On this page, results from our experiments are presented in a chronological order. This includes gels from gel electrophoresis of his-tag introducing PCRs and screening digestion, measured protein concentrations after cell lysis and protein extraction and images of western blot membranes. The results of the activity measurements can be found on the Measurements page.
Establishment of His-tagged gene block fragment by PCR
6 histidine residues were added by PCR with overhanging primers to each ordered block segment before the stop codon, as shown in Figure 1.
Figure 1. Schematic illustration of the establishing each his-tag block segment. Six histidine tag was inserted downstream of each nine different enzyme gene and upstream of stop codon to purify each enzyme protein later of this project.
The amplification was confirmed successful by electrophoresis, see Figure 2. The expected size of each His-tagged gene block fragments is 1507 bp (BphA1), 926 bp (BphB), 977 bp (BphC), 677 bp (BphD), 1009 bp (BphA2), 934 bp (PETase), 1949 bp (PueA), 1703 bp (PEG-DH) and 1984 bp (MHETase). For more details, check out Week 29 (July 17th) in the Notebook page!
Figure 2. Confirming the size of his-tagged block segments. Each his-tagged block
segment was constructed by PCR and the size of them were confirmed in a 1% agarose gel
electrophoresis. The gel picture shows from left: 1: GeneRuler Express DNA Ladder (SM1553) 2: BphA1,
3: BphB, 4: BphC, 5: BphD, 6: BphA2, 7: GeneRuler Express DNA Ladder, 8: PETase, 9: PueA, 10:
PEG-DH, 11: MHETase, 12: GeneRuler Express DNA Ladder. The length of each fragment correspond to the
expected lengths and successful amplification was thus confirmed by this electrophoresis.
Transformants screening
Each His-tagged block segment was inserted into pSB1C3 through Gibson Assembly and the resulting plasmid was transformed into DH5α. To screen the transformants colonies, the plasmids were extracted using Thermo Fisher’s miniprep kit, digested with restriction enzymes XhoI and SacI and run in a gel electrophoresis. The resulting gel is presented in Figure 3. Figure 3A shows the electrophoresis of BphA1, which was the only block segment for which the bands showing were not correct. Figure 3B shows all other digested block segments. Each block segment should have three fragments from this digestion as two restriction enzymes were used. Two of the fragments should be of the same length for all block segments, these are 116 bp and 776 bp. The last fragment should be different for each block segment and their corresponding expected lengths are 2708 bp (BphA1), 2162 bp (BphB), 2213 bp (BphC), 2165 bp BphD), 1913 bp (BphA2), 2117 bp (PETase), 3185 bp (PueA), 2939 bp (PEG-DH) and 3143 bp (MHETase). The plasmids with block segments for which the screening results were positive were sent for sequencing. For BphA1, BphB, BphC and BphA2, the sequencing results confirmed the right sequence. You can check how we got these results in our Notebook! We got positive colonies expressing BphB and PEG-DH (by sequencing we noticed that PEG-DH had deletion) in Week 31 (July 31st), for BphA1, BphC, and BpfA2 in Week 33 (August 10th), for BphD and PETase (but sequencing for them are negative) in Week 35 (August 26th) and for PueA (sequencing is negative) on August 27th the same week. You can see the corresponding negative bands for MHETase August 27th in the same week.
Figure 3. Confirming the size of digested plasmid for screening. Gels showing the lengths of the fragments resulting from digesting each his-tagged
block
segments with SacI and XhoI when run in a 1% agarose gel electrophoresis. The gel picture A shows
from
the left, 1: GeneRuler Express DNA Ladder (SM1553) 2: empty 3: BphA1 and picture B showed from left,
1: BphB, 2: BphC, 3: BphD, 4: BphA2, 5: GeneRuler Express DNA Ladder, 6: PETase, 7: PueA, 8: PEG-DH,
9: MHETase, 10: GeneRuler Express DNA Ladder. There should be three fragments each and two small
fragments
for all should be 116 bp and 776 bp. The last fragment length should be different by each strain and
they should be 2708 bp (BphA1), 2162 bp (BphB), 2213 bp (BphC), 2165 bp (BphD), 1913 bp (BphA2),
2117
bp (PETase), 3185 bp (PueA), 2939 bp (PEG-DH) and 3143 bp (MHETase) and all but MHETase were
confirmed
by this electrophoresis.
DC assay of extracted proteins
Once the cells had been lysed, the resulting protein concentrations were determined as presented in Table 1. The protein concentrations differ notably between the different block segments despite having been grown for the same amount of time and having the same RBS and promoter. This could be due to the metabolic burden inflicted by the expression of the enzyme, an effect that should be further evaluated to optimize the enzyme expression process.
| Block Segment | Protein Concentration [mg/ml] |
|---|---|
| BphA1 | 5.8999 |
| BphB | 3.7254 |
| BphC | 4.7667 |
| BphA2 | 6.0224 |
| NC | 6.9106 |
| PC | 1.9039 |
When examining the protein extracts in a western blot, in which the His-tags are utilized as binding spots for the antibodies, BphC (A3) was the only enzyme that showed a band at the expected length (32 kDa [1]), as seen in Figure 4. There seem to be some unspecific binding to some protein of approximately 36 kDa for all the different samples, but as this is present in the negative control as well it’s disregarded as background. Interestingly, also another unique band for BphC is clearly visible around 145 kDa. This band could be the result of misfolding and aggregate formation when BphC is expressed in E. coli. Since BphC originates from Pseudomonas sp, it is possible that our chassis lacks chaperones necessary for the correct folding of this specific protein leading to the aggregation. Read more about these results in the Notebook, Week 36 (September 3rd).
Figure 4. Western blot result. The western blot membrane after stain with antibodies that bind to his-tags. The plasmid backbone used for all block segments with a gene coding for RFP instead of a his-tagged enzyme was used as a negative control. The positive control was a his-tagged protein we got from another researcher in our lab. Expected lengths for the block segments are 52 kDa for BphA1, 29 kDa for BphB, 32 kDa for BphC and 23 kDa for BphA2. SpectraTM Multicolor Broad Range Protein Ladder (26634) was used as a size marker.
To further investigate the aggregation of BphC, new protein samples were extracted from cells that were in the exponential phase instead of the stationary phase. The same procedure was done for BphB (A2) in case the protein extraction at stationary phase was the reason that we couldn’t see any expression of the other enzymes. Table 2 shows the corresponding protein concentrations after the cell lysis.
| Block Segment | Protein Concentration [mg/ml] |
| BphB (OD600 = 0.5) | 2.7031 |
| BphB (OD600 = 1.2) | 5.6282 |
| BphC (OD600 = 0.3) | 1.5616 |
| BphC (OD600 = 0.9) | 2.4178 |
Figure 5. Western blot result. The western blot membrane after stain with antibodies that bind to his-tags of BphB and BphC at different growth phases (exponential and stationary). The plasmid backbone used for all block segments with a gene coding for RFP instead of a his-tagged enzyme was used as a negative control. The positive control is a protein solution containing a his-tagged protein we got from another researcher in our lab. SpectraTM Multicolor Broad Range Protein Ladder (26634) was used as a size marker.
- [1] “ bphC - Biphenyl-2,3-diol 1,2-dioxygenase - Pseudomonas sp. (strain KKS102) - bphC gene & protein.” https://www.uniprot.org/uniprot/P17297 (Accessed on Sep. 08, 2020).
