Vector design:The vector design of pET-24(+)_peptide was completed.

Transformation to TOP10: Ligated products were transformed into competent TOP10 cells through the heat shock method. As seen in Figure 2, the plate for pET-24d showed multiple colonies after overnight incubation on LB-Kan agar plates without any contamination.

Colonies are picked and inoculated: Two individual colonies were selected from the ligation plate, and each colony was inoculated into a separate tube containing LB broth. The tubes were incubated overnight at 37°C with shaking.

Recombinant plasmid purification: The recombinant plasmid was purified from the cell suspension using TIANprep Mini Plasmid Kit.

Transformation to BL21(DE3) The purified recombinant plasmid was transformed into competent BL21(DE3) cells using the heat shock method. As seen in Figure 3, the plate for pET24d showed multiple colonies after overnight incubation on LB-Kan agar plates; no contamination occurred.

Expression: BL21(DE3) cells carrying the recombinant plasmid pET-24d(+)-Ebp were induced with 0.2 mM IPTG and cultured at 25 °C overnight. Subsequently, the expressed peptide was extracted using lysis buffer and loaded onto a SDS PAGE gel to check for expression. However, the expected size of the peptide (2.4 kDa) was not observed in the SDS PAGE gel.

Vector design: The vector designs of pET-24d(+)-Ebp-Ssp-Mxe (ES) and pET-24d(+)-Scrb-Ssp-Mxe (SS) were completed.

Restriction digestion of insert: The PUC-Ebp-Ssp-Mxe and PUC-Scrb-Ssp-Mxe vectors were subjected to restriction digestion using XbaI and HindIII enzymes to generate compatible ends. Subsequently, DNA gel electrophoresis was performed on the restriction-digested products. Upon examination of the gel image (refer to Figure 6), fragments of the desired length (~1500 bp size) were observed in each lane. However, an additional third band was present, indicating the presence of an uncut vector. This is believed to be a result of inadequate reaction time, which did not allow for complete digestion.

Restriction digestion of vector: Restriction digestion of pET-24d(+) vector with XbaI and HindIII to generate compatible ends.

Gel purification of restriction-digested products: Promega gel purification kit was used to purify the digested DNA from the gel. Subsequently, the concentrations were determined using the Nanodrop Spectrophotometer.

Ligation reaction: The digested products were subjected to ligation using T4 DNA Ligase. The resulting ligated product was stored at -20°C in the refrigerator until the next transformation.

Transformation to TOP10: Ligated products were transformed into competent TOP10 cells through the heat shock method. As seen in Figure 7 and Figure 8, the plate for pET-24d showed multiple colonies after overnight incubation on LB-Kan agar plates without any contamination.

Colonies picking and inoculation: Five individual colonies were picked from the pET24d-Ebp-Ssp-Mxe ligation plate, and each colony was inoculated into a separate tube of LB broth. The tubes were incubated overnight at 37°C with shaking.

Recombinant plasmid purification: The recombinant plasmid was purified from the cell suspension using TIANprep Mini Plasmid Kit.

R.E. of recomb. plasmid S1 for colony selection: The recombinant plasmid was digested with XbaI and HindIII enzymes and subsequently subjected to gel electrophoresis. As shown in Figure 9, all lanes exhibited two bands with lengths of approximately 5000 bp and 1500 bp, equivalent to the sizes of pET and Ebp-Ssp-Mxe, respectively. The insert ES was present in the recombinant plasmid in the TOP10 cells.

Colony PCR: Eight individual colonies were picked from the pET-24d-Scrb-Ssp-Mxe ligation plate, and each colony was inoculated into a separate tube of LB broth, as well as a PCR tube containing the master mix. Colony PCR and gel electrophoresis were performed to confirm successful insertion. As seen in Figure 10, all lanes showed a fragment of the desired length (~1700 bp). The additional bands with a size of approximately 300 bp were believed to be primers.

Recombinant plasmid purification: TIANprep Mini Plasmid Kit was used to purify the recombinant plasmid in cell suspension.

Glycerol stock: Cells with pET24d-Ebp-Ssp-Mxe and pET24d-Scrb-Ssp-Mxe were stored in glycerol.

Transformation to BL21(DE3): The purified product was transformed into competent BL21(DE3) cells using the heat shock method. As shown in Figure 11 and Figure 12, multiple colonies were observed on LB-Kan agar plates after overnight incubation, indicating successful transformation, and no contamination was detected.

Colony sequencing result: Selected ES and SS plasmids were sent to BGI for sanger sequencing using the T7 promoter and T7 terminator. After aligning the designed sequence with the sequences sent for analysis, it was found that both the ES and SS sequences are correct.

Expression: The Ebp-Ssp-Mxe (ES) expression was induced with IPTG at 25°C overnight. After harvesting the cells through centrifugation and lysing them to obtain soluble and insoluble proteins, the samples were heated at 90°C for 5 minutes and run on an SDS-PAGE gel. As seen in Figure 13, a distinct band with high intensity, corresponding to the overexpressed protein, was observed at approximately 50 kDa molecular weight, indicating successful expression of the soluble protein.

Purification: Glycerol stocks of ES and SS were used to do protein expression in 100 ml of medium, to obtain a sufficient amount of the desired protein. The cells were then harvested by centrifugation (7500 rpm, 4°C, 15 min) and lysed using sonication (30 s+, 30 s-, Amp 60%, 10 min). Subsequently, the soluble protein in the lysate was obtained through centrifugation (8500 rpm, 4°C, 5 min). The soluble protein, which contained the intein splicing BioBrick, was loaded onto the chitin column for one-step purification and cyclization. The column was first equilibrated by the B1 buffer. After loading the sample, B1 buffer was used to wash the unbounded molecules,while buffer B2 and buffer B4 were used for intein 1 and 2 cleavage, by incubation overnight.

Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) The elution obtained from the purification was subjected to LC-ESI-MS analysis. The analysis results showed that the desired cyclic peptide was not detected.

Vector design: The vector designs of pET-24d(+)-Ebp-Npu-SsrA (EN), pET-24d(+)-Scrb-Npu-SsrA (SN) and pET-24d(+)-GFP-Npu-SsrA (GN) were completed.

Restriction digestion of insert: The PUC-Ebp-Npu-SsrA, PUC-Scrb-Npu-SsrA, and PUC-GFP-Npu-SsrA vectors were subjected to restriction digestion using XbaI and HindIII enzymes to generate compatible ends. Subsequently, DNA gel electrophoresis was performed on the restriction-digested products. Upon examination of the gel image (refer to Figure 18), fragments of the desired lengths (972 bp and 1653 bp) were observed in each lane. However, an additional third band was present, indicating the presence of an uncut vector. This is believed to be a result of inadequate reaction time, which did not allow for complete digestion.

Restriction digestion of vector: Restriction digestion of pET-24d(+) vector with XbaI and HindIII to generate compatible ends.

Gel purification of restriction-digested products: Promega gel purification kit was used to purify the digested DNA from the gel. Subsequently, the concentrations were determined using the Nanodrop Spectrophotometer.

Ligation reaction: The digested products were subjected to ligation using T4 DNA Ligase. The resulting ligated product was stored at -20°C in the refrigerator until the next transformation.

Transformation to TOP10: Ligated products were transformed into competent TOP10 cells through the heat shock method. As seen in Figure 19, Figure 20 and Figure 21, plates for pET24d showed multiple colonies after overnight incubation on LB-Kan agar plates without any contamination.

Colonies picking and inoculation: Five individual colonies were picked from the pET24d-Ebp-Ssp-Mxe ligation plate, and each colony was inoculated into a separate tube of LB broth. The tubes were incubated overnight at 37°C with shaking.

8. Recombinant plasmid purification: The recombinant plasmid was purified from the cell suspension using TIANprep Mini Plasmid Kit.

R.E. of recomb. plasmid EN for colony selection. Recombinant plasmids were digested by XbaI and HindIII, and run through gel electrophoresis. As seen in Figure 22, all lanes showed 2 bands with lengths ~5000bp and ~1000bp which were equivalent to pET and Ebp-Npu-Ssra size. Insert EN was present in the recombinant plasmid in TOP10 cell.

Colony PCR: Eight individual colonies were picked from the pET24d-Scrb-Ebp-Npu-Ssp and pET24d-GFP-Npu-SsR ligation plate, and each colony was inoculated into a separate tube of LB broth, as well as a PCR tube containing master mix. Colony PCR and gel electrophoresis were performed to confirm successful insertion.

Glycerol stock: Cells with pET-24d(+)-Ebp-Npu-SsrA, pET-24d(+)-Scrb-Npu-SsrA and pET-24d(+)-GFP-Npu-SsrA were stored in glycerol.

Transformation to BL21(DE3): The purified product is transformed into competent BL21(DE3) cells using the heat shock method. As shown in Figure 25, Figure 26, and Figure 27, the plates for pET24d-Ebp-NpuSsrA, pET24d-Scrb-NpuSsrA, and pET24d-GFP-NpuSsrA display multiple colonies after overnight incubation on LB-Kan agar plates, indicating successful transformation without any contamination.

Colony sequencing result: Selected EN, SN and GN plasmids were sent to BGI for sanger sequencing using the T7 promoter and T7 terminator. After aligning the designed sequence with the sequences sent for analysis, it was found that the sequences for EN, SN, and GN were correct.

Expression: EN and SN expressions were induced by IPTG at 25°C for 6 hours. After harvesting the cells through centrifugation and obtaining the soluble and insoluble proteins using a lysis buffer, the samples were run on a PAGE gel under two different conditions. For denatured samples, they were heated at 90°C for 5 minutes, treated with SDS, and then loaded onto the gel. For native samples, they were treated with 2X native gel loading buffer, not boiled, and then loaded onto the gel.

Purification: Glycerol stocks of EN, SN and GN were used to do protein expression in 100 ml of medium, to obtain a sufficient amount of the desired protein. The cells were then harvested through centrifugation with 7500 rpm for 15 minutes, subsequently lysed using sonication. The sonication cycles consisted of an on time of 20 seconds followed by an off time of 30 seconds. A total on time of 10 minutes and 60% Amplitude was employed. The soluble protein in the lysate was subsequently obtained through centrifugation with 8000 rpm for 5 minutes. The soluble protein, which contained the Npu splicing BioBrick, was loaded onto the chitin column for one-step purification and cyclization.

Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) After chitin column purification, the elution fractions from GN was subjected to LC-ESI-MS to determine the presence of the targeted eGFP protein. For GN, please refer to the result in Figure 33, where a peak at 26921 Da was observed. This peak aligned with the expected molecular mass of cyclic eGFP, which is 26923 Da, indicating that cyclic eGFP was detected in the elution fraction.

Chemical synthesis of EGFR peptide

BsAb binding affinity - ELISA

Functional assay - MTT