Team:Goettingen/Parts

Parts

On the way to construct our screening system, we used some existing parts, improved some of them, created new ones and then assembled them together. To get a quantitative output of how effective a certain substance is to disturb c-di-AMP homeostasis, we tried promoters of different strength in the famous Anderson promoter collection and we also characterized the promoters we used in our reporter system.

Parts we used in our project

Parts we modified/improved/created

<groupparts>iGEM013 Goettingen</groupparts>

Promoter Characterization

To construct our reporter systems, we needed promoters of different strength. The well-known Anderson promoter collection consists of nineteen constitutive promoters of different strength. Because of their specific strength, we chose four different promoters (Table 1).

Table 1. For our project, we were interested in four different promoters from the Anderson Collection. For each part, we used an intern number, which will be used in the text, as well.

The activity of these promoters was mainly characterized by measurement of the activity of a reporter protein expressed from these promoters. Protein activity is not only determined by the efficiency of transcription. In addition to this, translation efficiency contributes to the protein amounts in a cell. In particular, regarding fluorescent proteins like RFP or GFP, the growth phase might affect their fluorescence (see discussion below). A more direct way to determine the promoter activity is to assess the transcript levels. We were interested in how the mRNA levels of RFP placed downstream of the promoters from the Anderson collection change dependent on the promoter and how the protein activity compares to them.

For this, we transformed E. coli DH5α with the promoter parts present in the plasmid backbone BBa_J61002. As an empty vector control lacking a promoter and RFP, we used E. coli cells transformed with [http://partsregistry.org/Part:BBa_B0034 BBa_B0034(an RBS)] in plasmid Backbone pSB1A2. Subsequently, we characterized three clones of each strain by qRT-PCR and by monitoring the RFP fluorescence in different growth stages. In addition to this, we did fluorescence microscopy of exponentially growing cells (Fig. 1).

For qRT-PCR analysis (Fig. 2), E. coli cells were harvested at an OD_600nm of ca. 0.6 (exponential growth phase) and after 5 – 6 h when the OD_600nm had reached approximately 2 (stationary phase). The cells were incubated over night and a third sample taken the next morning ( > 20 h incubation, OD600nm was ca. 2 – 3). The relative mRNA levels of rfp were normalized to the rpoA levels. Then, the fold change (∆∆C_T) of the promoters compared to promoter 1 (the weakest) was calculated for each phase. The fold enrichments obtained for different growth phases could not be compared since the C_T values determined for rpoA suggested a generally altered transcription during the different growth stages.

Plate reader measurements served to determine the RFP fluorescence. The wells of a titer plate were inoculated with the clones and the RFP fluorescence and the OD_600nm measured over the time. (Fig 3).

Results

To characterize the promoters, we transformed E. coli DH5α with the promoter parts present in the plasmid backbone [http://partsregistry.org/Part:BBa_J61002 BBa_J61002]. As an empty vector control lacking a promoter and RFP, we used E. coli cells transformed with [http://partsregistry.org/Part:BBa_B0034 BBa_B0034] (an RBS) in plasmid Backbone pSB1A2. Subsequently, we characterized different clones (Clone 1 – 3) of these strains by qRT-PCR and by monitoring the RFP fluorescence in different growth stages. In addition to this, we did fluorescence microscopy of exponentially growing cells of clone 2 of each strain (Fig. 1). We noticed that the cells of the control strain did not fluoresce, while all strains transformed with a promoter plasmid exhibited a red fluorescence. The cells with promoter 1 and promoter 2 seemed to fluoresce less than those of promoter 3 and promoter 4.

Fig. 1. The E. coli cells shown above harbored an Anderson promoter plasmid or a control vector without RFP. The cells were cultured in broth LBAmp, harvested during exponential growth and analyzed by fluorescence microscopy. Left: Bright field (BF); Right: RFP; Abbreviations: P1 = [http:partsregistry/Part:BBa_J23117 BBa_J23117]; P2 = [http://partsregistry/Part:BBa_J23116 BBa_J23116]; P3 = [http://partsregistry/Part:BBa_J23110 BBa_J23110]; P4 = [http://partsregistry/Part:BBa_J23118 BBa_J23118]; P8 = [http://partsregistry/Part:BBB0034 BBa_B0034]; C2 = Clone 2.

For qRT-PCR analysis (Fig. 2), E. coli cells cultured in broth LB^Amp medium were harvested at an OD_600nm of ca. 0.6 (exponential growth phase) and when the OD_600nm had reached approximately 2 (stationary phase). The cells were incubated over night and a third sample taken the next morning (OD_600nm was ca. 2 – 3). The relative mRNA levels of rfp were normalized to the rpoA levels. Then, the fold change (∆∆C_T) of the promoters compared to promoter 1 (the weakest) was calculated for each phase. The fold enrichment obtained for different growth phases could not be compared since the CT values determined for rpoA suggested a generally altered transcription during the different growth stages. For the exponential phase (Fig. 2 A), the fold changes indicate that Promoters 1 and 2 were weak. For Promoter 2, the rfp mRNA levels appeared to be slightly higher than for Promoter 1. Promoter 3 led to even higher rfp mRNA levels. For stationary phase (Fig. 2 B) and overnight incubation (Fig. 2 C), the same tendency was observed. Only for Promoter 4 during stationary phase, the fold changes for the replicates provided contradicting results: While replicate 1 suggested that this promoter was stronger during stationary phase than promoter 3, the second replicate indicated the opposite. In addition to this, Promoter 4 seemed not to differ significantly in activity compared to Promoter 1 when considering replicate 2.

Fig. 2. The RNA from two E. coli clones of cells transformed with an Anderson promoter plasmid was subjected to qRT-PCR analysis. The C_T values of rfp determined by qRT-PCR were normalized to those of rpoA. The fold change of rfp transcript levels relative to the rfp transcript levels of promoter 1 were calculated. The bars indicate the fold changes. Two independent replicates are shown. Replica 1 = Clone 1; Replica 2 = Clone 3.

Plate reader measurements served to determine the RFP fluorescence. The wells of a titer plate were inoculated with the clones and the RFP fluorescence and the OD_600nm measured over the time (Fig 3). During the initial growth phase, all strains, including the non-fluorescent control strain, showed slightly elevated RFP/OD_600nm. For all clones 1, fluctuation during the first hours was observed, as well. During logarithmic growth of the strains, the RFP fluorescence dropped (minimum at 4 – 5 h). While the fluorescence remained low for the control strain, it increased for all promoter clones as soon as the stationary phase was reached (>5 h). After that, the fluorescence and optical density remained largely constant. The RFP fluorescence in stationary phase was strongest for Promoter 3 clones, followed by those of Promoter 4 and, then, Promoter 2. Promoter 1 clones showed the weakest RFP fluorescence.

Discussion

Since we used two of the promoters from the Anderson collection for our reporter system, we characterized them further. In doing so we compared three different methods to monitor promoter strength: fluorescence microscopy, determination of relative transcript levels of the rfp reporter gene by qRT-PCR and measurement of the fluorescence of the RFP protein. The fluorescence microscopy provided only a very rough impression of the different strength of the promoters. However, we could conclude, that RFP was expressed indicating that the promoters were functional. The weaker fluorescence observed for Promoter 1 and Promoter 2 indicated that these promoters might be less active than the other two.

The plate reader experiments allowed deeper insights into the strength of the different promoters. In these measurements, the observed fluctuations and elevated RFP/OD_600nm values during initial growth might result from the low cell number in LB medium and might therefore be not significant (compare: click here). The RFP fluorescence remained low during exponential growth. After 5 h, there was a strong increase in RFP fluorescence. At the same time, cell growth slowed down suggesting that both, reduced cell growth and stronger fluorescence might be correlated. In order to fluoresce, fluorescent proteins like GFP and RFP need oxygen (Remington, 2006). The oxygen RFP needs for fluorophore maturation could not have been available during log phase, since the cells consume the oxygen for enhanced cell growth. This possibility underlines the advantage of promoter characterization via transcript levels over promoter characterization via fluorophores. Still, fluorophore-depended characterization might be useful during stationary phase.

By qRT-PCR we were able to observe a tendency for the strength of the different promoters, though the exact fold changes did not seem to be reproducible. This lack of reproducibility might have resulted from the clones which differed in age. While the clones from replicate 1 were from the first transformation of the Biobrick vectors, the clones from replicate 2 originated from a new re-transformation of the plasmids. In general, our results suggested that Promoter 1 was the weakest, followed by Promoter 2, which was slightly stronger. Most interestingly, in E. coli DH5α, Promoter 3 seemed to be even stronger than Promoter 4, though it was shown to be weaker than promoter 4 before. However, our observations regarding Promoter 4 are difficult to interpret. First, this promoter led to contradicting results regarding the rfp fold changes during stationary phase, while the plate reader results independent of the clone indicated that it is weaker than Promoter 3. Second, qRT-PCR replicate 2 suggested that it is almost as weak as Promoter 4, which is in contrast to all other results obtained for this promoter. There are two possible explanations: (a) An experimental mistake resulted in insignificant fold changes; (b) Promoter 4 is subject to a complex regulation depending on the exact growth conditions. Considering this, it has to be noted that it is difficult to harvest the cells for RNA preparation at exactly the same OD_600nm. Hence, we took the samples for all strains at the same time when they had reached the approximate OD_600nm we were interested in. Since none of the two explanations could be ruled out, further characterization is necessary to obtain a more detailed view of the promoters and their regulation under different growth conditions. All in all, our results showed that it is useful to apply several methods for characterization. Taking the majority of the results of the three methods into account, we suggest that the order of the promoter activity in E. coli DH5α grown in LB^Amp is as follows: Promoter 1 [http://partsregistry.org/Part:BBa_J23117 BBa_J23117]< Promoter 2 [http://partsregistry.org/Part:BBa_J23116 BBa_J23116] < Promoter 4 [http://partsregistry.org/Part:BBa_J23118 BBa_J23118] < Promoter 3 [http://partsregistry.org/Part:BBa_J23110 BBa_J23110].

References

Roger Y. Tsien (1998) “The green fluorescent protein“, Annu. Rev. Biochem. Vol. 67, pp. 509–544

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