Defined (Standard) Promoters

In chapter 4.2 we found that alterations of the PylS synthetase promoter also effected the suppression efficiency of the amber codon inside a gene of a protein of interest and thereby the level of expression itself. The promoter of library clone 3E was among others the most powerful one and increased the levels of expressed PylS and histone H3 at least twofold (Figure 4.19). Hence, it was known that the promoter was stronger than the wild type glnS variant, which was classified as a promoter of moderate strength^[163].

Two questions arose then: Is it possible to enhance the performance of the system once more by the usage of promoters characterized as strong, versus weak, and how can the library promoter be categorized in comparison to defined standard promoters like the E. coli lac promoter or the lambda (λ) phage promoter? To this end a collection of different promoters was made whose strengths were already evaluated in literature. This set of promoters included endogenous E. coli promoters and their derivatives, coliphage promoters, as well as synthetic promoter variants. Their relative strength was mainly specified by so called Pbla-units^[164]. These units represent the ratio of RNA transcribed from a certain gene under the control of a promoter of interest with the transcripts of the β-lactamase gene (bla) under its endogenous promoter (Pbla). For instance, the promoter PA1

of the phage T7, with 76 ± 9 Pbla-units, was the strongest one found by Deuschle^[164] and thereby 76-fold more efficient than Pbla with 1 Pbla-unit. To avoid the need of a DE3 cell background, what means to be independent of the T7 RNA polymerase, no promoters of phage T7 were chosen. A list of the selected promoters together with their Pbla-units, if available, is shown in Table 4.1.

The sequences of the compiled promoters were cloned in front of the pylS gene (pCLA85;

this plasmid is described in Ch. 4.4.2) while exchanging the glnS promoter at the same time.

We tested the capacity of these defined standard promoters concerning the suppression efficiency in the same way as we did for the PylS promoter library (Ch. 4.2.1) by expressing a His6-tagged histone H3 with an amber stop codon at position R52. Thus, E. coli BL21 cells were transformed with the relevant pBK plasmids, including WT glnS (pCLA9) and library 3E promoter (pCLA24) as references, again in combination with the PylT and H3 R52TAG genes containing pCDF plasmid (pCLA32). The expression was performed with BocK and the addition of IPTG started the synthesis of H3 as well as of PylS if provided with a Plac derived promoter. All other promoters are constitutive and do not need an inducer. The comparative western blot is depicted in Figure 4.25.

Table 4.1: Relative in vivo strength of selected promoters.

The strength of the promoters (P) from various origins is given in Pbla-units. The sequences were obtained from the mentioned references. No Pbla-units were found for Plac1-6 Mt5 and Pcp25. Plac1-6 Mt5 was described to have a relative transcription efficiency of 112% and is consequently 56-fold stronger than Plac that exhibited only 2%^[165]. The synthetic promoter Pcp25 was merely characterized as “quite strong” without showing the data^[160]. The strength of Pλ found by Deuschle^[164] was corrected upwards by Knaus^[166].

Origin Promoter (P) Relative strength [Pbla-units]

Our initial observations suggested that the expression of the synthetase PylS worked for all the different promoters tested except Pcp25, Pλcon/N25DSR and PH207 ( Figure 4.25). For Pcp25 no signal could be detected, but the Pλcon/N25DSR promoter samples revealed a relatively intense band smaller than the expected size of 48 kDa probably due to degradation. The PH207 lane showed a very weak signal of the same reduced size.

The various promoters were in control of a broad spectrum of produced PylS, ranging from small amounts, in the case of the WT promoter, up to a multiple fold increases of these signals, as in the case of the phage T5 promoter PD/E20. The yield of PylS correlated with most of the relative promoter strengths given in Table 4.1. We noticed the lowest abundances for the glnS WT promoter, which was characterized as moderate^[163] before, and the library promoter Plib3E followed by the consensus sequence based Pcon, which had the fewest Pbla-units of all. Plac was the next strongest candidate referring to Pbla-units, what also coincided with the signal intensities on the blot. The modified lac promoter variant Plac1-6 Mt5 seemed to be more powerful than its precursor, but not to that extent as stated by Liu^[165]. The phage promoters appeared to be the overall strongest ones, as expected by Pbla -units. In previously performed experiments (not shown) Pλcon/N25DSR already turned out to be the most powerful promoter, according to the 65 Pbla-units, assuming that the mass of degraded PylS protein had a high transcription rate as a cause. However, despite its strength this promoter was not successful because no full-length PylS was produced in the

end. Regarding the blot, the promoter hybrid PtacI revealed higher PylS intensities as anticipated with respect to Pbla-units, arranging between Pλ and PD/E20.

We then observed a strong correlation of produced PylS and expressed histone H3 R52TAG, similar to the PylS library clones (Figure 4.19). This means, that with an increasing level of PylS the level of histone increased as well. Nevertheless, there seemed to be a certain limit since even a moderate increase of PylS (compare Pcon, lane 1, and PglnS, lane 10) was already beneficial, while yields of histone H3 were not further improved, if more PylS was expressed. In contrast to the current result Plac has been shown in previous trials (not shown) to mediate histone H3 amounts similar to Pcon.

Figure 4.25: Comparative analysis of defined standard promoters with WT glnS and library clone 3E promoter.

The suppression efficiency in histone H3 R52TAG (pCLA32) expression of systems under the control of defined standard promoters (pCLA62 to pCLA70) was compared to systems where the aaRS PylS was expressed under the control of the WT glnS (pCLA9) or library clone 3E promoter (pCLA24). 1 mM Bock was used as UAA and H3 expression was induced with 0.5 mM IPTG at OD600 = 0.8 as well as PylS that was dependent on Plac promoter and its derivatives. All samples were normalized by OD600. For the western blot whole cell extracts were separated with SDS PAGE (Ch. 3.2.2.3) and blotted onto a PVDF membrane (Ch. 3.2.2.4). Anti-His-antibody was used as primary antibody.

Referring to the two questions at the beginning of this chapter, the library promoter 3E and also the WT glnS promoter could be classified as weak, if compared to the power of the other standard promoters. Furthermore, it was possible to significantly improve the suppression efficiency of the given system by using strong promoters. However, we considered Pcon and Plac to use in later experiments (Ch. 4.4.6) because the former one provided an enhancement of the system comparable to the strongest promoters and the latter one offered the advantage of an inducible promoter, what could be essential if a product is (very) toxic to the host cell.