PylS Library

The glnS promoter of the pylS gene (pCLA86; this plasmid is described in Ch. 4.4.2) was randomized as described above. We determined a diversity of 5*10⁶ different clones for this library. For the separation of inactive clones from active ones the amplified DNA was combined with a reporter plasmid (pCLA4) to select on chloramphenicol (Ch. 3.2.4.1). A dilution of cells was plated that consisted of 5*10⁰, 5*10¹, 5*10² cells on agar plates with 0 µg/mL Cm and 5*10³, 5*10⁵, 5*10⁷ cells on plates with 25, 50 and 100 µg/mL Cm in the absence and presence of BocK. This was done for a clear discrimination of the effects from Cm and to obtain single clones for further investigations, e.g., broader range Cm assays. No growth could be observed on agar plates without Bock.

Cells were collected for the agar plates with BocK and the 5*10⁷ cell dilution in order to regain library plasmids from active clones and simultaneously remove reporter plasmids (Ch. 3.2.3.5). We chose the high cell count plates to assure the covering of the library diversity. Plasmid integrity was verified by restriction endonuclease digest (not shown).

As mentioned before, agar plates with low cell counts resulted in single colonies. In order to screen the diversity of the library 90 different clones from plates with 25, 50 and 100 µg/mL Cm were transferred onto fresh agar plates with a broader range of Cm, from 0 to 500 µg/mL, allowing a more precise differentiation of these clones (Figure 4.18).

Referring to Figure 4.18, a tendency of higher resistance to chloramphenicol from left to right could be observed. More cells on the right side of the agar plate survived the Cm concentration of 400 µg/mL in the presence of BocK, but also a Cm concentration of 75 to 100 µg/mL without BocK. This increase in resistance correlated with the amount of chloramphenicol that was existent in the original agar plates. Clones with the reference WT glnS promoter already showed a weaker growth at 250 µg/mL.

Next, we addressed changes on the promoters that have positive effects on suppression efficiency. For this purpose, ten out of the 90 single library clones were analyzed by western and northern blots. Since all clones survived a Cm concentration of 250 µg/mL in the presence of BocK, we only selected clones that also survived 400 µg/mL Cm, or those that could not survive low concentrations of Cm without BocK. These were the clones 1D, 2E, 3E, 4F, 6B, 7A, 8G, 10D, 11D and 12H, according to the grid in Figure 4.18. The pBK PylS library plasmid variants were separated from the reporter plasmid (Ch. 3.2.3.5; not shown).

Figure 4.18: Cm-Assay with single colonies from PylS library.

Cells were plated on agar plates containing Kan, Tet, increasing Cm and 1 mM BocK (A and B) or no BocK (C and D). A selection of two plates each is shown only. The clones 1A, 5A and 9A contained pCLA9 as a reference and clones 2A, 6A and 10A harbored pCLA86 as a reference. Clones in columns 1-4 originated from the library agar plate with 25 µg/mL Cm and 1 mM Bock, columns 5-8 from the plate with 50 µg/mL Cm and 1 mM BocK and columns 9-12 from the plate with 100 µg/mL Cm and 1 mM BocK.

The purified library plasmids (pCLA22 to pCLA31) were sequenced to assure that all selected clones differed in sequence, but exclusively in the promoter region. Changes in the expression rate of PylS should then be due to mutations in the. The sequencing showed that beside a few silent mutations only those parts of the promoters were mutated which were intended to be. A sequence alignment of all ten library clones revealed that none of them had the same sequence (Figure 8.1).

Lastly, we transformed E. coli BL21 cells with the isolated library plasmids in combination with a vector that harbored the genes for the cognate tRNA PylT and a His6-tagged histone H3 with an amber stop codon at position R52 (pCLA32). For suppression of the stop codon during the H3 expression the medium was supplemented with BocK. In the end, two samples of each culture were taken with one being used for comparative western blot analysis of expressed PylS and histone H3. The other was used for northern blot to investigate if the library also had an effect on this level of the translational apparatus (Figure 4.19).

Figure 4.19: Comparative analysis of PylS lib clones via western and northern blot.

For the investigation of the suppression efficiency of the library clones a histone H3 R52TAG (pCLA32) expression was performed. 1 mM Bock was used as UAA and H3 expression was induced with 0.5 mM IPTG at OD600 = 0.8. A selection of six different library clones (pCLA22 to pCLA27) was compared to WT PylS, which comprised the unchanged glnS promoter (pCLA9). 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 nitrocellulose membrane (Ch. 3.2.2.4). Anti-His-antibody was used as primary antibody. For the detection of the cognate tRNA PylT total RNA was extracted from a second aliquot (Ch. 3.2.3.14). Approximately 0.090 µg RNA were loaded for each sample. The DIG labeled probe C26 was utilized for hybridization and chemiluminescence signals recorded as described in Ch. 3.2.3.15. The upper band in the second row represents the aminoacylated form of PylT, whereas the lower band depicts the uncharged form.

We found that the PylS promoter library exhibited a variety of different promoter strengths. There were clones, for instance 3E, that had an increased expression efficiency of the synthetase PylS compared to the WT. On the other hand, we also observed clones, such as 4F, which displayed a strong decrease. Furthermore, the more PylS was expressed in the cells, the more PylT in the aminoacylated, slower migrating form, could be observed.

Finally, similar to the correlation of synthetase and cognate tRNA, we monitored a clear dependence on the amount of PylS produced and the suppression efficiency, visualized by

the amount of histone H3 R52TAG expressed. Hence, it was possible to influence the suppression efficiency of the amber stop codon with the aid of the PylS promoter library with an increase in PylS resulting in higher expression levels of H3.