3 Results and Discussion
3.2.1 M. mazei PylRS Library
As the libraries constructed from the M. barkeri PylRS did not seem very viable, a new library based on the PylRS from M. mazei was created within the framework of this thesis. To decide which residues should be mutated, the Trp analog [3,2]Tp was modeled into the M. mazei PylRS active site (Figure 32 a). Based on their proximity to [3,2]Tpa, the active site residues N346, C348, V401, W417, and G419 were chosen for randomization to NNS via site-saturation mutagenesis. The residues N346 and C348 are common targets for library design and frequently mutated in PylRS mutants selected for the incorporation of ncAAs205.The library was assembled via golden gate cloning493,494 on a pJZ plasmid with ampicillin resistance (backbone constructed during the master thesis). This cloning technique utilizes type IIs restriction endonucleases, which cut outside of their recognition sequence and thereby enable
“scarless” cloning. As the recognition sequence is cut off during digestion, cut sites can be placed anywhere, e. g. in the middle of genes, and are commonly introduced in the overhang of the PCR primers.
The quality of the library was surveyed via sequencing of the library mixture (Figure 32 b) as well as randomly picked single colonies (Figure 32 c). Some bias towards cytosine, especially at position G419 with the codon GGG can be seen. This is not unexpected, as GC base pairs form three hydrogen bonds and are therefore often favored in the annealing step of PCR reactions. At the beginning of the PCR only the wildtype, unmutated sequence is available as a template and thus due to stronger hydrogen bonding, those primers that contain many cytosines will preferentially anneal to the GGG codon of G419, producing C-rich templates for the following PCR cycles. Nevertheless, the sequencing results of the randomly picked colonies show good variety at the mutated positions. It should be pointed out that, with libraries of sizes commonly in the 106-108 range, sequencing of a few variants only offers a small snapshot and is not necessarily representative of the library. However, this small snapshot might at least reveal any major systematic errors that might have occurred during design and/or assembly.
As the results presented below do not reveal any obvious problems, the library was deemed satisfactory.
Figure 32 I M. mazei PylRS library. a) Crystal structure of M. mazei PylRS active site with bound ATP and modeled in [3,2]Tpa.
ATP (pink) and [3,2]Tpa (blue) are shown as sticks. Residues chosen for randomization are colored in yellow and also shown as sticks. PDB: 3VQV b) Chromatograph of the whole library sequencing. c) Sequencing results of seven randomly picked library members.
The completed library was then used in double-sieve selections to identify members capable of distinguishing between Trp and [3,2]Tpa. With NNS randomizations at 5 positions, the library has a theoretical size of 3 x 107 members. To ensure coverage of the entire library, freshly prepared electrocompetent cells were used for the first round of double-sieve selections. Transformation efficiencies were assessed by plating a dilution series of the recovered transformation on LB agar plates containing only the library plasmid’s selection marker (no double-sieve selection conditions). Counting of the single colonies of the dilution that produced distinguishable single colonies revealed that with 5 x 107 transformants library coverage was achieved.
To start selection with a low stringency a positive selection plasmid harboring only one stop codon in the chloramphenicol acetyl-transferase (CAT) gene was chosen. During this step, functional library
b)
1,210 1,220 1,230 1,240 1,250 1,260 1,270 1,273
2,447 2,457 2,467 2,477 2,487 2,497 2,507 2,510
Consensus
2,447 2,457 2,467 2,477 2,487 2,497 2,507 2,510
C T G N K S T T C N R S C A G A T G G G A T C G G G A T G C A C A C G G G A A A A T C T T G A A A G
1,420 1,430 1,440 1,450 1,460 1,470 1,480
2,657 2,667 2,677 2,687 2,697 2,707 2,717
Consensus
2,657 2,667 2,677 2,687 2,697 2,707 2,717
A T A A A C C C M C S A T A C C S G C A G G T T T C G G G C T C G A A C G C C T T C T A A A G G T
1,360 1,370 1,380 1,390 1,400 1,410 1,420 1,424
2,597 2,607 2,617 2,627 2,637 2,647 2,657 2,661
Consensus
2,597 2,607 2,617 2,627 2,637 2,647 2,657 2,661
G G A A C T T T C C T C T G C A K N S G T C G G A C C C A T A C C G C T T G A C C G G G A A T G G G
1,200 1,210 1,220 1,230 1,240 1,250 1,260 1,270 1,280 1,290 1,300 1,310 1,319
2,439 2,449 2,459 2,469 2,479 2,489 2,499 2,509 2,519 2,529 2,539 2,549 2,558
Consensus
2,439 2,449 2,459 2,469 2,479 2,489 2,499 2,509 2,519 2,529 2,539 2,549 2,558
G C T GTAGT T CG A GC A G A T G G G A T C G G G A T G C A C A C G G G A A A A T C T T G A A A G C A T A A T T A C G G A C T T C C T G A A C C A C C T G G G A A T T G A T T T C A A G A T C G
1,370 1,380 1,390 1,400 1,410 1,420 1,430 1,440 1,450 1,460 1,470 1,480 1,483
2,609 2,619 2,629 2,639 2,649 2,659 2,669 2,679 2,688 2,698 2,708 2,718 2,721
Consensus
2,609 2,619 2,629 2,639 2,649 2,659 2,669 2,679 2,688 2,698 2,708 2,718 2,721
T G C A GG CG T C G G A C C C A T A C C G C T T G A C C G G G A A T G G G G T A T T G A T A A A C C CCG G A T AAGCG C A - G G T T T C G G G C T C G A A C G C C T T C T A A A G G T T A A A
1,370 1,380 1,390 1,400 1,410 1,420 1,430 1,440 1,450 1,460 1,470 1,480 1,483
2,609 2,619 2,629 2,639 2,649 2,659 2,669 2,679 2,688 2,698 2,708 2,718 2,721
Consensus
2,609 2,619 2,629 2,639 2,649 2,659 2,669 2,679 2,688 2,698 2,708 2,718 2,721
T G C A GG CG T C G G A C C C A T A C C G C T T G A C C G G G A A T G G G G T A T T G A T A A A C C CCG G A T AAGCG C A - G G T T T C G G G C T C G A A C G C C T T C T A A A G G T T A A A
members are selected, as only colonies capable of suppressing the amber codon and expressing the resistance gene can survive in the presence of the selection marker chloramphenicol. As the selection plasmids harbored the same (low copy) origin of replication as the library plasmid (p15a), the ori on the selection plasmids was changed to ColE1. Plasmids with the same ori are thought to compete for replication factors, leading to one plasmid outgrowing its competitor495–497. Thus, the p15a ori’s on the selection plasmids were substituted with ColE1, which has a similar copy number, by golden gate cloning and the success of the substitution was verified via sequencing analysis.
The selection experiments were conducted on LB media and the stringency was increased with every positive selection by increasing the chloramphenicol (Cm) concentration from 37 µg/mL to 50 µg/mL to 70 µg/mL and finally 90 µg/mL. To follow the progress of the selection, the cells were streaked on selection plates in the presence of 0.1 mM [3,2]Tp, but also on control plates, where no [3,2]Tp was added. Comparison of colony numbers from the selection plates with those from the control plates gives a first indication, whether the mutants that are enriched with each round of selection charge the desired ncAA or one of the canonical amino acids. If mutants charging the desired ncAA are enriched, colonies on the selection plates should outnumber those on the control plates. For the first positive selection, the plates were incubated at 30 °C for 72 h to allow even slowly growing cells harboring less efficient PylRS variants to form colonies. Subsequent positive selections were incubated at 30 °C for 48 h. To ensure the screening of all library members, three transformations were conducted for the first positive selection. The transformations were pooled and spread on five large LB agar plates, whereby one plate served as a control plate without [3,2]Tp supplementation. As functional PylRS variants should be selected with each round of positive selection and thus the number of library members should decrease with each round, the number of transformations was likewise decreased with each round of positive selection. An overview of the experimental parameters is depicted in Table 1.
Table 1 I Overview of experimental parameters of the positive selections.
Round Media Cm [µg/mL]
[3,2]Tp [mM]
No. of transform.
No. of sel.
plates
No. of ctrl.
plates
1 LB 37 0.1 3 4 1
2 LB 50 0.1 2 3 1
3 LB 70 0.1 1 1 1
4 LB 90 0.1 1 1 1
After each positive selection, a negative selection was performed. Here, [3,2]Tp was not supplied and cells harboring PylRS mutants capable of charging any of the cAAs to their cognate tRNA can read through both amber codons, express the toxic barnase gene and die. Negative selections were incubated at 30 °C for only 14-15 h to avoid mutations of the barnase gene or inactivation of the selection plasmid. As barnase expression is extremely deadly, the selection pressure to avoid its expression is very high and thus the exposure was minimized. Expression of barnase was induced with 0.02 % arabinose and like the positive selections, the negative selections were conducted on LB media.
After each round of selection, the selection plasmids were digested to avoid carryover into the next round.
Figure 33 I Number of colonies on positive selection plates supplemented with 0.1 mM [3,2]Tp compared to control plates lacking [3,2]Tp. The colonies were counted using ImageJ, where every colony larger than 5 pixels was counted. The colony numbers per plate were normalized to the culture volume for better comparison. P1-4: positive selection 1-4, ctrl: control plates. P1 and P2 represent the mean of 4 and 3 plates, respectively, with the SD represented as an error bar.
Figure 33 shows the number of colonies on the selection plates compared to the number of colonies on the control plates after each round of positive selection (P1-4). The agar plates were scanned after incubation and colony numbers were counted using ImageJ, whereby every colony larger than 5 pixels was counted. It is of note that the first positive selection resulted in very diverse colony sizes with a few larger colonies (sel. Plate: 65 ± 12, ctrl.: 44) and numerous very small colonies (sel. plate:
1074 ± 337, ctrl.: 333 total), while the following positive selections yielded colonies of similar size. This phenomenon can likely be attributed to the longer incubation period of the first positive selection, which allowed even very slow-growing colonies to form. Representative agar plates of the positive selections are shown in the appendix (chapter 9.3, p. 163). Starting from the second round of positive selection, the number of colonies on the selection plates remains similar to the number of colonies on the corresponding control plates, while the colony number increases with each round of selection, even though the stringency is increased.
The overall increase of colony numbers after four rounds of positive selections and three rounds of negative selections indicate that certain functional PylRS variants were accumulated. However, while after the first round of positive selection the control plate exhibited only about 31 % of the colony numbers compared to the selection plates, this number increases to 95 % after the fourth positive selection, indicating that the selected variants are capable of charging one (or more) of the canonical amino acids. In order to examine whether [3,2]Tpa-charging mutants were enriched during selection, 30 colonies were picked and spotted on NMM19 –Trp plates with and without the non-canonical amino acid precursor [3,2]Tp and with increasing Cm concentrations (Figure 34). Minimal media lacking Trp were chosen, as the selection of a PylRS mutant capable of incorporating both, Trp as well as [3,2]Tp, was deemed likely due to their great structural similarity. In this case, the presence of Trp could complicate the read-out of this assay.
0 500 1000 1500 2000 2500 3000 3500 4000
number of colonies
P1 P2ctrl P3 P4
ctrl ctrl
ctrl
Cm
[µg/mL] - ncAA + 0.1 mM [3,2]Tp
37
70
120
Figure 34 I Screening for promising M. mazei PylRS library members. After the fourth positive selection, 30 colonies were resuspended in 50 µL sterile ddH2O and 2 µL of the suspension were spotted on NMM19 –Trp plates with and without 0.1 mM [3,2]Tp and with increasing Cm concentrations. The cells were incubated overnight at 37 °C.
Although all colonies can tolerate even high Cm concentrations there is no difference in growth between plates supplied with [3,2]Tp and plates without [3,2]Tp, suggesting that the selected PylRS mutants charge one of the canonical amino acids rather than [3,2]Tpa. Nevertheless, a few colonies from the fourth positive selection were sequenced in order to get an idea of what was enriched during selection. The sequencing results are summarized in Table 2.
As there was one sequence that was observed six times, a fluorescence assay was conducted to establish if this mutant is able to charge [3,2]Tpa (or Trp) to its cognate tRNA. BL21(DE3) cells were co-transformed with this PylRS HLLNQ mutant and a plasmid harboring the gene for sfGFP with an amber stop codon at position 2. In this experiment, sfGFP can only be successfully expressed and emit green light, when the amber stop codon is suppressed by the PylRS mutant. The experiment was conducted in a 96 well plate and fluorescence was detected by a plate reader. The cells were inoculated 1:100 and immediately induced with 1 mM IPTG.
Table 2 I Summary of the sequencing results of PylRS mutants isolated after the fourth positive selection. Six of eleven sequenced mutants have the same sequence (shown in bold).
Clone # Asn346 Cys348 V401 Trp417 Gly419
1 His Leu Leu Asn Gln
2 Gly Glu Ser His Asp
3 His Leu Leu Asn Gln
4 His Leu Leu Asn Gln
5 Gly Glu Ser His Asp
6 Asn Cys Val Trp Gly
7 Arg Gly Leu Pro Ala
8 Leu Gly Asp Val Leu
9 His Leu Leu Asn Gln
10 His Leu Leu Asn Gln
11 His Leu Leu Asn Gln
The tested media compositions are depicted in Table 3.
Table 3 I Media compositions tested in the fluorescence assay.
Media Amino acid/precursor Conc. [mM]
LB -
LB [3,2]Tp (precursor) 0.1
NMM19 - Trp [3,2]Tp (precursor) 1 NMM19 - Trp [3,2]Tp (precursor) 0.1 NMM19 - Trp [3,2]Tp (precursor) 0.03
NMM19 - Trp - -
NMM19 - Trp Trp (aa) 0.03
NMM19 - Trp Trp (aa) 0.1
NMM19 - Trp Trp (aa) 1
The absorbance at 600 nm as well as the fluorescence at 511 nm was detected overnight (Figure 35).
The absorption spectra (Figure 35 a) reveal that [3,2]Tp concentrations as high as 0.1 mM and 1 mM are likely toxic in minimal media, as no cell growth can be observed for these cultures. Interestingly, cells cultured in minimal media supplied with 0.03 mM [3,2]Tp seem to have a very long lag phase and only start growing after some 15 h of incubation at 37°C. The [3,2]Tp toxicity is less pronounced in rich media and cells supplemented with 0.1 mM in LB grow normally. Figure 35 b shows the fluorescence after 15 h of incubation, where most cultures had reached the stationary phase. However, as one culture had not reached the stationary phase yet and two cultures did not grow at all, fluorescence was not normalized to the absorption. No increase in fluorescence could be observed after 15 h of incubation, not even for NMM19 – Trp + 0.03 µM [3,2]Tp, where cell growth only started after 15 h.
There is no difference in fluorescence for cells cultivated in the absence of [3,2]Tp or Trp and those
supplemented with [3,2]Tp or Trp, indicating that the enriched PylRS mutant does not incorporate either of these two amino acids.
As suggested by phylogenetic analyses, the PylRS likely evolved from bacterial PheRS498, which have a structurally similar catalytic site. As a consequence, PylRS mutants that incorporate Phe are fairly easily selected499, which might have happened in this case as well. However, sequence comparison of the mutant enriched in this study with selected FRS mutants revealed only the C348L mutation as a match.
Especially the N346H mutation observed here differs greatly from the C348A or C348S mutations observed in the selected FRS mutants and cannot be observed in any other published PylRS mutants205,278. Thus, the identity of the amino acid incorporated by the HLLNQ mutant enriched here remains a mystery and was not investigated further.
Figure 35 I Fluorescence assay of sfGFP(R2TAG) with the PylRS HLLNQ mutant in media supplemented with and without Trp or [3,2]Tp. a) Absorbance at 600 nm. b) Fluorescence after 15 h of incubation.