MjYRS_AGGA Library

The glns promoter of the synthetase MjYRS (frameshift codon version = AGGA) was mutated next (pCLA72; this plasmid is described in Ch. 4.4.2.). Unfortunately, the standard touchdown PCR protocol resulted in two PCR products, with the minority having the expected size of approximately 3000 bp but the majority having a size of roughly 1200 bp.

This effect was suspected to be due to unspecific primer annealing, despite the touchdown, and needed to be optimized. Therefore, we eliminated the touchdown portion of the PCR, i.e., the first ten cycles, and applied a gradient of increasing annealing temperatures. The gradient covered 54.5 to 66.5 °C, in twelve steps. This modified inverse gradient PCR was set up twice, with one set being supplemented with 5% DMSO, which was thought to reduce the formation of secondary structures of template and primer DNA^[161,162].

Figure 4.20: Inverse gradient PCR for the MjYRS promoter library.

Two sets were tested with 12 different temperatures each, ranging from 54.5 to 66.5 °C (left to right; indicated by the black triangles). One set was supplemented with 5% DMSO. The expected size of the PCR product is approximately 3000 bp.

An increase of the annealing temperature caused a gain of the desired PCR product, as depicted in Figure 4.20. The addition of DMSO in combination with the high annealing temperature of 66.5 °C finally led to a shift from undesired to the desired PCR product of expected size.

Cells transformed with the product of the PCR using these optimized parameters yielded 1.5*10⁶ different clones. Inactive clones were removed as described above for the PylS library using the Cm reporter (pCLA6). Since MjYRS, in the version described here, decodes for tyrosine, no control plates without the “UAA” could be made. However, a decreasing number of colonies formed on increasing Cm concentrations. The library DNA was again

isolated from plates with 5*10⁷ cells and the isolation verified by restriction endonuclease digest (not shown).

The screening of the library diversity was the next step. To this end, we picked 30 different clones each from those single colony plates with 25, 50 and 100 µg/mL of Cm and transferred them onto fresh agar plates with a broader range of Cm, from 0 to 500 µg/mL, (Figure 4.21).

Compared to the PylS library clones no tendency of higher resistance to chloramphenicol from left to right could be noticed. In contrast to PylS, where the reference clones already showed weaker growth at 250 µg/mL Cm, the MjYRS WT promoter clones died more abruptly. None of the selected clones survived the highest concentration of 500 µg/mL.

Figure 4.21: Cm-Assay with single colonies from MjYRS_AGGA library.

Cells were plated on agar plates containing Amp, Tet and increasing Cm. A selection of two plates is shown only.

The clones 1A, 5A and 9A contained pCLA3 as a reference and clones 2A, 6A and 10A harbored pCLA72 as a reference. Clones in columns 1-4 originated from the library agar plate with 25 µg/mL Cm, columns 5-8 from the plate with 50 µg/mL Cm and columns 9-12 from the plate with 100 µg/mL Cm.

To investigate if the mutagenesis of the glnS promoter of the mjyrS gene had similar effects on the suppression efficiency as that of the mutant pylS promoter, ten clones out of the 90 were chosen. Since all the clones survived a Cm concentration of 250 µg/mL again, we considered how they behaved on the Cm400 plate. The DNA of the colonies 1D, 2F, 3B, 4A, 6D, 7E, 8G, 9F, 10B and 11E, according to the grid in Figure 4.21, was isolated, because these clones represented a range of growth behavior. Thus, they were supposed to be able to repeat the distinct promoter strengths achieved with the PylS library. Therefore, the reporter plasmid was removed as previously described.

Sequencing of the purified library plasmids (pCLA33 to pCLA42) confirmed the intended mutagenesis of the nucleotides surrounding the predicted -10 and -35 box, with the boxes

themselves remaining unaffected. A sequence alignment of all ten library clones revealed, that none of them had the same sequence (Figure 8.2).

For the classification of the library promoter strengths myoglobin with a frameshift codon at position S4 (pCLA43) was expressed. For this reason E. coli DH10B cells were transformed with the library plasmids pCLA33 to pCLA42 together with pCLA43, a vector that contained the genes for the cognate tRNA MjYT_UCCU and the His6-tagged myoglobin. Since the synthetase decodes for tyrosine no extra amino acid was added to the medium. Figure 4.22 shows the comparison of the ability to suppress the AGGA codon in case of myoglobin.

Regarding the blot in Figure 4.22 the expression levels of myoglobin fluctuated only in a small range, hardly distinguishable by eye. The quantification using ImageJ revealed a minimal signal intensity of 65% and a maximal intensity of 115% compared to wild type. In contrast to the PylS library (Ch. 4.2.1), the MjYRS_AGGA promoter library seemed to have only a small influence on the suppression efficiency. Correlations between expressed synthetase and the protein of interest were not possible since the aaRS was not detectable on the blots.

Figure 4.22: Suppression efficiency of MjYRS_AGGA lib clones.

The efficiency was determined by the expression of Myoglobin S4AGGA (pCLA43). Expression was induced with 0.2% arabinose (w/v) at OD600 = 0.8 and analyzed by western blot (lower panel) and the relative intensities measured by ImageJ (blue bars; upper panel; WT was set to 100%). The ten library clones (pCLA33 to pCLA42) were tested with respect to WT MjYRS_AGGA, which comprised the unchanged glnS promoter (pCLA72). For the western blot all samples were normalized by OD600, 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.