2. Sub-cloning of Ebp-Npu-SsrA (EN), Scrb-Npu-SsrA (SN) and eGFP-Npu-SsrA (GN) inserts to pET-24d(+)
Background:
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The inserts were synthesized by the IDT company and carried by a default pUC cloning vector.
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pUC vectors with desired insert (EN, SN and GN) were digested with XbaI and HindIII. Two fragments with different lengths were expected.
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EN & SN insert size: 972 bp.
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pUC plasmid backbone size: 2752 bp.
Observation:
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Two or three bands were observed at each lane. Sample 2 to 5 showed a band of ~900 bp in size. Sample 6 and 7 showed a band of ~1600 bp in size. All samples showed a band of ~2500 bp in size.
Conclusion:
Restriction digestion has been successfully performed. The Ebp-Npu-SsrA, Scrb-Npu-SsrA and eGFP-Npu-SsrA inserts are purified and ready for sub-cloning to pET-24d(+) plasmid. The presence of an extra third band in lane 3 and 6 are the uncut vector caused by incomplete digestion.
3. Transformation to TOP10
Background:
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The inserts (Ebp-Npu-SsrA, Scrb-Npu-SsrA and eGFP-Npu-SsrA) and pET-24d(+) plasmid were digested with XbaI and HindIII and ligated to form recombinant plasmids pET-24d(+)-Ebp-Npu-SsrA, pET-24d(+)-Scrb-Npu-SsrA and pET-24d(+)-eGFP-Npu-SsrA.
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Ligation products were transformed to TOP10 competent cells.
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Only bacteria received Kanamycin resistant gene in recombinant vector survived in LB agar plate containing kanamycin.
Observation:
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Multiple colonies were observed
Conclusion:
Successful transformation and ligation of pET-24d(+)-Ebp-Npu-SsrA, pET-24d(+)-Scrb-Npu-SsrA and pET-24d(+)-eGFP-Npu-SsrA to TOP10 cells are confirmed by the presence of transformants on the LB agar plate.
4.1 Restriction digestion of recombinant plasmid for colony selection
Background:
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Recombinant pET-24d(+)-Ebp-Npu-SsrA plasmid was cut by XbaI and HindIII to confirm the insert size.
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pET-24d(+) plasmid backbone size: 5207 bp.
Observation:
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All the samples showed bands of ~5000 bp and ~900 bp in size.
Conclusion:
Insert Ebp-Npu-SsrA has been successfully sub-cloned to the pET-24d(+) plasmid.
4.2 Colony PCR of recombinant plasmid for colony selection
Background:
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Recombinant plasmid was confirmed by colony PCR using the T7 universal primers. The target band was expected to be 168 bp longer than original insert size.
Observation:
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All the samples showed bands of ~1100 bp in size.
Concluding remarks:
Insert Scrb-Npu-SsrA has been successfully sub-cloned to the pET-24d(+) plasmid.
Background:
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Recombinant plasmid was confirmed by colony PCR using the T7 universal primers. The target band was expected to be 168 bp longer than original insert size.
Observation:
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All the samples showed bands of ~1800 bp in size
Conclusion:
Insert eGFP-Npu-SsrA has been successfully sub-cloned to the pET-24d(+) plasmid.
5 Transformation to BL21(DE3)
Background:
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pET-24d(+)-Ebp-Npu-SsrA, pET-24d(+)-Scrb-Npu-SsrA and pET-24d(+)-eGFP-Npu-SsrA were purified and transformed to BL21(DE3).
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Only bacteria received Kanamycin resistant gene in recombinant vector survived in LB agar plate containing kanamycin.
Observation:
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Multiple colonies were observed.
Conclusion:
Successful transformation of pET-24d(+)-Ebp-Npu-SsrA, pET-24d(+)-Scrb-Npu-SsrA and pET-24d(+)-eGFP-Npu-SsrA plasmids to BL21(DE3) cells is confirmed by the presence of transformed colonies on the LB agar plate.
6 Sequencing result
Background:
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Colonies were selected from the LB agar plate and sent for DNA sequencing.
Observation:
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No mutation was found in the peptide coding sequence of the selected plasmids.
Conclusion:
The DNA sequencing result confirmed the recombinant plasmid has no mutation. The recombinant plasmid, pET-24d(+)-Ebp-Npu-SsrA, pET-24d(+)-Scrb-Npu-SsrA and pET-24d(+)-eGFP-Npu-SsrA plasmids are ready for expression.
7 Expression
Background:
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After 0.2 mM of IPTG induction, cells were cultured at 25℃ overnight. Cells were harvested and lysed through sonication.
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After centrifugation, supernatant and pellet were obtained and indicated as soluble and insoluble groups, respectively.
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Denatured sample were mixed with 6X SDS loading buffer and denatured by heating at 90℃ for 5 mins.
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Native samples were mixed with 6X native gel loading buffer and were not denatured by heating.
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Samples were loaded to SDS-PAGE Gel and run for 60 min at 120 V.
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Expected molecular weight (MW) of Ebp-Npu-SsrA : 34.6 kDa
Observation:
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Compared to the uninduced samples, the induced samples clearly showed an extra band with high intensity in soluble fraction. The target protein is indicated by the red bracket (denatured samples: ~21 kDa; native sample: ~34 kDa).
Conclusion:
The distinct bands present in the IPTG-induced samples indicate successful expression of the target protein. The distinct band at ~34 kDa in the native gel matches the expected molecular weight (MW) of the Ebp-Npu-SsrA protein. The band at ~20 kDa in the denatured sample is assumed to be a fragment of the Ebp-Npu-SsrA protein due to premature intracellular cleavage of part of the intein from the target protein. The MW of the cleaved intein is around ~14 kDa. However, it cannot be seen in the gel photo due to sample overflow, which is indicated by the disappearance of the 15 kDa band in the MW ladder.
Background:
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After 0.2 mM of IPTG induction, cells were cultured at 25℃ overnight. Cells were harvested and lysed through sonication.
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After centrifugation, supernatant and pellet were obtained and indicated as soluble and insoluble groups, respectively.
●
Denatured samples were mixed with 6X SDS loading buffer and denatured by heating at 90℃ for 5 mins.
●
Native samples were mixed with 6X native gel loading buffer and were not denatured by heating.
●
Samples were loaded to SDS-PAGE Gel and run for 60 min at 120 V.
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Expected molecular weight (MW) of Scrb-Npu-SsrA : 34.6 kDa.
Observation:
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Compared to the uninduced samples, the induced samples clearly showed an extra band with high intensity in soluble fraction. The target protein is indicated by the red bracket (denatured samples: ~21 kDa and ~15 kDa; native sample: ~34 kDa).
The distinct bands present in the IPTG induced samples indicate successful expression of target protein. The distinct band at ~ 34 kDa in native gel matches the expected MW of Scrb-Npu-SsrA protein. The bands at ~21 kDa and ~14 kDa in denatured sample are fragments of Scrb-Npu-SsrA protein due to a pre-mature intra-cellular cleavage of part of the intein from the target protein.
Background:
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After 0.2 mM of IPTG induction, cells were cultured at 25℃ overnight. Cells were harvested and lysed through sonication.
●
After centrifugation, both supernatant and pellet were obtained and indicated as soluble and insoluble groups, respectively.
●
Samples were mixed with 6X native gel loading buffer and were not denatured by heating.
●
Samples were loaded to SDS-PAGE Gel and run for 60 min at 120 V.
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Expected molecular weight (MW) of eGFP-NPU-SsrA : 60 kDa.
Observation:
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The IPTG-induced samples clearly showed distinct band(s) in soluble fraction. The target protein is indicated by the red bracket (~60 kDa).
Conclusion:
The distinct bands present in the IPTG induced samples indicates successful expression of target protein. The distinct band at ~ 60 kDa matches the expected MW of eGFP-Npu-SsrA protein.
Observation:
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Strong fluorescence was observed in the eGFP-Npu-SsrA induced soluble group.
Conclusion:
eGFP is successfully expressed and the BioBrick is functional.
8 Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS)
Background:
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After chitin column purification, the elution fraction was subjected to LC-ESI-MS to determine the presence of the targeted eGFP protein.
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The calculated molecular mass of cyclic eGFP was 18 Da less than that of linear eGFP (26941 Da), resulting in a mass of 26923 Da.
Observation:
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A peak at 26921 Da was observed.
Conclusion:
Cyclic eGFP was detected in the elution fraction, indicating the successful purification using the chitin column and confirming that the eGFP has been modified into its cyclic form..
Owing to the time constraint, the purification and analysis of target peptide could not be finished within the competition period. Yet, the successful cyclization of eGFP control demonstrated that the Npu intein BioBrick is effective for cyclization of our target eGFP-binding peptide.
