BACKGROUND
the demands on a valuable expression system are, in a nutshell, to receive high product yields, to provide a stable and tightly regulatable expression and to ensure high plasmid stability.
the properties of such a system are determined by the combination of a specific organism with the desired gene, whereas the plasmid features play a crucial role, too.
the effects of two or more determinants can not easily be estimated by addition of singular effects, because combined features can cancel each other out.
therefore, several expression systems have to be tested and the conditions have to be adjusted.
owing to escherichia coli being a model organism for genetic studies, a multitude of well-established regulatable promoters are available.
a distinction is drawn between positively and negatively controlled regulatory mechanisms.
for many promoters, especially those involved in carbohydrate catabolism, both possibilities are implemented, which is true for the well characterized lac-operon for instance.
in other cases, such as the l-arabinose operon or the l-rhamnose operon, the expression is positively regulated.
these systems are often characterized by a slower response with very low basal transcriptional activity, which can be a great advantage for the production of proteins that are detrimental to the host cell.
the l-rhamnose system has successfully been used to express a variety of genes  <cit> .
this system often provides better results compared to other vectors, especially if the expression of a gene usually leads to a large moiety of insoluble protein.
l-rhamnose is taken up by the rhat transport system, converted to l-rhamnulose by an isomerase  and then phosphorylated by a kinase .
subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase  into dihydroxyacetone phosphate, which is metabolized in glycolysis, and l-lactaldehyde.
the latter can be oxidized into lactate under aerobic conditions and be reduced into l- <dig> -propanediol under unaerobic conditions.
the genes rhabad are organized in one operon which is controlled by the rhapbad promoter.
this promoter is regulated by two activators, rhas and rhar, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhabad.
if l-rhamnose is available, rhar binds to the rhaprs promoter and activates the production of rhar and rhas.
rhas together with l-rhamnose in turn binds to the rhapbad and the rhapt promoter and activates the transcription of the structural genes.
however, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids.
therefore, only the rhapbad promoter has to be cloned upstream of the gene that is to be expressed.
full induction of rhabad transcription also requires binding of the crp-camp complex  <cit> , which is a key regulator of catabolite repression.
in addition to transcriptional regulation, the degradation of messenger rna  as well as translation initiation appear to be important factors in controlling the level of gene expression.
most bacterial mrnas show a high turnover rate which allow to rapidly adjust gene expression to the specific needs of the cells.
rnase e is the principal endonuclease involved in mrna decay in e. coli.
the action of rnase e is favored by an accessible 5' terminus carrying a monophosphate residue  <cit> .
therefore, sequence independent thermodynamically stable 5'stem-loop structures protect mrna from endonucleolytic attack by rnase e as seen in ompa or t <dig> gene  <dig> mrna which show unusual long half-lifes  <cit> .
translation initiation is greatly enhanced in e. coli and other bacteria by the shine-dalgarno sequence  in mrna, located 5– <dig> base pairs upstream of the initiation codon  <cit> .
the canonical sequence  is complementary to a sequence close to the 3' end of the  <dig> s rrna.
numerous studies suggest, that mrna translation is less efficient when the sd sequence has a lower degree of complementarity to the  <dig> s rrna or a different distance to the start codon  <cit> .
since a read-through by the rna polymerase can lead to severe instability of the expression system, it is recommended to insert a transcription terminator downstream of the desired gene.
the vectors used in this study contain a rho-independent terminator from the rrnb operon, which forms a stem-loop structure.
besides the transcription termination, the stability of a vector can be affected by miscellanous parameters.
the multimerization of plasmids reduces the copy number per cell and leads to segregational instability, a phenomenon known as dimer catastrophe  <cit> .
multimers can be resolved to monomers by site-specific recombination via the xer-cer system of cole <dig>  <cit> .
additionally, the promoter pcer within cer directs the synthesis of the  <dig> nt transcript rcd , whose overexpression strongly inhibits the growth of cells on solid media, whereas in broth culture the growth is slowed down but not stopped  <cit> .
thus rcd might be part of a checkpoint which causes a delay in cell division until multimers are converted into monomers, and rcd seems to play a role in plasmid maintenance, which is functionally independent of dimer resolution  <cit> .
dimer-containing cells grow more slowly than their monomer-containing counterparts, and the appearance of rcd correlates with the inhibition of division of multimer-containing cells, perhaps in order to provide the opportunity to resolve the multimers  <cit> .
due to the fact that an antisense target on the e. coli chromosome could not be found, it has been suggested, that rcd might interact directly with a protein target  <cit> .
another parameter that can be of importance to the expression output is the number of plasmids contained by the cells.
in cole1-type plasmids an efficient regulation mechanism has been evolved, that helps to maintain a constant copy number by counteracting occasional deviations from the steady state level.
this inhibitor-target mechanism is based on the negative control of the frequency of replication initiation events, mediated by the interaction of two rna molecules, rnai and rnaii and rop , a protein consisting of  <dig> amino acids.
in this system rnaii is a preprimer whose processing into a primer by rnase h is inhibited by the hybridization with rnai.
additionally, the interaction of target and inhibitor is enforced by rop, and in absence of rop the copy number is quintupled  <cit> .
beside the possibility to ensure plasmid maintenance by an improved stability, this task can also be performed by killing or inhibiting all cells which have lost their plasmids.
beyond the application of antibiotics combined with resistance markers on the plasmids, this function can be fulfilled by so called addiction modules, which induce programmed cell death in case of loss.
this genetic system consists of two components, a stable toxin and an unstable antitoxin.
such systems were mainly found in low-copy plasmids of e. coli, where cured cells were killed because the unstable antidote is degraded faster than the toxin and leads to the postsegregational killing effect  of the escherichia coli f plasmid, which codes for a stable toxin  and a less stable antidote .
ccdb inhibits gyra, a subunit of the heterotetrameric dna gyrase consisting of gyra and gyrb, and thereby causes gyrase-dependent killing of the cells  <cit> .
this inactivation can be prevented and reversed in the presence of ccda protein.
the products of treating the inactive gyra-ccdb complex with ccda are free gyra and a ccdb-ccda complex  <cit> .
moreover, the formation of the complex might prevent ccda from being degraded by lon protease in an atp-dependent manner  <cit> .
though ccd is one of the best understood addiction systems, some key mechanisms of the regulation remain unclear, presumedly because the ccda-ccdb interaction and its stoichiometry is unexpectedly complex  <cit> .
eventually, upon plasmid loss, ccdb outlives ccda and kills the cell by poisoning gyra.
RESULTS
differences in egfp expression according to variations of genetic modules
in this study we tested the influence of a modification of the transcription initiation region, the presence of three genetic modules on the expression vectors and combinations thereof.
the l-rhamnose-inducible vector pjoe <dig>  had already included the first  <dig> base pairs  upstream of the aug start codon from the highly translated bacteriophage t <dig> gene  <dig>  this sequence included a sd sequence perfectly matching the e. coli canonical sequence but lacked the stem-loop structure.
in the first step, the original transcription initiation region of rhapbad on pjoe <dig>  was replaced by a modified t <dig> gene  <dig> untranslated leader sequence which included the stem-loop structure.
the difference to the original t <dig> gene  <dig> upstream sequence  <cit>  is a deletion of an  <dig> bp sequence between the stem-loop and sd which was replaced by the recognition site of restriction endonuclease aflii.
we have named the resulting promotor rhapbad-t7sl  and the corresponding plasmid pjoe <dig> , which was the source of the subsequent modifications.
in the next step, a ccdab cassette was inserted , one of the two cer sites was excised  and the rop site was deleted .
plasmids with all possible combinations of these modifications were constructed .
to compare the performance of the modified plasmids, the amount of egfp produced after induction in e. coli jm <dig> transformed with the individual plasmids was measured at intervals of  <dig> minutes .
the most significant enhancement was achieved by the conversion of rhapbad to rhapbad-t7sl, whereas the insertion of ccdab had only slight effects comparing pwa <dig> with pjoe <dig> .
the fluorescence received with pjoe <dig>  is threefold that of pjoe <dig>  referring to  <dig> μl of a cell suspension of  <dig>  od <dig> and it is quadrupled referring to the same volume of culture.
in the case of pwa <dig>  pwa <dig> and pwa <dig> ccdab even slightly decreased the expression of egfp.
the reduction of the two cer sites to one increased the measured fluorescence value, as recorded for pwa <dig>  pwa <dig>  pwa <dig> and pwa <dig>  compared to the corresponding plasmids with a second cer.
eventually, the removal of rop again increased the production of egfp, especially if combined with only one cer site, as it is the case for pwa <dig> and pwa <dig>  this effect is more apparent if the values relating to fluorescence per ml culture are compared, which is due to a slightly increased optical density of these cultures .
the enhanced fluorescence achieved with the Δrop plasmids correlates with an increased quantity of dna extracted from the corresponding cultures .
this observation is according to expectations, as rop is a regulator of copy number, and a deprivation of rop leads to an elevated copy number, in some cases concomitant with higher yields of plasmid-encoded protein.
the amount of plasmid dna extracted from strains with pwa <dig> and pwa <dig>  which in contrast to pjoe <dig>  and pwa <dig> only contain one cer site, is slightly shortened.
this might be due to the fact that cer not only is responsible for the resolution of multimers, which incidentally does not occur in reca strains like jm <dig>  but also the rcd hosted in cer can slow down growth of cells bearing multimers.
therefore, plasmids with two cer sites possibly mimic multimers, decelerate cell division and facilitate accumulation of plasmid dna.
comparison of plasmid stability
the usability of a plasmid is not only determined by the amount of protein produced in a specific combination of strain, gene and plasmid, but also by the stability of the expression vector.
especially if the induction is carried out on a large scale and during a longer period of process, plasmid loss can have a tremendous effect on total yield.
in order to test the influence of the variations of the genetic modules, we determined how many cells lost their plasmids during a prolonged induction.
if the cultures were induced at 30°c for  <dig> or  <dig> hours, which is roughly equivalent to  <dig> generations or  <dig> generations, respectively, no plasmid loss could be observed in none of the cultures .
but if the induction was carried out at 37°c for  <dig> hours, major differences between the plasmids could be detected.
the originating pjoe <dig>  and its two direct derivatives pjoe <dig>  and pwa <dig> were perfectly stable .
these results show, that the exchange of the original transcription initiation region by that of φ <dig> promoter from t <dig> and the insertion of the ccdab locus in addition had no negative effect on plasmid maintenance.
the elimination of one cer site producing pwa <dig> and pwa <dig> has led to a negligible raise of plasmid loss of 5–8% in approximately  <dig> generations, a faint disadvantage which is acceptable in regard to the enhanced expression.
in contrast, the deletion of rop added more instability to the vectors, for instance 25% of the cells bearing pwa <dig> had lost their plasmids.
furthermore, the combination of Δrop and ccdab increased the percentage of plasmid free cells to 32% .
this observation is not according to expectations, because the addiction module should mediate programmed cell death if the gene coding for the unstable antidote is lost.
moreover, the combination of one cer site and Δrop had a remarkably negative impact on plasmid persistence, as it resulted in a plasmid withdrawal of about 68%.
again, the addition of ccdab adversely affected the plasmid stability.
apparently, combination of the specific genetic modules cer, rop and ccdab, which are supposed to have a stabilizing impact on plasmids, does not simply lead to summable effects, but can contrarily destabilize vectors.
in general, the influence on plasmid maintenance of plasmid-borne elements and their interaction with the particular host strain has to be examined in individual cases.
variations in cell length after induction of genes in dependency of e. coli strains
when induction of egfp was carried out in the e. coli strains bw <dig> and bl <dig> rha- additionally to the experiments in jm <dig> mentioned above, lower values of fluorescence were observed .
however, the relations between the fluorescence obtained with the individual vectors were comparable.
but we noticed that in bw <dig> and bl <dig> rha- the optical density of the cultures increased significantly, though these strains are not able to use l-rhamnose as a carbon source, due to the lack of rhab.
examination by microscopy showed, that the reason for this raise was not a higher cell count, but an increased cell length .
as these differences were most significant in bl <dig> rha-, this strain was used to examine the effect on morphology in more detail .
arabinose and glucose supplement heightened the optical density but had no effect on cell length.
moreover, no changes could be observed in cells bearing the plasmids without egfp  under conditions of induction, or in cells carrying the plasmid pwa <dig> under non-induced conditions.
these experiments showed, that the increased length is not caused by the vector itself.
furthermore, the elongation was less distinct if egfp was expressed from pjoe <dig> , which also achieved lower fluorescence compared to pwa <dig> .
it is quite evident, that the cell-length effect in fact is dependent on the amount of egfp protein produced therein.
all strains showed an increase of cell length and optical density of the cultures under conditions of induction compared to cells without plasmid.
the standard deviation of length in cultures with prolonged cells was larger than in the control batches, which are more homogenous.
in cultures with rhab-positive cells the optical density was elevated more than in rhab-negative strains, an effect that is revealed in the comparison of w <dig> and bw <dig> or bl <dig> and bl <dig> rha-.
obviously, these differences were a result of metabolization of l-rhamnose.
in the e. coli k <dig> derivatives jm <dig>  w <dig> and bw <dig> the number of cells raised concomitantly to the optical densitiy if they were induced.
in bl <dig> and bl <dig> rha-, strains that belong to the e. coli b-type group, in contrast the number of cells declined if l-rhamnose was added.
this effect probably is in conjunction with the stronger impact on cell length, since cell division problems might occur.
influence of adjusted crp-binding site on expression of egfp
as crp is a key regulator for catabolite repression, the corresponding binding sites can be found in or near many promotors involved in carbohydrate catabolism.
for instance, the e. coli lac promoter dna site for crp differs from the consensus dna site at  <dig> of  <dig> positions.
it has been shown, that crp in vitro exhibits a 450-fold higher affinity for the consensus site than for the natural binding site  <cit> .
according to these observation, derivatives of pwa <dig> with modified crp-binding sites were constructed.
in pwa <dig>  four nucleotides out of eight differing from the consensus have been adjusted, whereas in pwa <dig>  one nucleotide in the most conserved region accidentally was deleted .
to test the influence of the mutations, the amount of egfp produced by e. coli jm <dig> transformed with the individual plasmids, grown in liquid medium supplemented with different concentrations of d-glucose and induced with l-rhamnose, was measured .
the amounts of egfp produced with pwa <dig>  were comparable to those produced with pwa <dig>  even under conditions of d-glucose addition no differences in production were detectable, neither in the total values reached at the end of the process nor in the production rates.
obviously, the adjustment implemented in pwa <dig>  did not weaken the sensitivity to catabolite repression mediated by crp.
however, the missing nucleotide in the crp-binding site of pwa <dig>  had a tremendous effect.
the fluorescence was shortened to about 10% of the reference magnitude achieved by pwa <dig>  but still it was repressed by d-glucose at a concentration of  <dig> %.
these results suggested that this poorly inducible promotor still is dependent on the binding of crp, but may have a lower affinity.
discussion
since many basic principles of genetics have been discovered using e. coli as a model organism, today its genetics is well characterized, and many details about the regulation of gene expression are described.
this knowledge made it possible to use its promoters in a cassette fashion, unaffected by the surrounding nucleotide context.
additionally, a large number of cloning vectors are available, which provides a long-range repertory of components out of which the most appropriate can be chosen.
the productivity of the expression system is influenced by many parameters, and besides the basic choice of an available expression system, these parameters can be tuned by the insertion or deletion of genetic modules.
in all organisms gene expression is regulated on various levels, at which mrna stability provides a key control device.
as several rnases are involved in mrna degradation, including endonucleases and 3'exonucleases, many sequences that act as stabilizers have been identified.
some of them only work under specific conditions, others stabilize mrnas of miscellanous sources under normal conditions and at high growth rates.
one well characterized example of a such a stabilizing element is a 21-bp fragment of the φ <dig> promoter of bacteriophage t <dig> which can potentially form an 8-bp stem loop  <cit> .
in our assays, the insertion of this stem loop quadrupled the amount of gfp that is produced in  <dig> h and fortunately had no measurable effect on plasmid maintenance.
this advantage is preserved even if different combinations of other modules are added, and it exhibits the most explicit improvement achieved by the modulation that were carried out in this study.
though we have not tested the actual stability of the mrna produced by our system, one can presume that this characteristic has been improved considerably and marks a significant step.
besides the stem-loop insertion into the rhap region we have tried to improve expression by modification of the - <dig> promotor region.
the sequence 'tagact' in pjoe <dig>  has been mutated into 'tataat', and the resulting plasmid pjoe <dig>  has been examined in an egfp expression assay as described herein.
unfortunately, this modulation resulted in a elevated basal level which under non-induced conditions attained the output of induced pjoe <dig>  cells, though it evidently fell behind in expression compared to pjoe <dig>  .
hence, this approach reveals that additional alterations of the already efficient rhapbad expression vectors are crucial, not only because they possibly do not advance yields, but also may disturb a well-balanced system by making it less regulable.
adjacent to plasmid inherent regulation mechanisms, the current metabolic status of the cells has an exceeding impact on the production of recombinant proteins as well.
for example, some promotors act mainly in the exponential phase of cell growth, whereas others are activated primarily in steady state cultures, and in addition the intracellular availability of nutrients further affects the activity of a given promotor.
generally, promotors assigned to operons associated with carbohydrate catabolism are controlled by crp via the intracellular camp levels.
as d-glucose lowers camp levels and thus inactivates binding of crp, but usually is added as a carbon source in minimal medium used for fermentations, it would be of interest to make such a promotor less sensitive towards low camp levels.
the approach tested in this study was to enhance the affinity of the crp-binding site.
unfortunately, the adjustment of the dna sequence according to the consensus sequence had no measurable effects.
the mutated plasmid still was inhibited by glucose in the same extend as the unmodified plasmid.
presumedly, the original binding site already shows a good affinity to crp and could not be improved by an adjustment with the consensus sequence.
on the other hand, the plasmid pwa <dig>  with the one base pair deletion in the crp binding site was clearly inferior.
it still was susceptible to glucose addition but obtained only about 10% of the yield compared to the original plasmid.
these results precisely show, that it is difficult to elevate protein production concomitantly with a low basal level and a tight regulation.
another way to elevate protein production is to increase the corresponding gene copies in the cells.
this was acchieved in this study by deleting the rop gene which is involved in controlling plasmid copy numbers.
the deletion increased the content of plasmid dna in the cells at least threefold as expected but there was no concomitant increase of egfp production.
this is not surprising since the egfp production in cells with pwa <dig> already amounts to about 20% of the total protein and even with the strongest expression systems there is a limit at about 30% of the total protein whatever gene is used.
to see the high-copy plasmid effect a reporter gene would be needed which is expressed at a lower level.
this might also answer the question if there is a sufficient supply of the activator rhas for high-copy plasmids from the single rhas gene copy on the chromosome.
when the rhars genes were introduced on a compatible plasmid with a moderate copy number  in cells containing already pwa <dig>  pwa <dig> or pwa <dig>  no significant increase in egfp production was observed upon induction with rhamnose .
previous results reported by wilms et al.
<cit>  showed that the introduction of the cer site of cole <dig> to plasmids reduces the appearance of multimers drastically.
this is especially true for reca-proficient strains like w <dig>  whereas the multimerization is inhibited in reca-deficient strains like jm <dig>  actually, the insertion of cer led to a tremendous stabilization.
over 90% of the cells were still carrying the plasmid, while less than 50% of the cells kept the plasmids without the cer-site when ampicillin was absent.
accidentally, in these cases two cer-sites in tandem had been ligated into the vectors.
the removal of one of them led to an increased egfp expression while the plasmid stability was only negligibly influenced.
the accompanying decrease in plasmid dna apparently did not adversely affect the protein yields, probably because it is the duplication of cer that causes problems to the cell division which are solved by removing one.
furthermore, one cer site sufficiently ensured the plasmid maintenance.
additionally, the bacterial plasmid addiction system ccdab was provided to some of the vectors in order to test its influence on the stability.
unexpectedly, ccdab did not deliver any improvement, as it did not stabilize the plasmid but in contrast increased plasmid loss at 37°c.
as the toxin ccdb kills cells that have lost their plasmids and therefore can not produce the unstable antidote ccda any more, apparently cells still carrying ccdab plasmids are at a serious disadvantage in cell growth.
it amounts to a situation where few cells that have successfully eliminated the plasmids start to overgrow cells that still express ccdab.
presumedly, the continous production of the toxin becomes such a great metabolic burden to the cells, that the evasion by plasmid loss outweighs the initial toxication.
thus it appears that the insertion of the addiction module in this case represents a set-back.
CONCLUSIONS
the initial plasmid pjoe <dig> , which already bears a well-working translation initiation signal of t <dig> gene  <dig> and the transcription terminator from the rrnb operon, has been mainly improved by insertion of a stem loop and by deletion of one cer site.
the resultant plasmid pwa <dig> therefore seems to perform best for egfp expression in our system.
further changes might be critical as they carry the risk of worsen it.
hence, it seems as there hardly will be any room for further improvements.

 the demands on a valuable expression system are, in a nutshell, to receive high product yields, to provide a stable and tightly regulatable expression and to ensure high plasmid stability. the properties of such a system are determined by the combination of a specific organism with the desired gene, whereas the plasmid features play a crucial role, too. the effects of two or more determinants can not easily be estimated by addition of singular effects, because combined features can cancel each other out. therefore, several expression systems have to be tested and the conditions have to be adjusted. owing to escherichia coli being a model organism for genetic studies, a multitude of well-established regulatable promoters are available. a distinction is drawn between positively and negatively controlled regulatory mechanisms. for many promoters, especially those involved in carbohydrate catabolism, both possibilities are implemented, which is true for the well characterized lac-operon for instance. in other cases, such as the l-arabinose operon or the l-rhamnose operon, the expression is positively regulated. these systems are often characterized by a slower response with very low basal transcriptional activity, which can be a great advantage for the production of proteins that are detrimental to the host cell. the l-rhamnose system has successfully been used to express a variety of genes. this system often provides better results compared to other vectors, especially if the expression of a gene usually leads to a large moiety of insoluble protein. l-rhamnose is taken up by the rhat transport system, converted to l-rhamnulose by an isomerase and then phosphorylated by a kinase. subsequently, the resulting rhamnulose-1-phosphate is hydrolyzed by an aldolase into dihydroxyacetone phosphate, which is metabolized in glycolysis, and l-lactaldehyde. the latter can be oxidized into lactate under aerobic conditions and be reduced into l- -propanediol under unaerobic conditions. the genes rhabad are organized in one operon which is controlled by the rhapbad promoter. this promoter is regulated by two activators, rhas and rhar, and the corresponding genes belong to one transcription unit which is located in opposite direction of rhabad. if l-rhamnose is available, rhar binds to the rhaprs promoter and activates the production of rhar and rhas. rhas together with l-rhamnose in turn binds to the rhapbad and the rhapt promoter and activates the transcription of the structural genes. however, for the application of the rhamnose expression system it is not necessary to express the regulatory proteins in larger quantities, because the amounts expressed from the chromosome are sufficient to activate transcription even on multi-copy plasmids. therefore, only the rhapbad promoter has to be cloned upstream of the gene that is to be expressed. full induction of rhabad transcription also requires binding of the crp-camp complex, which is a key regulator of catabolite repression. in addition to transcriptional regulation, the degradation of messenger rna as well as translation initiation appear to be important factors in controlling the level of gene expression. most bacterial mrnas show a high turnover rate which allow to rapidly adjust gene expression to the specific needs of the cells. rnase e is the principal endonuclease involved in mrna decay in e. coli. the action of rnase e is favored by an accessible 5' terminus carrying a monophosphate residue. therefore, sequence independent thermodynamically stable 5'stem-loop structures protect mrna from endonucleolytic attack by rnase e as seen in ompa or t gene mrna which show unusual long half-lifes. translation initiation is greatly enhanced in e. coli and other bacteria by the shine-dalgarno sequence in mrna, located 5– base pairs upstream of the initiation codon. the canonical sequence is complementary to a sequence close to the 3' end of the s rrna. numerous studies suggest, that mrna translation is less efficient when the sd sequence has a lower degree of complementarity to the s rrna or a different distance to the start codon. since a read-through by the rna polymerase can lead to severe instability of the expression system, it is recommended to insert a transcription terminator downstream of the desired gene. the vectors used in this study contain a rho-independent terminator from the rrnb operon, which forms a stem-loop structure. besides the transcription termination, the stability of a vector can be affected by miscellanous parameters. the multimerization of plasmids reduces the copy number per cell and leads to segregational instability, a phenomenon known as dimer catastrophe. multimers can be resolved to monomers by site-specific recombination via the xer-cer system of cole. additionally, the promoter pcer within cer directs the synthesis of the nt transcript rcd, whose overexpression strongly inhibits the growth of cells on solid media, whereas in broth culture the growth is slowed down but not stopped. thus rcd might be part of a checkpoint which causes a delay in cell division until multimers are converted into monomers, and rcd seems to play a role in plasmid maintenance, which is functionally independent of dimer resolution. dimer-containing cells grow more slowly than their monomer-containing counterparts, and the appearance of rcd correlates with the inhibition of division of multimer-containing cells, perhaps in order to provide the opportunity to resolve the multimers. due to the fact that an antisense target on the e. coli chromosome could not be found, it has been suggested, that rcd might interact directly with a protein target. another parameter that can be of importance to the expression output is the number of plasmids contained by the cells. in cole1-type plasmids an efficient regulation mechanism has been evolved, that helps to maintain a constant copy number by counteracting occasional deviations from the steady state level. this inhibitor-target mechanism is based on the negative control of the frequency of replication initiation events, mediated by the interaction of two rna molecules, rnai and rnaii and rop, a protein consisting of amino acids. in this system rnaii is a preprimer whose processing into a primer by rnase h is inhibited by the hybridization with rnai. additionally, the interaction of target and inhibitor is enforced by rop, and in absence of rop the copy number is quintupled. beside the possibility to ensure plasmid maintenance by an improved stability, this task can also be performed by killing or inhibiting all cells which have lost their plasmids. beyond the application of antibiotics combined with resistance markers on the plasmids, this function can be fulfilled by so called addiction modules, which induce programmed cell death in case of loss. this genetic system consists of two components, a stable toxin and an unstable antitoxin. such systems were mainly found in low-copy plasmids of e. coli, where cured cells were killed because the unstable antidote is degraded faster than the toxin and leads to the postsegregational killing effect of the escherichia coli f plasmid, which codes for a stable toxin and a less stable antidote. ccdb inhibits gyra, a subunit of the heterotetrameric dna gyrase consisting of gyra and gyrb, and thereby causes gyrase-dependent killing of the cells. this inactivation can be prevented and reversed in the presence of ccda protein. the products of treating the inactive gyra-ccdb complex with ccda are free gyra and a ccdb-ccda complex. moreover, the formation of the complex might prevent ccda from being degraded by lon protease in an atp-dependent manner. though ccd is one of the best understood addiction systems, some key mechanisms of the regulation remain unclear, presumedly because the ccda-ccdb interaction and its stoichiometry is unexpectedly complex. eventually, upon plasmid loss, ccdb outlives ccda and kills the cell by poisoning gyra.  differences in egfp expression according to variations of genetic modules in this study we tested the influence of a modification of the transcription initiation region, the presence of three genetic modules on the expression vectors and combinations thereof. the l-rhamnose-inducible vector pjoe had already included the first base pairs upstream of the aug start codon from the highly translated bacteriophage t gene this sequence included a sd sequence perfectly matching the e. coli canonical sequence but lacked the stem-loop structure. in the first step, the original transcription initiation region of rhapbad on pjoe was replaced by a modified t gene untranslated leader sequence which included the stem-loop structure. the difference to the original t gene upstream sequence is a deletion of an bp sequence between the stem-loop and sd which was replaced by the recognition site of restriction endonuclease aflii. we have named the resulting promotor rhapbad-t7sl and the corresponding plasmid pjoe, which was the source of the subsequent modifications. in the next step, a ccdab cassette was inserted, one of the two cer sites was excised and the rop site was deleted. plasmids with all possible combinations of these modifications were constructed. to compare the performance of the modified plasmids, the amount of egfp produced after induction in e. coli jm transformed with the individual plasmids was measured at intervals of minutes. the most significant enhancement was achieved by the conversion of rhapbad to rhapbad-t7sl, whereas the insertion of ccdab had only slight effects comparing pwa with pjoe. the fluorescence received with pjoe is threefold that of pjoe referring to μl of a cell suspension of od and it is quadrupled referring to the same volume of culture. in the case of pwa pwa and pwa ccdab even slightly decreased the expression of egfp. the reduction of the two cer sites to one increased the measured fluorescence value, as recorded for pwa pwa pwa and pwa compared to the corresponding plasmids with a second cer. eventually, the removal of rop again increased the production of egfp, especially if combined with only one cer site, as it is the case for pwa and pwa this effect is more apparent if the values relating to fluorescence per ml culture are compared, which is due to a slightly increased optical density of these cultures. the enhanced fluorescence achieved with the Δrop plasmids correlates with an increased quantity of dna extracted from the corresponding cultures. this observation is according to expectations, as rop is a regulator of copy number, and a deprivation of rop leads to an elevated copy number, in some cases concomitant with higher yields of plasmid-encoded protein. the amount of plasmid dna extracted from strains with pwa and pwa which in contrast to pjoe and pwa only contain one cer site, is slightly shortened. this might be due to the fact that cer not only is responsible for the resolution of multimers, which incidentally does not occur in reca strains like jm but also the rcd hosted in cer can slow down growth of cells bearing multimers. therefore, plasmids with two cer sites possibly mimic multimers, decelerate cell division and facilitate accumulation of plasmid dna. comparison of plasmid stability the usability of a plasmid is not only determined by the amount of protein produced in a specific combination of strain, gene and plasmid, but also by the stability of the expression vector. especially if the induction is carried out on a large scale and during a longer period of process, plasmid loss can have a tremendous effect on total yield. in order to test the influence of the variations of the genetic modules, we determined how many cells lost their plasmids during a prolonged induction. if the cultures were induced at 30°c for or hours, which is roughly equivalent to generations or generations, respectively, no plasmid loss could be observed in none of the cultures. but if the induction was carried out at 37°c for hours, major differences between the plasmids could be detected. the originating pjoe and its two direct derivatives pjoe and pwa were perfectly stable. these results show, that the exchange of the original transcription initiation region by that of φ promoter from t and the insertion of the ccdab locus in addition had no negative effect on plasmid maintenance. the elimination of one cer site producing pwa and pwa has led to a negligible raise of plasmid loss of 5–8% in approximately generations, a faint disadvantage which is acceptable in regard to the enhanced expression. in contrast, the deletion of rop added more instability to the vectors, for instance 25% of the cells bearing pwa had lost their plasmids. furthermore, the combination of Δrop and ccdab increased the percentage of plasmid free cells to 32%. this observation is not according to expectations, because the addiction module should mediate programmed cell death if the gene coding for the unstable antidote is lost. moreover, the combination of one cer site and Δrop had a remarkably negative impact on plasmid persistence, as it resulted in a plasmid withdrawal of about 68%. again, the addition of ccdab adversely affected the plasmid stability. apparently, combination of the specific genetic modules cer, rop and ccdab, which are supposed to have a stabilizing impact on plasmids, does not simply lead to summable effects, but can contrarily destabilize vectors. in general, the influence on plasmid maintenance of plasmid-borne elements and their interaction with the particular host strain has to be examined in individual cases. variations in cell length after induction of genes in dependency of e. coli strains when induction of egfp was carried out in the e. coli strains bw and bl rha- additionally to the experiments in jm mentioned above, lower values of fluorescence were observed. however, the relations between the fluorescence obtained with the individual vectors were comparable. but we noticed that in bw and bl rha- the optical density of the cultures increased significantly, though these strains are not able to use l-rhamnose as a carbon source, due to the lack of rhab. examination by microscopy showed, that the reason for this raise was not a higher cell count, but an increased cell length. as these differences were most significant in bl rha-, this strain was used to examine the effect on morphology in more detail. arabinose and glucose supplement heightened the optical density but had no effect on cell length. moreover, no changes could be observed in cells bearing the plasmids without egfp under conditions of induction, or in cells carrying the plasmid pwa under non-induced conditions. these experiments showed, that the increased length is not caused by the vector itself. furthermore, the elongation was less distinct if egfp was expressed from pjoe, which also achieved lower fluorescence compared to pwa. it is quite evident, that the cell-length effect in fact is dependent on the amount of egfp protein produced therein. all strains showed an increase of cell length and optical density of the cultures under conditions of induction compared to cells without plasmid. the standard deviation of length in cultures with prolonged cells was larger than in the control batches, which are more homogenous. in cultures with rhab-positive cells the optical density was elevated more than in rhab-negative strains, an effect that is revealed in the comparison of w and bw or bl and bl rha-. obviously, these differences were a result of metabolization of l-rhamnose. in the e. coli k derivatives jm w and bw the number of cells raised concomitantly to the optical densitiy if they were induced. in bl and bl rha-, strains that belong to the e. coli b-type group, in contrast the number of cells declined if l-rhamnose was added. this effect probably is in conjunction with the stronger impact on cell length, since cell division problems might occur. influence of adjusted crp-binding site on expression of egfp as crp is a key regulator for catabolite repression, the corresponding binding sites can be found in or near many promotors involved in carbohydrate catabolism. for instance, the e. coli lac promoter dna site for crp differs from the consensus dna site at of positions. it has been shown, that crp in vitro exhibits a 450-fold higher affinity for the consensus site than for the natural binding site. according to these observation, derivatives of pwa with modified crp-binding sites were constructed. in pwa four nucleotides out of eight differing from the consensus have been adjusted, whereas in pwa one nucleotide in the most conserved region accidentally was deleted. to test the influence of the mutations, the amount of egfp produced by e. coli jm transformed with the individual plasmids, grown in liquid medium supplemented with different concentrations of d-glucose and induced with l-rhamnose, was measured. the amounts of egfp produced with pwa were comparable to those produced with pwa even under conditions of d-glucose addition no differences in production were detectable, neither in the total values reached at the end of the process nor in the production rates. obviously, the adjustment implemented in pwa did not weaken the sensitivity to catabolite repression mediated by crp. however, the missing nucleotide in the crp-binding site of pwa had a tremendous effect. the fluorescence was shortened to about 10% of the reference magnitude achieved by pwa but still it was repressed by d-glucose at a concentration of %. these results suggested that this poorly inducible promotor still is dependent on the binding of crp, but may have a lower affinity. discussion since many basic principles of genetics have been discovered using e. coli as a model organism, today its genetics is well characterized, and many details about the regulation of gene expression are described. this knowledge made it possible to use its promoters in a cassette fashion, unaffected by the surrounding nucleotide context. additionally, a large number of cloning vectors are available, which provides a long-range repertory of components out of which the most appropriate can be chosen. the productivity of the expression system is influenced by many parameters, and besides the basic choice of an available expression system, these parameters can be tuned by the insertion or deletion of genetic modules. in all organisms gene expression is regulated on various levels, at which mrna stability provides a key control device. as several rnases are involved in mrna degradation, including endonucleases and 3'exonucleases, many sequences that act as stabilizers have been identified. some of them only work under specific conditions, others stabilize mrnas of miscellanous sources under normal conditions and at high growth rates. one well characterized example of a such a stabilizing element is a 21-bp fragment of the φ promoter of bacteriophage t which can potentially form an 8-bp stem loop. in our assays, the insertion of this stem loop quadrupled the amount of gfp that is produced in h and fortunately had no measurable effect on plasmid maintenance. this advantage is preserved even if different combinations of other modules are added, and it exhibits the most explicit improvement achieved by the modulation that were carried out in this study. though we have not tested the actual stability of the mrna produced by our system, one can presume that this characteristic has been improved considerably and marks a significant step. besides the stem-loop insertion into the rhap region we have tried to improve expression by modification of the - promotor region. the sequence'tagact' in pjoe has been mutated into'tataat', and the resulting plasmid pjoe has been examined in an egfp expression assay as described herein. unfortunately, this modulation resulted in a elevated basal level which under non-induced conditions attained the output of induced pjoe cells, though it evidently fell behind in expression compared to pjoe. hence, this approach reveals that additional alterations of the already efficient rhapbad expression vectors are crucial, not only because they possibly do not advance yields, but also may disturb a well-balanced system by making it less regulable. adjacent to plasmid inherent regulation mechanisms, the current metabolic status of the cells has an exceeding impact on the production of recombinant proteins as well. for example, some promotors act mainly in the exponential phase of cell growth, whereas others are activated primarily in steady state cultures, and in addition the intracellular availability of nutrients further affects the activity of a given promotor. generally, promotors assigned to operons associated with carbohydrate catabolism are controlled by crp via the intracellular camp levels. as d-glucose lowers camp levels and thus inactivates binding of crp, but usually is added as a carbon source in minimal medium used for fermentations, it would be of interest to make such a promotor less sensitive towards low camp levels. the approach tested in this study was to enhance the affinity of the crp-binding site. unfortunately, the adjustment of the dna sequence according to the consensus sequence had no measurable effects. the mutated plasmid still was inhibited by glucose in the same extend as the unmodified plasmid. presumedly, the original binding site already shows a good affinity to crp and could not be improved by an adjustment with the consensus sequence. on the other hand, the plasmid pwa with the one base pair deletion in the crp binding site was clearly inferior. it still was susceptible to glucose addition but obtained only about 10% of the yield compared to the original plasmid. these results precisely show, that it is difficult to elevate protein production concomitantly with a low basal level and a tight regulation. another way to elevate protein production is to increase the corresponding gene copies in the cells. this was acchieved in this study by deleting the rop gene which is involved in controlling plasmid copy numbers. the deletion increased the content of plasmid dna in the cells at least threefold as expected but there was no concomitant increase of egfp production. this is not surprising since the egfp production in cells with pwa already amounts to about 20% of the total protein and even with the strongest expression systems there is a limit at about 30% of the total protein whatever gene is used. to see the high-copy plasmid effect a reporter gene would be needed which is expressed at a lower level. this might also answer the question if there is a sufficient supply of the activator rhas for high-copy plasmids from the single rhas gene copy on the chromosome. when the rhars genes were introduced on a compatible plasmid with a moderate copy number in cells containing already pwa pwa or pwa no significant increase in egfp production was observed upon induction with rhamnose. previous results reported by wilms et al. showed that the introduction of the cer site of cole to plasmids reduces the appearance of multimers drastically. this is especially true for reca-proficient strains like w whereas the multimerization is inhibited in reca-deficient strains like jm actually, the insertion of cer led to a tremendous stabilization. over 90% of the cells were still carrying the plasmid, while less than 50% of the cells kept the plasmids without the cer-site when ampicillin was absent. accidentally, in these cases two cer-sites in tandem had been ligated into the vectors. the removal of one of them led to an increased egfp expression while the plasmid stability was only negligibly influenced. the accompanying decrease in plasmid dna apparently did not adversely affect the protein yields, probably because it is the duplication of cer that causes problems to the cell division which are solved by removing one. furthermore, one cer site sufficiently ensured the plasmid maintenance. additionally, the bacterial plasmid addiction system ccdab was provided to some of the vectors in order to test its influence on the stability. unexpectedly, ccdab did not deliver any improvement, as it did not stabilize the plasmid but in contrast increased plasmid loss at 37°c. as the toxin ccdb kills cells that have lost their plasmids and therefore can not produce the unstable antidote ccda any more, apparently cells still carrying ccdab plasmids are at a serious disadvantage in cell growth. it amounts to a situation where few cells that have successfully eliminated the plasmids start to overgrow cells that still express ccdab. presumedly, the continous production of the toxin becomes such a great metabolic burden to the cells, that the evasion by plasmid loss outweighs the initial toxication. thus it appears that the insertion of the addiction module in this case represents a set-back.  the initial plasmid pjoe, which already bears a well-working translation initiation signal of t gene and the transcription terminator from the rrnb operon, has been mainly improved by insertion of a stem loop and by deletion of one cer site. the resultant plasmid pwa therefore seems to perform best for egfp expression in our system. further changes might be critical as they carry the risk of worsen it. hence, it seems as there hardly will be any room for further improvements. 
starting from this plasmid, we constructed a set of expression vectors bearing different genetic modules like rop, ccdab, cer and combinations thereof, and tested the efficiency of expression and plasmid stability. the introduction of a stem loop into the translation initiation region of the rhapbad promoter resulted in the most significant improvement of egfp expression. the plasmid pwa containing the stem loop, one cer site and rop, attained high expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice. these features can be influenced by a variation of modifications and versatile genetic modules. the genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.
 a capable expression vector is mainly characterized by its production efficiency, stability and induction response. these features can be influenced by a variation of modifications and versatile genetic modules.  we examined miscellaneous variations of a rhapbad expression vector. the introduction of a stem loop into the translation initiation region of the rhapbad promoter resulted in the most significant improvement of egfp expression. starting from this plasmid, we constructed a set of expression vectors bearing different genetic modules like rop, ccdab, cer and combinations thereof, and tested the efficiency of expression and plasmid stability. the plasmid pwa containing the stem loop, one cer site and rop, attained high expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice.  we report the generation of variations of the rhapbad expression vector and characterization hereof. the genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage. 