This basic part is derived from plasmid pJL1, including a conserved DNA sequence of T7 promoter as 5'-taatacgactcactatagggaga-3'. The plasmid pJL1 is commonly used for the in vitro sfGFP expression of cell-free protein synthesis (CFPS). Hence, this part is used for the construction of three composite parts: sfGFP generator, Microcin H47 generator, and Microcin M generator, for CFPS in our project.
Figure 1. Schematic design of T7 promoter, generated by SnapGene
This basic part is derived from plasmid pJL1, including a conserved ribosome binding site (RBS) as 5'-aagaaggaga-3’. The plasmid pJL1 is commonly used for the in vitro sfGFP expression of cell-free protein synthesis (CFPS).
The plasmid design of this biological part is shown as Figure 3, assembled with iGEM standard backbone pSB1C3. To validate the correctness of the DNA sequence, results of Sanger sequencing show the successful assembly among T7 promoter, RBS (this part), and sfGFP.
Figure 2. Schematic design of RBS, generated by SnapGene
This basic part is derived from plasmid pJL1, including a DNA sequence for coding sfGFP (superfolder green fluorescent protein). The plasmid pJL1 is commonly used for the in vitro sfGFP expression of cell-free protein synthesis (CFPS).
Figure 3. Schematic design of sfGFP, generated by SnapGene
This basic part is derived from plasmid pJL1, including a conserved T7 terminator as 5'-ctagcataaccccttggggcctctaaacgggtcttgaggggttttttg-3'. The plasmid pJL1 is commonly used for the in vitro sfGFP expression of cell-free protein synthesis (CFPS).
Figure 4. Schematic design of T7 terminator, generated by SnapGene
This composite part is derived from plasmid pJL1, consisting of four basic parts: T7 promoter, ribosome binding site, coding sequence of superfolder green fluorescent protein, and T7 terminator. The plasmid pJL1 is commonly used for in vitro sfGFP expression via cell-free protein synthesis (CFPS), but an iGEM-standardized CFPS construction has not yet been commonly reported and characterized. Hence, this part is established to demonstrate the feasibility of CFPS in our project.
Figure 5. Schematic design of sfGFP, generated by SnapGene
Figure 6. Detailed assembly pattern of this composite part, including four basic parts: T7 promoter, RBS, sfGFP, and T7 terminator
For the characterization process of this part, follow five steps shown in Figure 7: (1) molecular cloning; (2) colony PCR; (3) sequencing; (4) plasmid extraction; (5) CFPS reaction.
Figure 7. Workflow for the construction and characterization of sfGFP.
Step 1: Molecular Cloning
To construct this part, we first acquired the linearized DNA fragments of both vector and insert fragments. Thus, we amplified vector pSB1C3 and inserted fragments using PCR. Agarose gel electrophoresis results (Figure 8) show the desired DNA bands as pSB1C3 (2070 bp) and inserted fragment (988 bp) used in this construction.
Figure 8. Agarose gel electrophoresis analysis of PCR products for molecular cloning.The DNA bands indicate one vector pSB1C3 and three inserted fragments. The first inserted fragment of sfGFP (988 bp) is used for the construction of this composite part, while the other two inserted fragments are used for zmicrocin H47 and Microcin M, respectively.
After obtaining the purified DNA products, we assembled these fragments using the Gibson Assembly strategy. We then transformed the reaction to competent E. coli Mach1-T1 cells and spread the transformants onto LB-agar plates containing 34 µg/mL chloramphenicol. Figure 9 shows that the E. coli transformants grew normally on LB-agar plates and were used for further experiments.
Figure 9. E. coli Mach1-T1 transformants on LB-agar plates, showing the expected phenotype for the construction of sfGFP.
Step 2: Colony PCR
To verify the constructions, we next performed colony PCR by using the reported protocol. For each construction (not only sfGFP, but also microcin H47 and microcin M), we selected four independent colonies from LB-agar plates and used primer pair VF2/VR to amplify the inserted DNA sequences. After that, we analyzed the DNA products by agarose gel electrophoresis. Results show that four PCR products match the desired DNA sizes of this construction (Figure 10).
Figure 10. Agarose gel electrophoresis analysis of PCR products for colony PCR. The four DNA products of this construction (of sfGFP) match the desired DNA size 1259 bp, annotated by green√.
Step 3: Sequencing
Consequently, we picked the desired PCR products for sequencing. Results of Sanger sequencing show the successful construction of this part (Figure 11), which means that the plasmid could be used for the following experiments.
Figure 11. Validation of DNA sequence by Sanger sequencing, generated by SnapGene.
Step 4: Plasmid Extraction
After we acquired the correct plasmids, we then tried to extract and purify the plasmids for the following CFPS reactions. By using the plasmid extract kit, we gained the purified plasmids and analyzed them by agarose gel electrophoresis. Results in Figure 12 show that the extracted plasmids are clean, consisting of two conformations: linear and supercoiled. To further evaluate the plasmid sizes, we digested the three plasmids by EcoRI (restriction enzyme). After digestion, the three plasmids show the expected linearized comformation and match the desired DNA sizes. These results indicate the successful construction and extraction of this composite part.
Figure 12. Agarose gel electrophoresis analysis of plasmid extracts for three plasmid constructions. The plasmids without EcoRI digestion show two conformations: linear (larger DNA sizes) and supercoiled (smaller DNA sizes), while after EcoRI digestion, the plasmids are linearized and show expected DNA sizes refer to DNA sequences. As for this part, the digested plasmid meet the desired DNA size 3015 bp.
Step 5: CFPS Reaction
After plasmid extraction, CFPS reactions were performed, producing sfGFP (Figure 13).
Figure 13. E. coli-based CFPS reaction for sfGFP production.
Before CFPS reaction, we initially established a standard curve for the conversion of "sfGFP (µg/mL)—Fluorescence (a.u.)" (Figure 14).
Figure 14. Standard curve of "sfGFP yield-Fluorescence" conversion.
Subsequently, following the commonly used CFPS protocol, we successfully produced sfGFP yielding 1051(±84) µg/mL, which achieved acceptable yield in this research area. Meanwhile, the green color of expressed sfGFP could be clearly observed under the bright field, also the green fluorescence could be detected and imaged by imager (Figure 15).
Figure 15. Results of E. coli-based CFPS reaction for sfGFP production. The sfGFP expression could be easily visualized under the bright field, and the green fluorescence could be detected by imager. Furthermore, the yield exceeded 1000 µg/mL, which has reached the acceptable result of E. coli-based CFPS reactions in this research field. The plasmid (this construction) was added as 200 ng into 15 µL CFPS reactions, and the other experimental operations followed the reported protocol.
By following these steps, the composite part was successfully constructed and characterized, demonstrating the feasibility of CFPS for the in vitro production of proteins (e.g., sfGFP). This CFPS system was further utilized for producing two antimicrobial peptides in our project, Microcin H47 and Microcin M.
This composite part is derived from plasmid pJL1, consisting of four basic parts: T7 promoter, ribosome binding site, coding sequence of antimicrobial peptide Microcin H47, and T7 terminator. The plasmid pJL1 is commonly used for the in vitro protein expression of cell-free protein synthesis (CFPS), and the iGEM-standardized CFPS construction has been constructed and characterized yet in our project. To further expand the application of CFPS systems, this composite part is designed to demonstrate the feasibility of in vitro antimicrobial peptide (AMP) synthesis by CFPS for extended useful purposes.
Figure 16. Schematic design of microcin H47, generated by SnapGene
Figure 17. Detailed assembly pattern of this composite part, including four basic parts: T7 promoter, RBS, Microcin H47, and T7 terminator.
For the characterization process of this part, follow five steps shown in Figure 18: (1) molecular cloning; (2) colony PCR; (3) sequencing; (4) plasmid extraction; (5) CFPS reaction.
Figure 18. Workflow for the construction and characterization of this part.
Step 1: Molecular Cloning
To construct microcin H47, we first need to acquire the linearized DNA fragments of both vector and inserted fragments. Thus, we amplified vector pSB1C3 and inserted fragments by using PCR. Results of agarose gel electrophoresis showing the desired DNA bands (Figure 19) as pSB1C3 (2070 bp) and inserted fragment (511 bp) used in this construction. Note that the PCR template of inserted fragment was derived from the previously reported research article.
Figure 19. Agarose gel electrophoresis analysis of PCR products for molecular cloning. The DNA bands indicate one vector pSB1C3 and three inserted fragments. The second inserted fragment of Microcin H47 (511 bp) is used for the construction of this composite part, while the other two inserted fragments are used for sfGFP and Microcin M, respectively.
When we got the purified DNA products, then we assembled these fragments by using Gibson Assembly strategy. Next, we transformed the reaction to competent E. coli Mach1-T1 cells and spread the transformants onto LB-agar plates containing 34 µg/mL chloramphenicol. As shown in Figure 20, the E. coli transformants could normally grow on LB-agar plates and be used for the following experiments.
Figure 20. E. coli Mach1-T1 transformants on LB-agar plates, showing the expected phenotype refer to the construction of this part.
Step 2: Colony PCR
To verify the constructions, we next performed colony PCR by using the reported protocol. For each construction (not only this part, but also sfGFP and Microcin M), we selected four independent colonies from LB-agar plates and used primer pair VF2/VR to amplify the inserted DNA sequences. After that, we analyzed the DNA products by agarose gel electrophoresis. Results show that three PCR products match the desired DNA sizes of this construction, and the other one was the empty vector of pSB1C3 as false positive (Figure 21).
Figure 21. Agarose gel electrophoresis analysis of PCR products for colony PCR. The three DNA products of microcin H47 match the desired DNA size 782 bp, annotated by green √, while the other one is the empty vector of pSB1C3 as false positive that annotated by red ×.
Step 3: Sequencing
Consequently, we picked the desired PCR products for sequencing. Results of Sanger sequencing show the successful construction of this part (Figure 22), which means that the plasmid could be used for the following experiments.
Figure 22. Validation of DNA sequence by Sanger sequencing, generated by SnapGene.
Step 4: Plasmid Extraction
After we acquired the correct plasmids, we then tried to extract and purify the plasmids for the following CFPS reactions. By using the plasmid extract kit, we gained the purified plasmids and analyzed them by agarose gel electrophoresis. Results in Figure 23 show that the extracted plasmids are clean, consisting of two conformations: linear and supercoiled. To further evaluate the plasmid sizes, we digested the three plasmids by EcoRI (restriction enzyme). After digestion, the three plasmids show the expected linearized conformation and match the desired DNA sizes. These results indicate the successful construction and extraction of this composite part.
Figure 23. Agarose gel electrophoresis analysis of plasmid extracts for three plasmid constructions. The plasmids without EcoRI digestion show two conformations: linear (larger DNA sizes) and supercoiled (smaller DNA sizes), while after EcoRI digestion, the plasmids are linearized and show expected DNA sizes refer to DNA sequences. As for this part, the digested plasmid meets the desired DNA size 2538 bp.
Step 5: CFPS Reaction
Once the plasmid was successfully constructed and extracted, we performed the CFPS reactions for demonstrating the feasibility of in vitro AMP Microcin H47 expression (Figure 24).
Figure 24. Schematic diagram of E. coli-based CFPS reaction for AMP Microcin H47 production.
Subsequently, following the commonly used CFPS protocol and Western-Blot analysis protocol, we successfully produced Microcin H47 (7.4 kDa) with acceptable soluble fraction (Figure 25).
Figure 25. Western-Blot analysis of E. coli-based CFPS reaction for AMP Microcin H47 production. The in vitro expressed Microcin H47 (c-terminal 6×HisTag, 7.4 kDa) could be detected with a soluble fraction. The plasmid (this construction) was added as 200 ng into 15 µL CFPS reactions, and the other experimental operations followed the reported protocol.
By doing these, the composite part was successfully constructed and characterized, demonstrating the feasibility of CFPS reaction for the in vitro production of antimicrobial peptide Microcin H47. Hence, this CFPS system was further utilized for the in vitro production of another antimicrobial peptide Microcin M in our project.
This composite part is derived from plasmid pJL1, consisting of four basic parts: T7 promoter, ribosome binding site, coding sequence of antimicrobial peptide Microcin M, and T7 terminator. The plasmid pJL1 is commonly used for the in vitro protein expression of cell-free protein synthesis (CFPS), and the iGEM-standardized CFPS construction has been constructed and characterized in our project. To further expand the application of CFPS systems in iGEM competition, this composite part is designed to demonstrate the feasibility of in vitro antimicrobial peptide (AMP) synthesis by CFPS for extended useful purposes.
Figure 26. Schematic design of this part, generated by SnapGene.
Figure 27. Detailed assembly pattern of this composite part, including four basic parts: T7 promoter, RBS, Microcin M, and T7 terminator.
For the characterization process of this part, follow five steps shown in Figure 28: (1) molecular cloning; (2) colony PCR; (3) sequencing; (4) plasmid extraction; (5) CFPS reaction.
Figure 28. Workflow for the construction and characterization of this part.
Step 1: Molecular Cloning
To construct this part, we first need to acquire the linearized DNA fragments of both vector and inserted fragments. Thus, we amplified vector pSB1C3 and inserted fragments by using PCR. Results of agarose gel electrophoresis show the desired DNA bands (Figure 29) as pSB1C3 (2070 bp) and inserted fragment (562 bp) used in this construction. Note that the PCR template of inserted fragment was derived from the previously reported research article.
Figure 29. Agarose gel electrophoresis analysis of PCR products for molecular cloning. The DNA bands indicate one vector pSB1C3 and three inserted fragments. The third inserted fragment of microcin M (562 bp) is used for the construction of this composite part, while the other two inserted fragments are used for sfGFP and microcin H47, respectively.
Step 2: Colony PCR
To verify the constructions, we next performed colony PCR by using the reported protocol. For each construction (not only this part, but also sfGFP and microcin H47), we selected four independent colonies from LB-agar plates and used primer pair VF2/VR to amplify the inserted DNA sequences. After that, we analyzed the DNA products by agarose gel electrophoresis. Results show that four PCR products match the desired DNA sizes of this construction (Figure 31).
Figure 31. Agarose gel electrophoresis analysis of PCR products for colony PCR. The four DNA products of this construction match the desired DNA size 833 bp, annotated by green √.
Step 3: Sequencing
Consequently, we picked the desired PCR products for sequencing. Results of Sanger sequencing show the successful construction of this part (Figure 32), which means that the plasmid could be used for the following experiments.
Figure 32. Validation of DNA sequence by Sanger sequencing, generated by SnapGene.
Step 4: Plasmid Extraction
After we acquired the correct plasmids, we then tried to extract and purify the plasmids for the following CFPS reactions. By using the plasmid extract kit, we gained the purified plasmids and analyzed them by agarose gel electrophoresis. Results in Figure 33 show that the extracted plasmids are clean, consisting of two conformations: linear and supercoiled. To further evaluate the plasmid sizes, we digested the three plasmids with EcoRI (restriction enzyme). After digestion, the three plasmids show the expected linearized conformation and match the desired DNA sizes. These results indicate the successful construction and extraction of this composite part.
Figure 33. Agarose gel electrophoresis analysis of plasmid extracts for three plasmid constructions. The plasmids without EcoRI digestion show two conformations: linear (larger DNA sizes) and supercoiled (smaller DNA sizes), while after EcoRI digestion, the plasmids are linearized and show expected DNA sizes referring to DNA sequences. As for this part, the digested plasmid meets the desired DNA size 2589 bp.
Step 5: CFPS Reaction
Once the plasmid was successfully constructed and extracted, we performed the CFPS reactions to demonstrate the feasibility of in vitro AMP Microcin M expression (Figure 34).
Figure 34. Schematic diagram of E. coli-based CFPS reaction for AMP Microcin M production.
Subsequently, following the commonly used CFPS protocol and Western-Blot analysis protocol, we successfully produced Microcin M (9.8 kDa) with acceptable soluble fraction (Figure 35).
Figure 35. Western-Blot analysis of E. coli-based CFPS reaction for AMP Microcin M production. The in vitro expressed Microcin M (c-terminal 6×HisTag, 9.8 kDa) could be detected with a soluble fraction. The plasmid (this construction) was added as 200 ng into 15 µL CFPS reactions, and the other experimental operations followed the reported protocol.
Through these methodologies, the composite part for the production of antimicrobial peptide Microcin M was successfully constructed and characterized, confirming the efficacy of cell-free protein synthesis (CFPS) for in vitro peptide expression. This CFPS system not only validates the potential for Microcin M synthesis but also lays the groundwork for the subsequent in vitro production of the antimicrobial peptide Microcin H47 within our project framework.
(1) In 4 L of deionized water, add 20 g of NaHCO3 and bring to a boil. Then, add 20 g of mulberry silk cocoons and simmer for 30 minutes.
(2) Repeat the above step once.
(3) Rinse the degummed mulberry silk in deionized water to remove various ions, wring it out, and place it in a 60 °C oven to dry.
Figure 36. The degrummed mulberry silk after being dried.
(1) Prepare a 9.3 mol/L LiBr solution: Weigh 40.39 g of LiBr powder, slowly add it to 25 mL of deionized water while stirring, and then add deionized water to make up to 50 mL.
(2) Weigh 5 g of degummed mulberry silk, add it to the LiBr solution, and gently stir with a glass rod to ensure the silk is completely submerged in the solution.
(3) Add a magnetic stir bar, heat in an oil bath at 60 °C, and stir at 1000 rpm for 1 hour.
(4) Dialysis: Pour the silk protein solution into a dialysis bag and dialyze in deionized water for 3 days.
Figure 37. Degummed mulberry silk dissolved in the LiBr solution.
Figure 38. The silk protein solution in the dialysis bag.
(1) Mix silk protein with functional components: silk protein-cell-free peptide system, silk protein-nisin system, silk protein-antibiotic system, and pure silk protein system.
- (i) Silk protein-cell-free peptide system: Mix H47 peptide and M7 peptide with the silk protein solution in a volume ratio of 1:1.
- (ii) Silk protein-nisin system: Dissolve nisin powder at a concentration of 40 mg/g in the silk protein solution.
- (iii) Silk protein-antibiotic system: Add 1 μL of Kana antibiotic to 1 mL of silk protein solution.
(2) Take 200 μL from each of the above systems and add them to the microneedle mold, removing bubbles in a vacuum oven (30 min, twice) to ensure that the solution fully penetrates the microneedle holes.
(3) Freeze in a -20 °C refrigerator for 2 hours, then freeze-dry for 12 hours.
Figure 39. Adding the three systems to the microneedle mold.
Figure 40. Counting for the rate of absorbance in different conditions targeting different bacteria.
As long as we find a more effective microcin that allows E.coli to produce more powerful antimicrobial peptides, we will immediately validate the results through a series of biochemical assays related to protein synthesis, enzyme activity, electrophoresis analysis, and plasmid embedding. If our findings pass these assays, we will proceed to test their efficacy for AMP synthesis in E.coli.
Additionally, we will continue searching for more environmentally friendly materials for microneedles and focus on how to increase the rate of absorbance of microneedles under different conditions.
Figure 41. Finding more appropriate materials to make microneedles.