Caso 10. Insumo: PA392390-Producto final: 230990- Destino final Nueva Zelanda 45

The enzymatic activity of the purified Ms02 DPCK enzyme was determined by performing in vitro enzyme reactions, followed by analysis via HPLC. The 50 µl reaction mixtures were incubated for 1 h at 37˚C and consisted of the following: 50 mM Tris-HCl buffer (pH 7.6), 20 mM KCl, 10 mM MgCl2, 1 mM Tris(2-carboxyethyl)phosphine, 1.8 mM phosphoenol-

85 pyruvate, 0.04 U/µl pyruvate kinase, 1.5 mM ATP, 0.25 mM DePCoA and 0.1 mg/ml DPCK enzyme. Subsequently, the derivatisation and HPLC analysis were performed as described by Goosen et al. [231]. The purified DPCK enzyme of E. coli was used as positive control.

4.4 Results

4.4.1 Amplification, cloning and site-directed mutagenesis of msDPCK

4.4.1.1 Amplification and isolation of msDPCK using Ms02 genomic DNA

The msDPCK gene (579 bp) could be successfully amplified at three of the five tested annealing temperatures (50˚C, 55˚C, 60˚C) by using Ms02 genomic DNA as template, which can be observed on the agarose gel analysis of the PCR products (Figure 4.4 A). Additionally, the agarose gel analysis showed that each of the annealing temperatures produced a single PCR product with no non-specific binding.

Consequently, the Ta of 55˚C was selected for large-scale amplification and subsequent

purification as the PCR product at this Ta produced a thick and bright band at the expected

size (~600 bp), which indicated a high concentration of product. A Ta of 50˚C also produced

a thick and bright band; however, the selected Ta of 55˚C was closer to the estimated Tm of

±58˚C (according to the IDT specification sheet) and was, therefore, the preferred choice. The subsequent pooled PCR products were successfully purified producing a single band at the expected size (~600 bp, Figure 4.4 B). The estimated concentration of the purified msDPCK PCR product was determined to be 273.7 ng/µl.

Figure 4.4 2% Agarose gels showing the PCR product of the amplification of the msDPCK gene

using the Ms02 genomic DNA as template. (A) Optimisation of annealing temperatures (Ta). The

orange box indicates the expected position of the amplified product. M: 100 bp DNA ladder marker, Lane 1-2: Ta = 50˚C, Lane 3-4: Ta = 55˚C, Lane 5-6: Ta = 60˚C, Lane 7-8: Ta = 65˚C, Lane 9-10:

Ta = 70˚C. (B) Pooled and purified PCR product. M: 100 bp DNA ladder marker, Lane 1: Open,

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4.4.1.2 Cloning of msDPCK PCR product into pET28a(+) with an N-terminal 6xHis-tag for

subsequent SDM

Colony PCR was performed (using Primer set 1 in Table 4.1) in order to analyse the E. coli JM109 colonies that formed after transformation with the ligation reaction of the RE digested products (digested with the NdeI and XhoI REs). The agarose gel analysis of the colony PCR products (Figure 4.5) confirmed the presence of the msDPCK gene insert within 14 of the 17 tested colonies. The positively confirmed colonies can be identified by the presence of a single bright band at the expected size (~600 bp). Conversely, the colonies that contained no insert (Lane 1, 2 and 5 in Figure 4.5) could be identified by the lack of a PCR product band.

The presence of the msDPCK gene insert within the positively confirmed colonies, thus, suggests that the RE digestion, ligation and subsequent transformation of the pET28a(+)-msDPCKn plasmid were successful. Furthermore, the colony PCR negative control (Lane 18 in Figure 4.5) did not result in any PCR product band, which further strengthens the reliability of the colony PCR analysis.

Figure 4.5 2% Agarose gel of the colony PCR products (using Primer set 1) after RE digestion,

ligation and transformation of the pET28a(+)-msDPCKn plasmid into E. coli JM109 cells. M: 100 bp DNA ladder marker, Lane 1-17: PCR products of the colonies tested whereof the colonies of Lane 1, 2 and 5 tested negative for the msDPCK gene insert, Lane 18: Negative control.

4.4.1.3 Site-directed mutagenesis of msDPCK

After digestion with DpnI, the mutated plasmid product of the SDM PCR was transformed into E. coli JM109 cells, and analysed by colony PCR (results not shown). Six positively tested colonies were selected for O/N cultures and the plasmid isolations of these cultures were, subsequently, analysed by sequencing PCR. Of the six analysed plasmid inserts, two

87 inserts were identified to have the desired mutated sites while retaining an otherwise correct and complete msDPCK gene sequence.

The full sequence alignment of the msDPCK gene and the sequenced inserts of the two plasmid isolations (Supplementary Figure 2.1) is shown in Appendix 2, whereas a condensed version showing segments of this sequence alignment is depicted in Figure 4.6. The desired mutated sites can be observed at position 561 and 567 in the sequence alignment, which indicates that the TGA codon (red box in Figure 4.6) was mutated to produce a TGG codon. Thus, the SDM PCR was successful in these two particular plasmid isolations.

Figure 4.6 A condensed sequence alignment showing segments of the sequencing results

subsequent to the SDM of the msDPCK gene insert. The msDPCK gene, along with the sequenced inserts of the isolated plasmids from colony 1 and 6 are shown, respectively. The red boxes indicate the TGA codons in the msDPCK gene. The black brackets indicate the start and end position of the section of the sequence that is not shown.

Subsequently, both of these isolated plasmids were subjected to RE digestion with NdeI and XhoI, followed by the isolation of the SDM_msDPCK gene inserts. The respective isolated inserts were then ligated into new pET28a(+) vectors and the resulting plasmids transformed into E. coli JM109 cells, which was analysed by colony PCR using primers of the T7 primer set (Figure 4.7).

Accordingly, only half of the 12 tested colonies were confirmed positive for the correct insert, which can be seen by the single colony PCR product band (~750 bp) in Lane 3, 4, 5, 6, 9 and 12 of the agarose gel analysis (Figure 4.7). The amplified product is slightly larger in size since the primers of the T7 primer set (Table 4.3) was used; thus, amplifying part of the

88 pET28a(+) vector, along with the SDM_msDPCK gene insert. This suggests that the RE digestion, ligation and succeeding transformation of the SDM_pET28a(+)-msDPCKn plasmid were successful within these colonies. Moreover, the reliability of the colony PCR is strengthened by the lack of any colony PCR product band in the negative control lane of the agarose gel analysis (Lane 13).

Figure 4.7 2% Agarose gel of the colony PCR products (using the T7 primer set) after RE digestion,

ligation and transformation of the SDM_pET28a(+)-msDPCKn plasmid into E. coli JM109 cells. M: 100 bp DNA ladder marker, Lane 1-12: PCR products of the colonies tested whereof the colonies of Lane 3, 4, 5, 6, 9 and 12 tested positive for the correct msDPCK gene insert, Lane 13: Negative control.

Two of the six colonies that were confirmed to be positive for the correct SDM_msDPCK gene insert were used to make O/N cultures for the purpose of plasmid isolation. These respective plasmid isolations were then transformed into Invitrogen™ E. coli BL21 Star™ (DE3) cells, which was analysed by colony PCR. Overnight cultures of three positively confirmed colonies were made, followed by plasmid isolations, which were used for sequencing PCR. The sequence alignment of the sequencing PCR results (Supplementary Figure 2.2) provided the final confirmation of a successful SDM. The SDM_pET28a(+)-msDPCKn plasmid isolated from the third colony was, subsequently, used in following experiments since it displayed no sequence variation in its pET28a(+) vector component or its msDPCK gene insert component (apart from the desired SDM mutations).

4.4.2 Amplification and sub-cloning of SDM_msDPCK

4.4.2.1 C-terminal 6xHis-tag plasmid (pET28a(+)-msDPCKc)

The Ta optimisation PCR revealed an optimal Ta of 50˚C for the amplification of the

SDM_msDPCK gene insert using the SDM_pET28a(+)-msDPCKn plasmid as template. The SDM_msDPCK gene insert was successfully amplified and purified (estimated concentration of 357.3 ng/µl), which was succeeded by RE digestion (using XhoI and NcoI), ligation and

89 transformation (into E. coli JM109 cells). The subsequent agarose gel analysis of the colony PCR products (Figure 4.8) confirmed the presence of the SDM_msDPCK gene insert in all of the tested colonies, which can be seen by the single amplified PCR product band (~600 bp). The negative control of the PCR also strengthened the reliability of the colony PCR since no product band can be observed in Lane 11.

Figure 4.8 2% Agarose gel of the colony PCR products (using Primer set 2) after RE digestion,

ligation and transformation of the pET28a(+)-msDPCKc plasmid into E. coli JM109 cells. M: 100 bp DNA ladder marker, Lane 1-10: PCR products of the tested colonies where all colonies tested positive for the SDM_msDPCK gene insert, Lane 11: Negative control.

4.4.2.2 Modified MBP-6xHis-tag plasmid (pMALcHT-msDPCK)

The PCR amplification of the SDM_msDPCK gene insert using the SDM_pET28a(+)- msDPCKn plasmid as template was successfully performed at a Ta of 50˚C, which was

determined to be the optimal Ta by PCR optimisation tests. The purity of pooled and purified

PCR amplification products was confirmed by agarose gel analysis (Lane 3 in Figure 4.9 A) and had an estimated concentration of 301.2 ng/µl. The products of the RE digestion (using EcoRI and SalI) was analysed before ligation (Figure 4.9 A). The isolated pSPr022 plasmid (Lane 1) was digested, the P. falciparum ACP gene insert removed and the resulting plasmid (pMALcHT) was loaded in Lane 2. A single product band of the pMALcHT plasmid (orange box in Lane 2) can be seen at a smaller size, compared to that of the isolated pSPr022 plasmid and, therefore, suggests the removal the insert. Moreover, the single product band indicates that the pMALcHT plasmid is pure. Similarly, this can be observed for the RE digestion product of the purified PCR product (red box in Lane 4). Thus, the RE digestion was successful for both the amplified SDM_msDPCK gene insert and the pSPr022 plasmid. Subsequently, the successive ligation and transformation was performed and analysed by colony PCR (using the primers of Primer set 3). All of the tested colonies were confirmed to

90 be positive for the SDM_msDPCK gene insert, which can be seen by the single product band (Lane 1-6) between 500 bp and 600 bp according to the marker (Figure 4.9 B). The lack of a product band in the negative control (Lane 7) also gave added support for the reliability of the colony PCR. Additionally, the inserts of three positively confirmed colony plasmid isolations were sequenced. This provided additional confirmation of the correct gene insert, including the presence of the desired TGG mutations (results not shown).

Figure 4.9 Agarose gels of the analyses subsequent to the amplification of the SDM_msDPCK gene

insert (using Primer set 3). (A) 1% Agarose analysis of the RE digestion products before ligation. The orange box indicates the pMALcHT plasmid (pSPr022 after digestion and removal of P. falciparum ACP gene insert). The red box indicates the SDM_msDPCK gene insert. M: 1 kb DNA ladder marker, Lane 1: Isolated pSPr022 plasmid, Lane 2: pMALcHT plasmid after dephosphorylation, Lane 3: Pooled and purified PCR product. Lane 4: Purified PCR product after RE digestion. (B) 2% Agarose analysis of the colony PCR products (using Primer set 3) after ligation and transformation of the pMALcHT-msDPCK plasmid into E. coli JM109 cells. M: 100 bp DNA ladder marker, Lane 1-6: PCR products of the tested colonies where all colonies tested positive for the SDM_msDPCK gene insert, Lane 7: Negative control.

4.4.3 Transformation of plasmids prior to expression

4.4.3.1 N-terminal 6xHis-tag plasmid (SDM_pET28a(+)-msDPCKn)

The results of the transformation of the SDM_pET28a(+)-msDPCKn plasmid into Invitrogen™ E. coli BL21 Star™ (DE3) cells is reported in Section 4.4.1.3. This transformation was successful, which was confirmed by the colony PCR, as well as sequencing PCR (Supplementary Figure 2.2).

4.4.3.2 C-terminal 6xHis-tag plasmid (pET28a(+)-msDPCKc)

The isolated pET28a(+)-msDPCKc plasmid was successfully transformed into Invitrogen™ E. coli BL21 Star™ (DE3) cells. This was confirmed by colony PCR, as well as PCR

91 sequencing of the isolated plasmid inserts of two positively tested colonies using the forward and reverse primer of the T7 primers for the respective colonies. The sequence alignment of the sequencing results (Figure 4.10) indicates that the inserts of both selected colonies are correct and also properly ligated into the pET28a(+) vector with no stop codon (red box), which is as expected since the 6xHis-tag is at the C-terminal end.

4.4.3.3 Modified MBP-6xHis-tag plasmids (pMALcHT-msDPCK and pRK586)

The successful transformation of the pMALcHT-msDPCK plasmid into Invitrogen™ E. coli BL21 Star™ (DE3) cells was confirmed by colony PCR using Primer set 3 (Table 4.1). All of the colonies that were tested exhibited a single PCR product at the expected size (Figure 4.11 A). Subsequently, these E. coli cells were transformed with the pRK586 plasmid, which was analysed by colony PCR using the same primers as before. Even though these primers only confirmed the presence of the SDM_msDPCK gene insert (Figure 4.11 B), it was assumed that the pRK586 plasmid is also present in all of the positively tested colonies since the transformed cells were plated on a LB agar plate supplemented by two different antibiotics. Therefore, in order for the colonies to grow, both plasmids had to be successfully transformed to provide the necessary antibiotic resistance.

Figure 4.11 2% Agarose gels of the colony PCR products (using Primer set 3) after transformation

into Invitrogen™ E. coli BL21 Star™ (DE3) cells. M: 100 bp DNA ladder marker, Lane 1-10: PCR products of the tested colonies where all colonies tested positive for the SDM_msDPCK gene insert, Lane 11: Negative control. (A) Colony PCR following transformation of pMALcHT-msDPCK (single plasmid). (B) Colony PCR following transformation of pRK586 (double plasmid).

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Figure 4.10 Sequence alignment of the PCR sequencing results following the transformation of the pET28a(+)-msDPCKc plasmid into Invitrogen™ E. coli BL21 Star™ (DE3) cells. The red box indicates the expected lack of a stop codon in the sequenced plasmid inserts. The black box indicates the 6xHis-tag.

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4.4.3.4 Molecular chaperone expressing plasmid (pG-JKE8)

The pG-JKE8 plasmid was transformed into Invitrogen™ E. coli BL21 Star™ (DE3) cells that consisted of the previously transformed SDM_pET28a(+)-msDPCKn plasmid. The colony PCR analysis (Figure 4.12) only confirmed the presence of the SDM_msDPCK gene insert in the tested colonies. The transformation of the pG-JKE8 plasmid was, however, presumed to be successful due to the growth of positively tested colonies on an LB agar plate supplemented with two different antibiotics.

Figure 4.12 2% Agarose gels of the colony PCR products (using Primer set 1) after transformation of

the pG-JKE8 plasmid into Invitrogen™ E. coli BL21 Star™ (DE3) cells containing the SDM_pET28a(+)-msDPCKn plasmid. M: 100 bp DNA ladder marker, Lane 1-10: PCR products of the tested colonies where all colonies tested positive for the SDM_msDPCK gene insert.