PylT and MjYT_UCCU Library

The last two promoters that had to be changed were the lpp promoters of the pylT and mjYT_UCCU genes. The modification of the pylT promoter (pCLA79; this plasmid is described in Ch. 4.4.2) resulted in a library that held 5*10⁵ different clones. The mutagenesis on the M. jannaschii tRNA promoter (pCLA82; this plasmid is described in Ch. 4.4.2) yielded a diversity of 1.2*10⁶ distinct clones.

For the removal of inactive clones via Cm reporter assay the tRNA libraries had to be combined with their cognate aaRS. This is because they were cloned and mutated on the reporter plasmids themselves. The PylT library was used for transformation together with the PylS (pCLA1) and the MjYT_UCCU library together with the MjYRS_AGGA (pCLA3). Cells were plated as before (Ch. 4.2.1) with no extra UAA added to the agar plates for the M. jannaschii system and Bock added for the M. barkeri pair. The quantity of colonies generally decreased while increasing the Cm concentration. The DNA of both libraries was recovered and checked for correct sequence identity. In the case of PylT a strong contamination with the parental reporter plasmid (pCLA4) was found, which ran higher on an agarose gel (bands at 5625 and 4701 bp) than the mutagenized version (bands at 3928 and 1163 bp) used for making the library (data not shown). Before repeating the whole procedure, the usage of the restriction enzyme NdeI, which only cuts the parental plasmid, helped to clean the PylT library DNA (that still included the inactive clones). Thus, both isolated libraries showed the correct sizes as confirmed by restriction enzyme digest (not shown).

We selected a total number of 96 different clones of each library from the single colony plates with 25, 50 and 100 µg/mL of Cm to transfer them on fresh agar plates for the broad range Cm reporter assay (0 to 500 µg/mL). The results for the PylT library are shown in Figure 4.23 and for the MjYT_UCCU library in Figure 4.24.

The control plates without BocK in Figure 4.23 showed that most of the PylT library clones were not able to survive concentrations of Cm higher than 75 µg/mL and all of them died at 150 µg/mL. In the presence of BocK all clones could resist 250 µg/mL, but only a few colonies in column I withstood 400 µg/mL. These clones originated from the Cm100 single colony plate, a trait already observed for the PylS library, but less distinctive.

Comparable to the MjYRS library, the MjYT_UCCU library clones displayed a wide range of growth efficiencies over the whole plate, independently from the original Cm concentration (Figure 4.24). Most of the clones resisted a Cm concentration of 400 µg/mL, but none of

them survived 500 µg/mL. A few colonies became less dense at lower concentrations, e.g., the clones B3 and B6 already at 150 µg/mL (not shown) and the clones H6 and J2 at 250 µg/mL.

Figure 4.23: Cm-Assay with single colonies from PylT 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. Clones in columns A-D originated from the library agar plate with 25 µg/mL Cm and 1 mM Bock, columns E-H from the plate with 50 µg/mL Cm and 1 mM BocK and columns I-L from the plate with 100 µg/mL Cm and 1 mM BocK.

Figure 4.24: Cm-Assay with single colonies from MjYT_UCCU library.

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

Clones in columns A-D originated from the library agar plate with 25 µg/mL Cm, columns E-H from the plate with 50 µg/mL Cm and columns I-L from the plate with 100 µg/mL Cm.

As before, ten out of the 96 clones of each tRNA library were selected to investigate effects on the suppression efficiency, due to changes in the lpp promoter, with western and northern blots. The clones A4, C2, E7, F5, H4, I3, I6, J7, K2 and L5, according to the grid in Figure 4.23, were chosen for the PylT library and their DNA separated from the synthetase plasmid (Ch. 3.2.3.5). Full separation was assured by the use of the restriction enzymes NdeI and StuI, which only cut the pBK synthetase plasmids (pCLA1 and pCLA3) but not the modified tRNA containing reporter plasmids (pCLA79 and pCLA82). The same was done for MjYT library clones B3, B8, D4, E1, G4, G7, I2, I8, K6 and L1, according to the grid in Figure 4.24. This procedure failed for PylT clone H4 and for MjYT clone G4, thus only nine clones per library were available (data not shown).

Finally, the sequencing of all purified tRNA library plasmids (pCLA44 to pCLA61) revealed that for both libraries, beside a few minor mutations, only those nucleotides surrounding the predicted -10 and -35 box were altered. The sequence alignment of each library illustrated and confirmed the diversity of the selected clones, with all of them being divergent (Figure 8.3 and Figure 8.4).

In conclusion, promoter libraries for each component of the two tRNA/aaRS pairs originating from the organisms M. barkeri and M. jannaschii have been prepared. The PylS library revealed a strong dependence of expressed synthetase on the expression of the protein of interest. When more synthetase was expressed it leads to an increased expression level for the desired protein. The MjYRS library did not display such a clear depenence. Individual studies of the tRNA library clones with western and northern blots have not been done, yet. Both libraries were used in combination with the synthetase libraries, as described in a later chapter (Ch. 4.4.4).