The Construction of The Modular Genetic Tool

The construction of the modular genetic tool started with the incorporation and/or the exchange of the numerous unique restriction sites according to Figure 4.26-B. To this end, a series of twelve different QuikChanges (Ch. 3.2.3.10) was performed that mutated the basic pBK synthetase plasmids pCLA9 (PylS His6-tagged “gap” variant) and pCLA3 (MjYRS_AGGA) as well as the pREP plasmids for the tRNAs PylT (pCLA4) and MjYT_UCCU (pCLA6). Table 4.2 shows a list of all applied QCs including plasmids that were used for mutagenesis. Every cloning step was checked for success via restriction digest using the appropriate enzymes (data not shown) and sequencing data revealed that all twelve QCs were successfully mutated.

Table 4.2: Overview of all QuikChanges performed in order to construct the modular genetic tool.

Twelve different QCs were made in total. The number of the QC is aligned with the used template, the purpose of the QC and the resulting plasmid. Except for pCLA77 and pCLA80 all produced plasmids were sequenced.

Details of the construction (primers and sequencing primers) can be found in Table 8.3.

QC Template Purpose Product

#

MjYRS_AGGA 1 pCLA3 C-terminal His6-tag on MjYRS_AGGA

pCLA71 2 pCLA71 SalI restriction site behind MjYRS

terminator

pCLA72

PylS

3 pCLA9 NdeI restriction site in front of PylS start codon changed to KpnI

pCLA73 4 pCLA73 PstI restriction site behind PylS

stop codon changed to SacI

pCLA74 5 pCLA74 SalI restriction site in front of

PylS promoter

pCLA75 6 pCLA75 NotI restriction site behind PylS

terminator 8 pCLA77 NotI restriction site in front of

PylT promoter

pCLA78 9 pCLA78 MfeI restriction site behind PylT

terminator

pCLA79

MjYT_UCCU 10 pCLA6 deletion of genes for T7 RNA Polymerase, AraC and GFP

pCLA80 11 pCLA80 MfeI restriction site in front of

MjYT_UCCU promoter

pCLA81 12 pCLA81 XhoI restriction site behind

MjYT_UCCU terminator

pCLA82

The extent of the mutagenesis in the preparation of the modular genetic tool holds a certain amount of risk because one or more mutations could influence the efficiency of the system negatively. Thus, chloramphenicol reporter assays were made to test the final plasmids pCLA72, pCLA76, pCLA79 and pCLA82 as well as the intermediate steps. Most of the plasmids revealed the same suppression efficiency as the basic plasmid (data not shown), except for PylS (Figure 4.27). Independent of the plasmid used for transformation, the cells were not able to survive Cm concentrations higher than 25 µg/mL. However, cells that harbored the unchanged basic plasmid (pCLA9) were still able to grow on agar plates with 400 µg/mL chloramphenicol.

Figure 4.27: Cm-Assay with final pBK PylS plasmid and intermediate steps from preparation for the modular genetic tool.

The catalytic activity of PylS His6 “gap” on the basic plasmid (pCLA9) was compared to PylS on the mutated plasmids pCLA73 to pCLA76 from QC#3-6 (Table 4.2). Therefore a chloramphenicol reporter assay (Ch. 3.2.4.1) was performed and cells were plated on agar plates containing Kan, Tet, increasing Cm and 1 mM BocK. Only cells with the basic plasmid were able to survive Cm concentrations higher than 25 µg/mL.

Since the activity already decreased after the first QC that exchanged the NdeI restriction with KpnI and the resulting plasmid served as precursor for the following QCs, we needed to determine if the catalytic activity of PylS itself was disturbed or if there was another cause for this effect. For this reason QC#5 and #6 (Table 4.2) were repeated using the basic plasmid (pCLA9) as template this time (QC#5B: pCLA83 and #6B: pCLA84), in order to incorporate the corresponding sites in a different order. Additionally, two further clones of QC#3 were tested in a subsequent Cm reporter assay (Figure 4.28).

Figure 4.28: Cm-Assay to detect the stage responsible for PylS activity loss.

The catalytic activity of PylS His6 “gap” on the basic plasmid (pCLA9) was compared to PylS on the mutated plasmids pCLA73, pCLA83 and pCLA84 from QC#3, #5B and #6B (two clones each). Therefore a chloramphenicol reporter assay (Ch. 3.2.4.1) was performed and cells were plated on agar plates containing Kan, Tet, increasing Cm and 1 mM BocK. Only cells with the plasmid from QC#3 were not able to survive Cm concentrations of 25 µg/mL.

It could be stated that the incorporation of the restriction sites SalI and NotI alone did not perturb the suppression efficiency of PylS as depicted in Figure 4.28. In contrast, the two other clones of QC#3 tested, showed the same phenotype as before. Hence, the replacement of the NdeI site upstream of the PylS start codon by KpnI seemed to be the reason for the PylS activity loss.

It was necessary to exchange, or at least to destroy, the NdeI site at the PylS gene for the construction of the modular genetic tool because the gene for MjYRS_AGGA also possesses an NdeI site. We then asked two questions. First, is it possible to restore the PylS activity on pCLA76 by mutating the KpnI site back to NdeI? Second, is it possible to restore the activity and destroy the NdeI site simultaneously? A series of six different QCs was applied to pCLA76, one to restore the NdeI site and the other ones to restore the site while introducing point mutations (pCLA85 to pCLA90). The NdeI-restore mutants were again screened via Cm reporter assay (Figure 4.29).

Figure 4.29: Cm-Assay of the PylS NdeI restore mutants.

The catalytic activity of PylS His6 “gap” on the basic plasmid (pCLA9) was compared to PylS on the mutated plasmids pCLA73 (QC#3), pCLA85 (NdeI restore) and pCLA86 to pCLA90 (NdeI restore Point Mutations (PM) 1-5).

Therefore a chloramphenicol reporter assay (Ch. 3.2.4.1) was performed and cells were plated on agar plates containing Kan, Tet, increasing Cm and 1 mM BocK. Only cells with the plasmid from QC#3 were not able to survive Cm concentrations of 25 µg/mL.

The activity of PylS could be almost restored by reinstalling the NdeI site in front of the PylS start codon. The PylS activity of the point mutation variants of NdeI was similar to the fully reconstituted NdeI site, as shown in Figure 4.29. Moreover, the point mutated NdeI sites were not digested by the restriction enzyme NdeI any more (data not shown). Due to the

highest PylS activity of all point mutation clones, pCLA86 (NdeI-restore_PM1) was chosen for further experiments.

Since all needed restriction sites were cloned into the basic pBK and pREP vectors, according to Figure 4.26-B and Table 4.2, we performed digests in a preparative scale (Ch. 3.2.3.2; Table 4.3) to cut out the genes for both tRNA/aaRS pairs and combined it on a pCDF Duet-1 backbone (Novagen; pCLA91).

Table 4.3: Overview of preparative digests performed in order to construct the modular genetic tool.

Plasmids for inserts and backbones were digested with the same enzymes. The insert is equal to the component mentioned in the table. The plasmid pCLA86 had to be cut by three restriction enzymes to assure the full separation of backbone and insert on a subsequent agarose gel. Cutting with SalI and NotI only would yield bands with sizes of 1555 and 1707 bp, which are difficult to excise from gel precisely.

Plasmid Enzymes Component Product

Insert Backbone

pCLA72 pCLA91 BamHI + SalI MjYRS_AGGA pCLA92

pCLA86 pCLA92 SalI + NotI (+ XhoI) PylS pCLA93

pCLA79 pCLA93 NotI + MfeI PylT pCLA94

pCLA82 pCLA94 MfeI + XhoI MjYT_UCCU pCLA95

All four components could be successfully transferred into the pCDF Duet-1 vector. Plasmid integrity was verified by restriction enzyme digests (Figure 4.30) and sequencing.

Figure 4.30: Restriction digests of the final modular genetic tool.

The final plasmid pCLA95 that contains all four components (digests 1, 3, 5 and 7) was compared to the insert plasmids (Table 4.3) pCLA72 (digest 2), pCLA86 (digest 4), pCLA79 (digest 6) and pCLA82 (digest 8). Enzymes and sizes: BamHI and SalI (1 = 5618 and 1206; 2 = 1820 and 1206); SalI and NotI (3 =5269 and 1555; 4 = 1555, 890 and 817); NotI and MfeI (5 = 6612 and 212; 6 = 4879 and 212); MfeI and XhoI (7 = 6506 and 318; 8 = 4776 and 318).