at both ends. To circularize the linear DNA fragments, the PCR products were first treated with the restriction enzyme PstI (the corresponding cleavage sites were generated by the primers at both ends of the PCR product), and then the fragment ends were ligated together. This generated the vectors pRhokS-2 and pRhotS-2 ( ).

Generation of the R. capsulatus expression strain B10S-T7

Furthermore, a special R. capsulatus T7 expression strain was generated. In this expression strain (strain name: R. capsulatus B10S-T7), the T7 RNA polymerase gene, which is under the control of a Rhodobacter-specific fructose-inducible promoter ( _Pfru , Duport et al., 1994), was isolated by homologous recombination in integrates the chromosomally located recA gene of R. capsulatus. The bacterial strain thus has two particular properties which are of crucial importance for heterologous expression: (1) The integration of the T7 RNA polymerase gene into the chromosome has produced a genetically stable expression host which is highly reproducible as well as controllable Expression of phage polymerase allowed. (2) By integrating the T7 RNA polymerase gene into the recA gene, the R. capsulatus strain B10S-T7 has a recA mutation, completely preventing homologous recombination events between expression vectors and host chromosome.

Construction of the mutagenesis vector pRc-T7

To generate a stable R. capsulatus-based T7 expression system, the T7 RNA polymerase gene should be integrated by homologous recombination into the chromosomal recA gene of the bacterium. For this purpose, the plasmid pRc-T7 was constructed as follows: (1) In the vector pIC20R (Marsh et al., 1984) a 1128 bp DNA fragment was first cloned, which is a part of the gene recA from R. capsulatus including entire promoter region. For this, the corresponding DNA fragment was previously amplified specifically with a PCR (primer: recA-up: 5'-G GAATTC CAGCGCGACGGTGGTGAAGG-3 ', recA-down: 5'-G GAATTC CGCATTGCCGCCCGAGGTC-3'). The cloning of the resulting DNA fragment was carried out via the EcoRI interfaces, which flank the PCR product due to the PCR primer used (the EcoRI sites are underlined in the primer sequences mentioned above). The recombinant vector was designated pIC20R-recA '. (2) In parallel, the T7 RNA polymerase gene was PCR amplified so that the PCR product at the 5 'end underlined an NdeI site (primer: Pol1-up: 5'- CATATG AACACGATTAACATCGCT-3', NdeI site ) and at the 3 'end a PstI site (primer: Pol1-low: 5'- CTGCAG TTACGCGAACGCGAAGTCCGA-3'; XhoI site underlined). These interfaces were used to clone the T7 RNA polymerase gene into the vector pFRKII (Duport et al., 1994). This cloning step produced a cassette which, in addition to a selectable resistance marker (Gm resistance gene), carries the T7 RNA polymerase gene, which is now under the control of the R. capsulatus fructose promoter. (3) The 5.7 kb P _fru > T7 polymerase gene GmR cassette thus generated was isolated as a HindIII fragment from the recombinant pFRKII derivative and cloned into the unique HindIII site of pIC20R-recA ', so that both 500 bp of the homologous recA sequence are located upstream and downstream of the cassette. (4) The entire 6.8 kb insert fragment (recA :: P _fru> T7 polymerase-Gm ^R) was last on the flanking EcoRI Cloned into the mobilizable suicide vector pSUP401 (Simon et al., 1983). The resulting mutagenesis construct was named pRc-T7 and is in shown.

Insertion of the T7 RNA polymerase gene into the R. capsulatus chromosome

To generate the R. capsulatus T7 expression strain B10S-T7, the plasmid pRc-T7 was first transferred to the R. capsulatus wild-type strain B10S. Subsequently, the T7 RNA polymerase gene was stably integrated into the chromosome by means of a double recombination event via the homologous DNA segments of the recA gene ( ).

Plasmid integration ( ) was subsequently isolated from the stable interposon insertion ( ) based on the detectable antibiotic resistance of the produced Rhodobacter clones:
In a single crossover, the entire plasmid pRc-T7 is integrated into the chromosomal gene recA. In this way, both the gentamicin resistance gene and the kanamycin resistance gene enter the chromosome of R. capsulatus B10S. However, the arrangement of the individual elements of the plasmid differs depending on the recombination site. If, on the other hand, a recombination event occurs between the homologous regions of both possible recombination sites, only the interposon cassette with the Gm resistance gene is inserted into the chromosomal recA gene. The rest of the plasmid, including kanamycin resistance, is lost. The recombinant R. capsulatus strains identified as putative positive R. capsulatus B10S-T7 strains by the antibiogram were then further characterized. By means of various PCR detection methods, the specific integration of the T7 polymerase gene into the chromosomally localized recA gene has been demonstrated (data not shown). The resulting R. capsulatus T7 expression strain was subsequently characterized functionally.

Characterization of the T7 expression strain R. capsulatus B10S-T7

Heterologous expression of a cytoplasmic protein in R. capsulatus

The functionality of the R. capsulatus T7 expression system was tested in two ways: first, it was determined by means of immunological detection methods whether the T7 RNA polymerase could be expressed fructose-dependent in the R. capsulatus strain B10S-T7 ( ). On the other hand, the functionality of the expression vector pRhotHi-2 was checked by cloning the gene of the "yellow fluorescent protein" (YFP) into the Rhodobacter expression vector and then using the T7 RNA polymerase-dependent expression of the YFP gene immunological methods qualitative ( ) and by the specific YFP fluorescence quantitatively ( ) was detected.

In addition, the expression level of the R. capsulatus T7 expression system was compared with that of the P _Km- dependent system (pRhok vectors). In order to determine whether oxygen exerts an influence on the R. capsulatus expression system, the cultivation of the different R. capsulatus expression strains was carried out under both aerobic and anaerobic conditions ( and ).

The results can be summarized as follows: (i) The YFP-encoding target gene can be probed with both the P _Km and the T7 expression vectors pRhokHi-2 and pRhotHi-2 under phototrophic growth conditions ( , Lane 2-6; , black bars) and under aerobic heterotrophic conditions ( , Lane 9-13; gray bars) are expressed. In the represented YFP fluorescence is a direct measure of the accumulation of the fluorescent reporter. By the P _T7- dependent expression of the YFP gene, a two to three times higher expression rate could be achieved. (ii) The T7 RNA polymerase and the reporter protein YFP accumulate in the R. capsulatus T7 expression strain only under inducing conditions ( Lanes 4, 6, 11 and 13; + I) in significant amounts, whereas the P _Km- dependent expression of YFP is constitutive, as expected, and thus without the addition of an inducer.

(iii) Complete induction of T7 polymerase-dependent reporter gene expression can be achieved by adding 8 mM fructose (data not shown). (iv) Two to four hours after induction, maximum expression level is reached in R. capsulatus (data not shown).

Heterologous expression of a membrane protein in R. capsulatus

To check the extent to which the R. capsulatus Expression Toolbox can be used to express heterologous membrane proteins and insert them under phototrophic growth conditions into the ICM of the The bop gene (encoded for the membrane-bound bacterio-opsin with seven transmembrane helices) from the archaea Halobacterium salinarum (Dunn et al., 1981) was cloned into the vector pRhotHi-2 and subsequently transferred into the R. capsulatus expression strain B10S-T7 ,

To test whether R. capsulatus expresses and accumulates bacterio-opsin under phototrophic culture conditions, cell-free protein extracts of strain R. capsulatus B10S-T7 were generated with the plasmid-encoded bop gene (pRhotHi-2-bop). The detection of the Bop protein was carried out using an antiserum (anti-His ₆ antibody, Fermentas GmbH, St. Leon-Roth Germany) by Western blot analysis ( ).

This in The result of this study illustrates that bacteriopsin in R. capsulatus only accumulates B10S-T7 when a suitable inducer has been added to the medium (distinct signals with a molecular weight of about 30 kDa in lane 2 and 3). The figure also shows that Bop is mainly detectable in the cell membrane and in membrane components (lane 3). Less bacterio-opsin accumulates in inclusion bodies (lane 2). No bop was detectable in the soluble cell constituents (lane 4).

For the isolation of membrane vesicles from the membrane fraction ( , Lane 3), an aliquot was fractionated by sucrose density gradient centrifugation. For this, a continuous sucrose gradient of 10 to 60% sucrose (w / w) was prepared and ultracentrifuged with cell-free membrane components for 17 hours at 175000 g. Subsequently, fractions of 1 ml each were taken. In these fractions, the protein content was determined by means of Bradford analysis and the sucrose content in the refractometer. This in The result of these investigations shows the highest total protein content in membrane fractions 28 to 33 and a significantly lower total protein content in fractions 2 to 5.

The results thus clearly demonstrate that (i) heterologous membrane proteins can be expressed using the new Rhodobacter Expression Toolbox. (ii) The membrane protein accumulated in the cells is integrated into the intracytoplasmic membrane to a large extent upon phototrophic culture of the cells and can be (iii) identified by sucrose density gradient centrifugation in defined membrane fractions.

Generation of broad-host-range vectors for in vivo fluorescence labeling of Gram-negative bacteria based on the pRhot / k vectors

In vivo fluorescent reporters are used in molecular biology for a wide range of different applications. They can be used to study regulatory processes as well as to analyze the localization, interaction and folding of proteins. In addition, fluorescent reporters are used to mark living cells so that they can be identified and localized with simple methods. Based on the newly developed pRho expression vectors, which have a broad host range and thus replicate in different Gram-negative bacteria, new fluorescence labeling vectors were generated. For this purpose, three different genes coding for FMN-based fluorescent proteins (FbFP) were cloned into the expression vectors pRhokHi-2 and pRhotHi-2. In contrast to conventional GFP family of fluorescent proteins, FbFPs have the advantage that they can be used universally in aerobic and anaerobic biological systems (Drepper et al., 2007). After generation of the recombinant pRho vectors (details are described in the Materials & Methods section), the fluorescent proteins BsFbFP, PpFbFP and EcFbFP were expressed in E. coli and subsequently the in vivo fluorescence was determined on the Luminescence Spectrometer LS50B fluorescence photometer. Based on the specific FbFP fluorescence, it was shown that both the PKm-dependent ( ) as well as the PT7-dependent ( ) Expression of FbFP proteins leads to detectable in vivo fluorescence.

The functionality of the fluorescent labeling vectors could also be detected in the Gram-negative bacteria Pseudomonas putida and R. capsulatus using the example of pRhokHi-2-PpFbFP and pRhotHi-2-PpFbFP (data not shown).

Construction of the vectors pRhok / t-YFP, pRhok / t-bop and pRhok / t-FbFP

Construction of the YFP expression vectors pRhokHi-2-YFP and pRhotHi-2-YFP

For the synthesis of P _T7 and P _{aph II} -dependent YFP-pRho expression vectors, the gene YFP was amplified by means of PCR with specific oligonucleotide starter molecules (YFPUP: 5'-GGA ATT CCA TAT GGT GAG CAA GGG CGA GGA GCT-3 ', _Tm = 73 ° C; YFPDWN: 5'-CGG GAT CCT TAC TTG TAC AGC TCG TCC ATG C-3 ', _Tm = 69 ° C; MWG Biotech AG, Ebersberg, Germany). Through the oligonucleotide starter molecules, an NdeI restriction site was added to the PCR product at the 5 'end and a BamHI restriction site at the 3' end. The sequence of the NdeI site simultaneously contains the start codon AUG of the YFP gene. The DNA template used for YFP was the recombinant plasmid pFF19-EYFP (Timmermans et al., 1990), which codes for YFP, inter alia. The flanking restriction sites allowed the recloning in the context of further work.

Previously, however, the amplified DNA fragment was cloned blunt-end into the SmaI restriction site of the sequencing vector pSVB10. The E. coli strain DH5α was then transformed with the resulting recombinant plasmids pSVB10 * IYFP and pSVB10 * IIYFP and incubated overnight at 37 ° C. The cloning success was confirmed by suitable test restrictions (data not shown) and the DNA sequence of the cloned fragment was checked by sequencing (Sequiserve GmbH, Vatersteffen, Germany).

After successful sequencing, the YFP reporter gene was excised with the restriction endonucleases NdeI and BamHI in a double restriction from the pSVB10 derivatives and cloned into the NdeI and BamHI restriction sites of the P _Km expression vector and the P _T7 expression vector, respectively. The resulting recombinant plasmids are named pRhokHi-2-YFP and pRhotHi-2-YFP. The protein YFP synthesized by means of the expression plasmids pRhokHi-2-YFP and pRhotHi-2-YFP consists of 239 amino acids and carries no C-terminal hexahistidine peptide.

Construction of the bop expression vectors pRhokHi-2-bop and pRhotHi-2-bop

In order to enable constitutive (aphII promoter-dependent) and T7 RNA polymerase-dependent synthesis of bacteriophysin (Bop), the corresponding bop gene was transformed into the expression plasmids pRhokHi-2 (constitutive expression) and pRhotHi-2 (T7 RNA polymerase-dependent expression) cloned. For this purpose, bop was amplified by polymerase chain reaction (PCR) with specific oligonucleotide starter molecules (5'-TTT CAT ATG TTG GAG TTA TTG CCA ACA GCA GTG GAG-3 ';5'-AAA CTC GAG GTC GCT GGT CGC GGC CGC-3'; MWG Biotech AG, Ebersberg, Germany). Through the oligonucleotide starter molecules, an NdeI restriction site was added to the PCR product at the 5 'end and an XhoI restriction site at the 3' end. The sequence of the NdeI site simultaneously contains the start codon AUG of the bop gene. As the DNA template for the bop gene, the recombinant plasmid PAWG / P served (T5) His ₁₀ bop (Dyczmons, NG, 2006).

After successful PCR, the blunt-ended amplified DNA fragment was cloned into the SmaI restriction site of the sequencing vector pSVB10. Subsequently, the E. coli strain DH5α was transformed with the resulting recombinant plasmids pSVB10-bopI and pSVB10-bopII and incubated overnight at 37 ° C. The cloning success was checked by suitable test restrictions (data not shown) and the DNA sequence of the cloned fragment was confirmed by sequencing (Sequiserve GmbH, Vaterstetten, Germany).

After successful sequencing, the bop gene was excised in a double restriction from the pSVB10 derivatives using the restriction endonucleases NdeI and XhoI and inserted into the corresponding cleavage sites of the aphII promoter-dependent expression vector pRhokHi-2 and the T7 RNA polymerase-dependent expression vector pRhotHi -2 cloned. The resulting recombinant plasmids are named pRhokHi-2-bop and pRhotHi-2-bop, respectively.

The recombinantly synthesized bacterio-opsin protein pRhokHi-2-bop and pRhotHi-2-bop consists of 270 amino acids (including hexahistidine peptide) and is synthesized as a preprotein. The amino acids 1 to 13 serve as so-called leader sequence, which are removed by post-translational processing.

In contrast, the wild type preprotein from Halobacterium salinarum consists of 261 amino acids, whose 13 N-terminal amino acids are removed by post-translational processing. The wild-type protein bacterio-opsin thus consists of 248 amino acids.

Construction of the FbFP expression vectors pRhokHi-2-FbFP and pRhotHi-2-FbFP

To enable constitutive (aphII promoter-dependent) and T7 RNA polymerase-dependent synthesis of the in vivo fluorescent markers BsFbFP, PpFbFP and EcFbFP, the corresponding reporter genes were transformed into the expression plasmids pRhokHi-2 (constitutive expression) and pRhotHi-2 (T7 RNA polymerase-dependent expression) cloned. The BsFbFP and PpFbFP coding genes were present in the vector pET28, whereas the EcFbFP gene had been previously cloned into the pBluescript vector (Drepper et al., 2007). The genes EcFbFP (409 bp) and PpFbFP (449 bp) were isolated with NdeI and XhoI from the original vectors and cloned into the pRho vectors via the corresponding cleavage sites. Since the BsFbFP gene (790 bp) has an additional XhoI site, the third fluorescent reporter gene was cloned into the broad host range expression vectors via the NdeI and BamHI sites.

Ligation of DNA fragments

For the ligation of DNA fragments and correspondingly hydrolyzed vector DNA, T4 DNA ligase (Fermentas) was used. The enzyme was used according to the manufacturer's instructions in the buffer supplied and incubated either at RT for 2 h or at 16 ° C. overnight. In a reaction volume of 10 to 20 μl, vector DNA and the DNA to be inserted were used in a ratio of 1: 3 to 1: 5.

After the reaction, the ligase was inactivated by incubation at 65 ° C for 10 minutes.

transformation

Production of transformation-competent cells

After the E. coli strain to be transformed was grown overnight in 5 ml of LB medium to a stationary growth phase, 1 ml of the culture was inoculated into 100 ml of LB medium previously provided with 2 ml of Mg ²⁺ mix. At 37 ° C, the culture was incubated until it reached an OD ₅₈₀ of 0.4 to 0.6. From that moment on, the cells had to be kept at 4 ° C. Mg ²⁺ mix MgCl ₂ 500 mM MgSO4 500 mM

The cells were pelleted for 20 minutes at 7,000 rpm in a refrigerated centrifuge and then resuspended in 50 ml of TMF buffer (precooled to 4 ° C). Transformation buffer (TMF) CaCl2 100 mM RbCl2 50 mM MnCl2 40 mM

After incubation on ice for 30-60 minutes, the cells were again centrifuged for 20 min at 7,000 rpm and 4 ° C, after which they were resuspended in 10 ml TMF + 20% (v / v) glycerol (4 ° C).

200 μl aliquots of culture were prepared and stored at -80 ° C.

Heat shock transformation of E. coli cells with plasmid DNA

200 μl of transformation-competent cells were mixed with 100 μl of ice-cold TMF buffer and a previously prepared ligation batch or 1-5 μl of isolated plasmid DNA). As a negative control, an approach without addition of DNA was always treated in parallel.

There was at least a 30-minute incubation on ice, after which the cells were exposed to heat shock (42 ° C for 1.5 to 2.5 min). Immediately after the heat shock, the batch was mixed with 700 μl of LB medium (without antibiotic). The cells were then dependent on that by the plasmid mediated antibiotic resistance for several hours (usually 2 h, with Gm resistance for 4 h) incubated at 37 ° C on a breeding roller for phenic expression. After this time, the culture was centrifuged for 5 min at 13,000 rpm, 800 ul supernatant removed and resuspended the cells in the remaining supernatant. This suspension was then plated on selective LB agar.

Preparation of plasmid DNA

The preparation of plasmid DNA was carried out according to the principle of alkaline lysis. In the absence of time, the Perfectprep Plasmid Mini Kit from Eppendorf was used instead of the method described below; In each case, the HiSpeed Plasmid Midi Kit from QIAGEN was used to prepare larger amounts of plasmid DNA.

1.5 to 3 ml of an E. coli overnight culture were pelleted by centrifugation at 13,000 rpm for 5 minutes and then resuspended in 100 μl of solution 1. This was followed by 5 min incubation on ice. Then 200 .mu.l of solution II were added, the mixture was inverted until complete lysis of the bacteria and then incubated again on ice for 5 min. Then the mixture was mixed carefully with 150 .mu.l of solution III and incubated on ice for 10 min. Solution I (pH 8.0) glucose 50 mM Tris-HCl 25 mM EDTA 10mM Solution II NaOH 0.2 M SDS 1% (w / v) Solution III (pH 4.8) 5 M potassium acetate 60 ml acetic acid 8.5 ml

By centrifugation at 13,000 rpm and 4 ° C. for 10 minutes, denatured chromosomal DNA and proteins as well as cell debris were sedimented. The supernatant was transferred to a new Eppendorf tube and the same amount of isopropanol (pre-cooled to 4 ° C) was added. This was followed by a 10 minute incubation on ice, after which centrifugation was again carried out at 13,000 rpm and 4 ° C. for 10 minutes.

The supernatant was discarded and the pellet washed three times with 1 ml of 70% ethanol (4 ° C). After the last washing step, as much of the ethanol residue as possible was removed and the pellet was dried for half an hour at 37 ° C., after which it was resuspended in 35 μl of TE buffer + RNase A (from Sigma, final concentration 20 μg / ml). TE buffer (pH 8.0) Tris-HCl 10mM EDTA 1 mm

This was followed by a 15 minute incubation at 37 ° C. The isolated plasmid DNA was stored at -20 ° C.

Methods for the characterization of DNA

Hydrolytic Cleavage of DNA by Restriction Endonucleases

For the hydrolytic cleavage of DNA by restriction endonucleases (restriction), 1 enzyme unit (unit) per μg of DNA was used by the respective restriction enzymes (Fermentas). To optimal To ensure reaction conditions, the buffers recommended by the manufacturer for the respective enzymes (or enzyme combinations in double digestion) were used. The cleavage was carried out at the temperature indicated for the enzyme for 1 to 2 hours. The inactivation of the enzymes after the reaction was carried out by the addition of 5 ul DNA sample buffer.

agarose gel electrophoresis

Agarose gel electrophoresis was used to determine the size of hydrolyzed DNA molecules, to estimate the quantity of isolated DNA, and to prepare DNA fragments for preparative isolation.

In this process, a separation of different sized DNA molecules occurs. Due to the negative charge of the sugar-phosphate backbone of the DNA migrates to the cathode when an electrical voltage, wherein conformation and molecular weight for a different migration speed of different sized DNA fragments provide. Further influence on the migration speed have the applied voltage and the concentration of the agarose gel.

To prepare the agarose gel, agarose (to a final concentration of 0.6 to 1% as needed) was boiled in 0.5X TBE buffer. TBE buffer 5-fold (pH 8.3): Tris-HCl 89 mM borate 89 mM EDTA 2.5mM

This solution was poured while hot into a gel chamber, a gel comb (with sufficiently large teeth depending on the amount of DNA sample to be applied) used and ethidium bromide (0.5 ug per ml of agarose TBE solution) was added. Ethidium bromide intercalates into the helix of double-stranded DNA and emits visible light upon exposure to UV light, indicating the location of the DNA bands.

The samples were spiked with 1/5 volume DNA sample buffer. DNA sample buffer (5x): EDTA 100 mM glycerol 43% (v / v) BPB 0.05% (w / v)

After polymerization of the gel, the gel comb was removed, the gel placed in a gel electrophoresis chamber and overlaid with 0.5X TBE buffer. The DNA samples were filled in the gel pockets. In addition, a DNA marker (Gibco 1 kb DNA Ladder from Invitrogen) was applied to determine the size of the bands after gel run. Then a voltage of 135 V was applied until the desired separation was achieved (usually 15 to 30 minutes depending on the concentration of the gel).

Finally, the gel was photographed with the Eagle-Eye II camera (Stratagene). The DNA was made visible by irradiation with UV light (λ = 254-366 nm), through which the ethidium bromide bound to the DNA emits light in the visible range (λ = 590 nm).

DNA concentration determination

To determine the concentration of DNA, 5 μl of the prepared DNA sample (s) were applied together with the "O'GeneRuler 1 kb ladder" DNA marker (Fermentas) on an agarose gel. The strength of the band in the DNA sample was compared to the band strengths in the DNA marker and the DNA concentration in the samples was estimated from the quantities given by the manufacturer.

Elution of DNA fragments from agarose gels

After the DNA sample was hydrolyzed with restriction enzymes and separated on an agarose gel, the desired DNA band was cut out of the gel with a scalpel and the DNA was eluted by means of the Perfectprep Gel Cleanup Kit from Eppendorf.

Amplification of DNA by Polymerase Chain Reaction (PCR) with Turbo-Pfu Polymerase

In polymerase chain reaction (PCR) specific DNA regions are amplified and enriched in vitro. Starting from a template (the DNA with the region to be amplified) and for the two ends of the DNA region specific oligonucleotides (so-called starter molecules or primers) synthesized a thermostable DNA-dependent DNA polymerase the desired DNA region.

For this purpose, first the template is denatured, whereupon the primers can hybridize to their complementary sequences. In the following elongation, the DNA polymerase extends the oligoprimers in the 5'g3 'direction, thus completing the missing DNA strand. Since this process occurs on both strands of template DNA, the DNA doubles with each cycle. Usually, 30 to 40 cycles are run through.

The turbo-Pfu polymerase was used to carry out the PCR. This polymerase has a 3'g5 'exonuclease activity (a so-called "proof-reading function"), thereby minimizing the risk of mutations of the amplified DNA by misincorporated nucleotides. PCR approach (50 μl): A. least. 36.1 μl Template DNA 1.0 μl Upper Primer 1.0 μl Lower primer 1.0 μl dNTPs 0.5 μl PCR buffer 5.0 μl DMSO 5.0 μl Turbo Pfu polymerase 0.4 μl

Turbo-Pfu polymerase, PCR buffer and dNTPs were from the Turbo-Pfu polymerase kit (Stratagene). The reactions were carried out in the eppendorf Eppendorf PCR machine ep gradient S.

The following program was used: Initial denaturation 98 ° C 10 min denaturation 98 ° C 1 min hybridization su 1 min elongation 72 ° C su Terminal elongation 72 ° C 10 min

The steps denaturation, hybridization and elongation were repeated in 30 cycles.

The hybridization temperature depended on the primers used; if the optimum temperature was not known, a temperature gradient was applied if necessary. The duration of the elongation was dependent on the length of the DNA segment to be amplified (1 min per 2 kb).

Analysis of the amplified DNA was performed by plating 5 μl of each PCR on an agarose gel.

Generation of StrepII tag variants by means of Quick change PCR

The vectors pRhokHi-2 and pRhotHi-2 were used with the described primers under the conditions described in a polymerase chain reaction as a template to the linear precursors of To generate vectors pRhokS-2 and pRhotS-2 (6723 bp). The samples of this polymerase chain reaction were treated with the enzyme DpnI. (This is a restriction enzyme whose target sequence is a statistically common 4-mer (GATC), but the enzyme has the ability to recognize this target sequence only if the adenosine has a methyl group (DNA methylation), this is exclusive in the case of the DNA used as template). By this step one loses the original and unmodified variant (pRhokHi-2 and pRhotHi-2) of the vectors.

In a further step, the vectors were treated with the restriction enzyme PstI, whereby the recognition sequence of this enzyme was introduced into the vector via the primer overhang. The samples were then separated by agarose gel electrophoresis and the corresponding DNA fragment (6690 bp) purified. This was followed by transformation with competent E. coli DH5α cells. These cells were plated for selection on LB agar plates with the antibiotic kanamycin (50 μg / ml).

Single colonies were picked from these plates and 100 ml of LB medium were inoculated with the antibiotic kanamycin (50 μg / ml). After culturing for 24 h at 37 ° C. and 120 rpm on a shaker, the cells were harvested and the vectors pRhokS-2 and pRhotS-2 were isolated therefrom.

bacterial growth

antibiotics

All antibiotics were filter sterilized before use (Millipore membrane filter, pore diameter 0.2 μm). The antibiotic aliquots were stored at -20 ° C. Tab. 3 Antibiotics used Antibiotic (abbreviation) Concentration in LB medium Concentration in PY medium Concentration in RCV medium source Ampicillin (amp) 100 μg / ml - - Serva Kanamycin (Km) 50 μg / ml 25 μg / ml 25 μg / ml Serva Streptomycin (Sm) 50 μg / ml 200 μg / ml 200 μg / ml GERBU Biotechnik GmbH Gentamicin (Gm) 10 μg / ml 4 μg / ml 4 μg / ml Serva Spectinomycin (Sp) 100 μg / ml 10 μg / ml 10 μg / ml calbiochem

Cultivation of E. coli

Cultivation of E. coli was carried out on LB agar plates or in LB liquid medium at 37 ° C. For the selection or stabilization of plasmids, an adequate antibiotic was optionally added. The incubation period was at least 12 hours. LB full medium (liquid) tryptone 10.0 g yeast extract 5.0 g NaCl 5.0 g A. least. ad 1000 ml Autoclaving (20 min, 200 kPa, 121 ° C) LB agar: tryptone 10.0 g yeast extract 5.0 g NaCl 5.0 g agar 15.0 g LB medium ad 1000 ml Autoclaving (20 min, 200 kPa, 121 ° C)

Liquid cultures were inoculated either from single colonial stock or transformation plates or from a 1/500 final volume freezing or preculture, and then either in test tubes on a breeding roller (up to a culture volume of 5 ml) or in Erlenmeyer flasks (culture volume at most 1 / 10th of the vessel volume) on an incubation shaker at 130 rpm.

Cultivation of R. capsulatus

The phototrophic cultivation of R. capsulatus was carried out on solid medium (PY agar) or in RCV liquid medium. Optionally, a suitable selection pressure was applied. The cultures were incubated for two to three days at 30 ° C. PY agar: Bacto peptone 10.0 g yeast extract 0.5 g agar 15.0 g A. least. Ad 1000 ml Autoclaving (20 min, 200 kPa, 121 ° C) Additions after autoclaving: 1M MgCl ₂ 2 ml 1 M CaCl ₂ 2 ml 0.5% FeSO ₄ -HCl (see below) 2.4 ml RCV minimal medium: 10% DL malate (pH 6.8) 40.0 ml 1% EDTA 2.0 ml 20% MgSO4 1.0 ml Trace element solution (see below) 1.0 ml 7.5% CaCl 2 1.0 ml 0.5% FeSO4-HCl (see below) 2.4 ml 0.1% thiamine 1.0 ml A. least. ad 1000 ml Autoclaving (20 min, 200 kPa, 121 ° C) Addition after autoclaving (to 1000 ml RCV): 10% (NH ₄ ) ₂ SO ₄ 10.0 ml 1 M phosphate buffer, pH 6.8 (see below) 9.6 ml 0.5% FeSO ₄ -HCl: FeSO ₄ 0.5% (w / v) 32% hydrochloric acid 1 ml A. least. Ad 200 ml Autoclaving (20 min, 200 kPa, 121 ° C) phosphate buffer (pH 6.8): KH ₂ PO ₄ 81.3 g K ₂ HPO ₄ 78.7 g A. least ad 500 ml Autoclaving (20 min, 200 kPa, 121 ° C) trace element solution for RCV: MnSO4 × 1H2O 0.4 g H3BO3 0.7 g Cu (NO 3) 2 × 3H 2 O 0.01 g ZnSO4 × 7H2O 0.06 g Na2 MoO4 × 2H2O 0.02 g A. least. ad 250 ml Autoclaving (20 min, 200 kPa, 121 ° C)

Anaerobic, photoheterotrophic cultivation:

For growing on PY agar plates, special anaerobic incubation pots were used using the Gas-Pack Anaerobic System (by BBL), which provides an anaerobic environment.

When grown in liquid culture, the culture volume was placed in vessels which could be sealed airtight with a septum. After inoculation, the vessel was sealed and then injected into the vessel with an injection needle through this septum for 5 to 10 minutes, while air was allowed to escape through a second injection needle to avoid overpressure.

The incubation was carried out in bright light (six bulbs a 60 W, equivalent to 2500 lux).

Aerobic, heterotrophic cultivation:

Aerobic cultivation on PY agar plates was carried out by storing the plates with the bacteria in the dark.

For growth in liquid medium, 10 ml of RCV medium was added to 100 ml Erlenmeyer shake flasks. The cultures were then incubated on an incubation shaker (130 rpm) in the dark.

Cultivation of R. capsulatus expression cultures

For the expression of heterologous genes, the expression strains of R. capsulatus were first inoculated from preferably fresh stock plates in a suitable culture volume (depending on the number of later main cultures, usually 1-2 ml per planned main culture) RCV liquid medium (optionally under selection pressure) and incubated under anaerobic conditions (even if later major cultures should be attracted aerobically).

From these precultures, major cultures (test cultures) in the desired volume of the same medium were inoculated to an OD ₆₆₀ of 0.2. The induction of R. capsulatus B10S-T7, if necessary, was done with 8 mM fructose. The main cultures were incubated as needed under anaerobic or aerobic growth conditions.

Storage of bacterial strains

The storage of the bacteria for short periods (maximum two weeks) was carried out on agar plates of the appropriate medium at 4 ° C (E. coli) or room temperature (R. capsulatus).

For longer term storage, freezing cultures were prepared:

Freezing cultures of E. coli:

1.3 ml of cell suspension from an overnight culture were mixed with 10 ul DMSO and frozen at -20 ° C.

Freezing cultures of R. capsulatus:

R. capsulatus freezing cultures were prepared from anaerobic PY agar plates by first resuspending a larger amount of cell material in 3 ml of RCV medium. 1.5 ml of the cell suspension were centrifuged at room temperature for 10 min at 13,000 rpm and the pellet subsequently resuspended in 1 ml of PYG buffer. This suspension was distributed to two Eppendorf tubes and the volume of the culture determined. Both cultures were mixed 1: 1 with 87% glycerol. Storage took place at -20 ° C. PYG-buffer: Bacto peptone 3.0 g yeast extract 3.0 g 20% MgCl2 2.5 ml 7.5% CaCl 2 4.0 ml A. least. ad 1000 ml Autoclaving (20 min, 200 kPa, 121 ° C)

Determination of optical density

The determination of the optical density (OD) was carried out after suitable dilution in distilled H ₂ O by photometric measurement with a spectrophotometer. The zero value was also set with H ₂ O.

For E. coli, a wavelength of 580 nm was used. An OD ₅₈₀ of 1 corresponds to approximately 2 x 10 ⁹ cells per ml of culture. In R. capsulatus the measurement was at 660 nm; An OD ₆₆₀ of 1 corresponds to approximately 3 × 10 ⁸ cells per ml of culture volume.

Conjugative transfer of mobilizable plasmids from E. coli to R. capsulatus

Plasmids possessing a mobilization cassette (MOB site) can be transferred from the conjugation competent E. coli strain S17-1 to R. capsulatus. This is done by the method of filter crossing.

The recipient R. capsulatus was transferred from a fresh phototrophically grown stock plate into 5 ml RCV medium and resuspended. From this suspension, 1 ml was placed in a sterile 2 ml Eppendorf tube. Several single colonies of the donor cultured fresh on selective LB agar overnight are resuspended in 1 ml of PY-Fe, with particular care being taken to avoid damaging donor pili. From the donor suspension, 0.5 ml was added to the recipient suspension. PY-F (liquid): Bacto peptone 10.0 g yeast extract 0.5 g A. least. Ad 1000 ml Additions after autoclaving: 1M MgCl ₂ 2 ml 1 M CaCl ₂ 2 ml

The donor-recipient mixture was centrifuged for 10 min at 13,000 rpm and room temperature, and the supernatant was removed to a small residue in which the pellet was resuspended. Then, the cell suspension was plated on a nitrocellulose filter (cellulose acetate, 0.2 μm, 25 mm diameter, Whatman Schleicher & Schuell) placed on a PY agar plate without antibiotic.

After incubation overnight at 30 ° C in the dark, the filters were resuspended in 1 ml of RCV medium without antibiotic. From this suspension, 200 μl was plated on PY agar. The incubation was carried out under phototrophic conditions.

Protein Biochemical Methods & Fluorescence Measurements

Protein biochemical methods

Total protein isolation from R. capsulatus to detect T7 polymerase and YFP

After culturing the expression cultures, the OD _{660 was} determined and 1 OD cells were harvested by centrifugation at 13,000 rpm for 10 minutes.

The pellet of each culture was first washed in 0.125 M Tris-HCl (pH 6.8) and resuspended after a new centrifugation step in 100 μl SDS sample buffer. This mixture was boiled for 12 min at 99 ° C and 1000 rpm. SDS sample buffer 0.5 M Tris-HCl pH 6.8 1.25 ml 87% glycerol 1.2 ml 10% SDS 3.0 ml β-mercaptoethanol 0.5 ml 0.5% BPB 0.6 ml H2O 2.65 ml

If the samples are not to be immediately applied to an SDS acrylamide gel, they can be stored at -20 ° C; in this case, the samples had to be re-boiled for 10 minutes after thawing.

Denaturing SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

During denaturing protein cell electrophoresis, SDS binds to the proteins and, through its negative charge, abolishes the intrinsic charge of the proteins. As a result, the SDS-protein complexes separate only according to their molecular size, but not according to their charge.

The SDS-PAGE was performed either with NUPAGE 4-12% Bis-Tris ready gels from Invitrogen (electrophoresis apparatus: XCell SureLock ^™ Electrophoresis Cell) with a transit time of 35 min at 200 V, or in a BioRad mini-protein gel chamber.

In the latter case, the separating gel was poured after assembly of the gel chamber, and this immediately overlaid with the collecting gel. Thanks to the glycerine in the separating gel solution, a two-phase system was formed, and the two gels barely mingled. A gel comb with sufficiently large teeth was used for the planned protein samples, and the gel was left to polymerize for about 30 minutes. 12% SDS separating gel (quantities for 2 mini-gels) acrylamide 4 ml glycerin 3.4 ml 4 × separation gel buffer (1.5 M Tris-HCl, pH 8.8) 2.5 ml 10% SDS 0.1 ml 10% APS 50 μl TEMED 5 μl 5% SDS collection gel (amounts for 2 mini-gels): acrylamide 0.83 ml 4 × collecting gel buffer (0.5 M Tris-HCl, pH 6.8) 1.25 ml 10% SDS 50 μl A. least 2.9 ml 10% APS 50 μl TEMED 5 μl

After polymerization, the gel was placed in the electrophoresis chamber (Mini-Protean Gel Chamber II ^™ from Biorad) and filled with 1X SDS running buffer. 10 × SDS running buffer (pH 8.3): Tris-HCl 30.3 g glycine 144.0 g SDS 10.0 g H2O ad 1000 ml

After the gel comb was removed, the protein samples were applied to the gel. In addition, a protein marker (Page Ruler ^™ Prestained Protein Ladder from Fermentas) was applied to later estimate the size of the proteins detected. The electrophoresis was initially at 100 V until the protein samples had run into the separating gel, but then with 200 V until the blue marker had reached the lower gel edge.

Transfer of Proteins to PVDF Membranes (Western Blot)

After separation of proteins by SDS-PAGE, the proteins were transferred to a PVDF membrane (Biorad) by Western blotting.

When self-cast gels were used, the membrane was first equilibrated in methanol for 1 min, then in distilled water for 5 min, and finally in 1x Dunn carbonate buffer for 10 min. 10 × Dunn carbonate buffer (pH 9.45): NaHCO3 8.4 g Na2CO3 3.18 g H2O ad 1000 ml 1 × Dunn carbonate buffer (prepare shortly before use): 10 × Dunn carbonate buffer 100 ml methanol 200 ml H2O 700 ml

To transfer the proteins, the blot was built up in a transfer apparatus by stacking the various elements in the following sequence as free of air bubbles as possible: sponge, 2 × Whatman paper, gel, membrane, 2 × Whatman paper, sponge. A maximum of two blots could be housed in a transfer chamber ((mini) trans-blot electrophoretic transfer cell from Biorad).

The transfer took place at 150 mA for 15 min and at constant current of 300 mA in a 1-fold Dunn carbonate buffer for another 20 min.

When using NUPAGE ready gels, the membrane was also equilibrated in methanol for 1 min and then loaded in transfer buffer (NUPAGE). The gels were also placed in transfer buffer after gel run and opening the plastic wrap. The blot was then set up according to the manufacturer's instructions in the XCell SureLock ^TM electrophoresis chamber. The transfer was at 25 V for 1 h.

Immunodetection of proteins

After transfer of the proteins to a PVDF membrane, it was blocked overnight at 4 ° C in TEST + 3% milk powder to saturate free binding sites of the membrane. TBST buffer Tris 50 mM NaCl 150 mM MgCl2 1 mm Tween 20 0.2%

The following day, the membrane was incubated for 1 hour at 30 ° C with gentle shaking in TEST, which was mixed with the appropriate first antibody in a suitable dilution (see below). This was followed by 2 washes with TEST for 30 min each. Thereafter, a solution of TEST with the matching to the first antibody second antibody in a suitable dilution (see below) was added to the membranes. Thereafter, several washes in TEST (30 min, 15 min, 4 x 5 min) followed to remove unbound antibody. Finally, the chemiluminescent detection could be performed. Detection was either with Amersham's Enhanced Chemoluminescence (ECL) Western Blotting Detection System according to the manufacturer's instructions or with "home-made" ECL solutions.

If the detection was carried out with self-applied ECL solutions, 0.3 μl of 35% hydrogen peroxide were initially introduced for each membrane in a 2 ml Eppendorf tube. For the detection, 1 ml of solution A (50 mg of luminol in 500 ml of 100 mM Tris / HCl pH 8.6) and 100 μl of solution B (11 mg of p-coumaric acid in 10 ml of DMSO) were added to the hydrogen peroxide and mixed by inverting. The solution was then applied to the membrane and distributed using a pipette.

The proof was carried out either on the luminograph of the company EG & G Berthold or on the STELLA system of the company raytech.

After detection of chemiluminescence, the membranes were placed in amido black solution for 5 to 10 minutes with gentle shaking to stain all protein bands. Thereafter, the membranes were designed to dry in air, discoloration is not necessary. Amidoblack staining solution: Amidoblack 0.1% (w / v) ethanol 45% (v / v) acetic acid 10% (v / v)

Used antibodies

Detection of YFP: YFP was detected by the antibody rabbit anti-GFP (BD Biosciences, dilution 1: 10,000); the second antibody was goat anti-rabbit (Amersham, 1: 5,000).

Detection of T7 RNA polymerase: T7 RNA polymerase was detected using T7 RNA polymerase monoclonal antibody (Novagen, 1: 10,000 dilution). The associated secondary antibody was goat anti-mouse (Biorad, 1: 3,000).

Detection of proteins with His ₆ tag: This was detected with anti-His (C-term) -HRP conjugate antibodies. These antibodies are conjugated to horseradish peroxidase, which allows chemiluminescent detection. Therefore, no second antibody is needed in this case.

In vivo fluorescence measurement for YFP

To determine the fluorescence emission of the fluorescent reporter protein YFP, the optical density of the expression cultures was determined, and 0.5 OD cells were harvested by centrifugation (10 min, 13,000 rpm). These were then taken up in 1 ml of 100 mM Tris-HCl pH 8.0.

If the test cultures were grown aerobically, they could be used immediately for fluorescence measurements. On the other hand, samples from anaerobically cultivated cultures had to be shaken for at least two hours with the Eppendorf tube open, room temperature and 1,000 rpm, so that the oxygen in the air could cause the autocatalytic formation of the fluorophore.

The sample was filled with 750 μl for measurement in quartz cuvettes (F3, Hellma company). The measurement was carried out with the luminescence spectrometer LS 50B from Perkin Elmer.

For this purpose, the sample was excited at a wavelength of 488 nm and the emitted fluorescence spectrum recorded over the range 515 to 615 nm. The fluorescence maximum of YFP is 529.5 nm. Other parameters: excitation slit 12.5; emission slit 15; increment 0; Excitation at 488 nm; scan speed: 240; Number of scans: 1.

After the measurement the determined values were extrapolated to an OD of 1 and 1 ml sample volume.

Detection of FbFP-mediated in vivo fluorescence

The fluorescence measurement was carried out using a fluorescence photometer Luminescence Spectrometer LS50B from Perkin Elmer. For the fluorescence measurement it is necessary that the cells are present with an optical density of 0.5. After the cultures have been adjusted to this density, in the case of E. coli and P. putida, the cells are centrifuged for 2 min at 13,000 rpm and in the case of R. capsulatus for 10 min at 13,000 rpm. After centrifugation, the supernatant is removed and the pellet for fluorescence measurement to the original volume with 100 mM Tris-HCl (pH 7) filled. 750 μl of these samples were then taken and transferred to QS quarter microcuvettes (Hellma). The gap settings on the fluorescence photometer were 12.5 nm (excitation slit) and 15.0 nm (emission slit). The FbFP proteins were excited at 450 nm and their fluorescence emission between 480 nm and 600 nm detected. The documentation of the spectra was done with the fluorescence data manager software from Perkin Elmer, the evaluation with Microsoft Excel.

The respective fluorescence intensity as "relative fluorescence intensity" was calculated from the measured fluorescence emission at a wavelength of 495 nm per cell density OD = 1 and 1 ml of cell suspension.

Synthesis of bacterio-opsin in the phototrophic bacterium R. capsulatus

For the synthesis of bacterio-opsin (bop) in the phototrophic organism Rhodobacter capsulatus, the E. coli donor strain S171 was first transformed with the expression plasmid pRhotHi2-bop. Subsequently, the plasmid was introduced by conjugative transfer into the expression host R. capsulatus B10S-T7 and propagated photo-heterotrophically on PY-solid medium under selection pressure. From this PY solid medium, appropriate cell colonies were harvested and used to inoculate precultures in RCV liquid medium. After three days of photoheterotrophic growth, these precultures were used to inoculate test cultures with a cell number corresponding to an optical density of 0.5 at 660 nm. The cultivation took place photoheterotrophically for 24 hours. To induce bop expression, fructose was added to the RCV medium of the test cultures at a final concentration of 8 mM. As a control, test cultures were used to which no inducer was added.

To check whether R. capsulatus expresses and accumulates bacterio-opsin under the culture conditions described above, cell-free protein extracts of strain R. capsulatus B10S-T7 were generated and fractionated with the plasmid-encoded bop gene (pRhotHi-2-bop). The Bop protein was detected using an antiserum (Anti-His ₆ (C-Term) antibody, Fermentas GmbH, St. Leon-Roth, Germany) by Western blot analysis.

Preparation of cell-free protein extracts from R. capsulatus:

For the production of cell-free protein extracts (whole cell protein extracts, GZE), the respective R. capsulatus strains were propagated for three days photoheterotrophically under selective pressure on PY solid medium. From this PY solid medium, appropriate cell colonies were harvested and used to inoculate precultures in RCV liquid medium. After three days of photoheterotrophic growth, these precultures were used to inoculate test cultures with a cell number corresponding to an optical density of 0.5 at 660 nm. The cultivation took place photoheterotrophically until the late logarithmic growth phase.

1.5 ml of each of the test cultures were sedimented (10 min, 16000 g, 20 ° C.) and the cell pellet was resuspended in 1 ml of collecting gel buffer. After re-centrifugation (10 min, 16000 g, 20 ° C), the cell pellet was resuspended in 150 μl of SDS sample buffer. The batch was incubated for 15 min at 99 ° C under agitation and then centrifuged (5 min, 16000 g, 20 ° C). The supernatant was used for denaturing SDS-polyacrylamide gel electrophoresis.

Fractionation of R. capsulatus samples:

• (1) Zentrifugation bei 2370 g für 3 min bei 20°C. Das Sediment wurde in 100 μl SDS-Probenpuffer aufgenommen und stellt Fraktion 1 dar (Zelltrümmer und Einschlusskörper).
• (2) Der Überstand wurde bei 18300 g für 30 min bei 20°C zentrifugiert. Das Sediment wurde in 100 μl SDS-Probenpuffer aufgenommen und stellt Fraktion 2 dar (Zellmembranen und Membranbestandteile).
• (3) Der Überstand stellt Fraktion 3 dar (lösliche Zellbestandteile). Die löslichen Proteine wurden mittels Fällung mit Trichloressigsäure (TCA) präzipitiert.

For the fractionation of R. capsulatus samples was proceeded as for the production of cell-free protein extracts. However, after the cell pellet was resuspended in 1 ml of collecting gel buffer, the samples were lysed by ultrasound (2 x 2.5 min, 50 cycles / min, 30% power, Bandin Sonoplus, UW2070, MS72, Bandelin electronic GmbH & Co. KG, Berlin , Germany). Then two centrifugation steps were carried out to generate three fractions:

• (1) Centrifuge at 2370 g for 3 min at 20 ° C. The sediment was taken up in 100 μl SDS sample buffer and represents fraction 1 (cell debris and inclusion body).
• (2) The supernatant was centrifuged at 18300 g for 30 min at 20 ° C. The sediment was taken up in 100 μl SDS sample buffer and represents fraction 2 (cell membranes and membrane constituents).
• (3) The supernatant represents fraction 3 (soluble cell components). The soluble proteins were precipitated by precipitation with trichloroacetic acid (TCA).

For this purpose, 1 volume of protein solution (fraction 3) and 0.1 volume of TCA (70%) were mixed, briefly vortexed and incubated at 20 ° C for 10 min. Subsequently, the batch was centrifuged at 16000 g, 5 min at 20 ° C. The sediment was washed with acetone (80%). The supernatant was discarded and the sediment was dried in a vacuum centrifuge (Concentrator 5301, Eppendorf AG, Hamburg, Germany) and taken up in 100 μl SDS sample buffer.

For the fractionation of liter cultures for use in sucrose density gradients, the complete culture was sedimented at 16000 g for 30 min at 4 ° C. The pellet was dissolved in Tris-HCl buffer (100 mM, pH 7.0, 1 tablet Complete Protease Inhibitor per 1000 ml buffer), so that a total volume of 30 ml was achieved. Subsequent cell digestion is performed using a French Pressure Cell Disruptor (Thermo Electron Corporation) in an FA-031 Pressure Cell at 1500 psi. Each culture was subjected to four passes. The digested cells were fractionated as described above but not taken up in SDS sample buffer. The sediment, representing fraction 2 (cell membranes and membrane components), was taken up in 2 ml Tris-HCl buffer (100 mM, pH 7.0, 1 tablet Complete Protease Inhibitor per 1000 ml buffer, 2% sucrose) and sent to sucrose density gradient centrifugation ,

Saccharose density gradient centrigugation of R. capsulatus membrane fractions:

For the isolation of membrane vesicles photoheterotrophic liter test cultures of R. capsulatus were fractionated. Fraction 2 was used for sucrose density gradient centrifugation. The cell number used corresponded approximately to an optical density of 1000 at 660 nm per density gradient. Continuous sucrose gradients of 10 to 60% sucrose were poured using a Peristaltic Pump P-1 (Pharmacia Fine Chemicals) and a 21A Multiphor II gradient mixer (LKB). The membrane fraction was then applied to the gradient and ultracentrifuged at 175,000 g and 4 ° C for 17 hours. After centrifugation was complete, the density gradient was removed in 1 ml aliquots. Subsequently, the sucrose concentration was analyzed in a refractometer (Optech Abbe refractometer) and the Bradford protein concentration. In addition, the extracted aliquots were examined immunologically with specific antibodies (anti-His ₆ - (C-term) antibodies, Fermentas GmbH, St. Leon-Roth Germany) on their content of bacterio-opsin.

Quantitative determination of Bradford protein concentration:

To quantitate the protein concentration after sucrose density gradient centrifugation, the Bradford test (Bradford, 1976) was used. This is a photometric method for the quantitative determination of proteins in the range of 1-200 μg / ml protein using the triphenylmethane dye Coomassie-Brilliant-Blue G-250. By complexing with proteins, the dye is stabilized in its blue, unprotonated, anionic sulfate form with an absorption maximum of 595 nm.

For exact protein concentration determination, it was necessary to measure a calibration series with defined protein concentrations. For this purpose, three calibration samples each with 0.02 mg / ml, 0.04 mg / ml, 0.05 mg / ml, 0.08 mg / ml and 0.10 mg / ml bovine serum albumin (BSA) in distilled water. prepared from a calibration solution (2 mg / ml albumin standard, Pierce). Subsequently, 0.1 ml of each calibration sample was mixed with 0.9 ml Bradford reagent and analyzed photometrically (Shimadzu UV / VIS 160, Shimadzu GmbH, Duisburg, Germany) at 595 nm after an incubation time of 5 min at 20 ° C.

After measuring all calibration samples, the procedure was comparable to the protein solutions of unknown concentration to be analyzed. From a measured absorbance of 0.4, the sample was adequately diluted. From the linear relationship of absorbance and protein concentration in the absorbance measurement range from 0 to 0.4, the protein concentration of the unknown samples can be calculated from the calibration data.

The Bradford reagent was prepared as follows:
100 mg of Coomassie Brilliant Blue G-250 were dissolved in 50 ml of ethanol (absolute) in a 1000 ml light-tight bottle. Then 100 ml of ortho-phosphoric acid (85.5% strength) were added and stirred for 1 h before being distilled with distilled water. was made up to 1000 ml. After stirring for 5 minutes, the solution was allowed to stand for one day (lowering of suspended solids) or filtered and used as described above.

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This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

Claims (12)

A method of expressing heterologous genes, the method comprising the steps of: - preparing at least one expression vector; Cloning at least one heterologous target gene into the expression vector; - producing a Rhodobacter capsulatus expression strain, whereby the expression strain by incorporating a T7 RNA polymerase gene is under the control of Rhodobacter-specific promoter P _fru, in the recA gene of Rhodobacter capsulatus - chromosome is produced; Introducing the recombinant expression vector into the cells of the expression strain; and expression of the heterologous target gene in the cells of the expression strain. A method according to claim 1, characterized in that the vector pBBR22b is modified to produce the expression vector. A method according to claim 2, characterized in that the vector pBBR22b is modified by deletion of the lacI gene. A method according to claim 2 or 3, characterized in that the vector pBBR22b is modified by insertion of the aphII gene of the Tn5 transposon, which is under the control of the Km promoter. Method according to one of claims 1 to 4, characterized in that the heterologous target gene is cloned in the reading direction of a T7 promoter in the expression vector. A method according to claim 4 or 5, characterized in that the heterologous target gene is cloned in the reading direction of the Km promoter of the aphII gene in the expression vector. A method according to claim 4 or 5, characterized in that the heterologous target gene opposite to the reading direction of the Km promoter of the aphII gene is cloned into the expression vector. Expression strain comprising cells of the bacterium Rhodobacter capsulatus, which are modified by integration of a T7 RNA polymerase gene, which is under the control of the Rhodobacter-specific promoter P _fru , into the recA gene of the Rhodobacter capsulatus chromosome. Kit for the heterologous expression of at least one gene comprising at least one modified pBBR22b vector with a polylinker downstream of a T7 promoter and additionally the expression strain according to claim 8. Cell culture comprising cells of the expression strain according to claim 8. Use of the method according to any one of claims 1 to 7, the expression strain according to claim 8, the kit according to claim 9 and / or the cell culture according to claim 10 for fluorescence labeling of cells. Use of the method according to any one of claims 1 to 7, the expression strain according to claim 8, the kit according to claim 9 and / or the cell culture according to claim 10 for overexpression of the target gene under aerobic or anaerobic conditions.

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