Team:Tunghai TAPG/Results
Overview
JJ01 is our core subject, it is an antimicrobial peptide that has been verified that it has excellent antibacterial effect and lower hemolysis. We discovered there are three key proteins could help us to produce antimicrobial peptide effectively.
Goal:
- Improved the engineering bacteria provided by iGEM by adding four proteins which could be optimized--- eGFP (enhanced green fluorescent protein), SUMO (small ubiquitin-like modifier), sec VMA intine and 6xHis tag.
- 2. Using eGFP as an identifier to monitor the yield of the proteins.
Results:
- Execute the production of antimicrobial peptide JJ01.
- Verify the effect of antimicrobial peptide JJ01.
Results
Design the Vector
Improved the engineered bacteria that provided by iGEM by adding four special proteins with different effects. Adding His 6x which can bind to metal divalent ions, and facilitate the purification of the target protein. The proteins with His tag can be purified by divalent metal ions affinity chromatography column under non-denaturing conditions. Then addition of SUMO is to avoid the possible toxicity of our target proteins to the bacterial host and increase its solubility as well. And using eGFP as a reporter to help us quickly screen for successful transformation of colonies and also allow us to quickly monitor the protein production. In order to obtain the pure target peptide, we have added the Intein. which is a self-cleavable polypeptide chain under specific conditions.
Fig.1. Successful PCR 1% Agarose gel electrophoresis.
- Lane 1: 100 bp DNA Ladder
- Lane 2: sample 1 (SUMO: 297 bp)
- Lane 3: sample 2 (eGFP: 717 bp)
- Lane 4: sample 3 (Intein: 1534 bp)
Figure 1. Three DNA fragments were produced by PCR for the major parts in the recombinant plasmid followed by the agarose gel electrophoresis analysis. We could notice the size of each fragment is very close to our expectation.
We have designed a vector that contains eGFP(enhanced green fluorescent protein), SUMO (small ubiquitin-like modifier), and sec VMA intein, then using PCR to amplify these fragments followed by agarose gel electrophoresis. Fig.1 demonstrates the size of each PCR product is similar to that of our expected.
Fig.2. Successful PCR 0.7 % Agarose gel electrophoresis
- Lane 1: 1kb DNA Ladder
- Lane 2: sample1 (vector with Intein:4784 bp)
- Lane 3: sample2 (vector without Intein:3250 bp)
Figure 2.Two vectors were selected for PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis. The size of each fragment is very close to our expectation and their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan.
Ligation with BsaⅠ-HFv2 enzyme and T7 DNA Ligase, then using PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis. The size of each fragment is very close to our expectation and their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan. Heat shock transformation into chemically component E. coli (DH5α) was performed and cells were streaked on a plate to obtain isolated colonies. The colonies were selected by PCR amplification followed by the agarose gel electrophoresis analysis. And their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan.
Plasmid construction
We have designed the target gene (JJ01) and contracted with commissioned Mission Biotech for JJ01 DNA synthesis as well as sequencing later on. Because protein cleavage and purification are commonly not easy and enzymes for protein cleavage are quit expensive. we therefore constructed two plasmids, one of which contains sec VMA Intine for easy purification. We learned from the Journal of Engineering that the addition of Intein is not always conducive to soup up the expression of the recombinant protein due to the large molecular weight of Intein. This result is consistent with our experimental results, which inferred that the addition of Intein in the vector made the target protein to be at the precipitation. Later on, we will commission it to a Bio company for protein purification with HPLC.
Fig.3. Successful PCR 4% Agarose gel electrophoresis.
- Lane 1:50 bp DNA Ladder
- Lane 2-4: sample 1-3 (target: 33 bp)
Figure 3. target gene (JJ01) were used PCR to carry on a small amount of expansion, afterward, Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size.
JJ01 antibacterial peptide was composited by 11 amino acids. We reverse transcript the JJ01 antibacterial peptide from amino acid according to genetic code. We then can design the primers for PCR to amplify the DNA fragment, followed by the agarose gel electrophoresis analysis.
Fig.4. Checkout plasmid 4817 bp 0.7% Agarose gel electrophoresis.
- Lane 1: sample 1 (plasmid without Extraction)
- Lane 2: sample 2 (plasmid with Extraction)
- Lane 3: 1 kb DNA Ladder
Figure 4. Plasmids were used PCR to carry on a small amount of expansion, afterward, Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size and the content is low but, the relative purity is higher.
The ligation reaction was carried out using the cleavage enzyme BsaI-HFv2 and ligase T7 DNA Ligation, and the ligated product was subjected to DNA extraction using a Gene-Spin TM 1-4-3 DNA Purification Kit, and plasmids were used PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis with the proper size. Although the intensity is weak, the purity is relatively higher.
Fig.5. Checkout E.coli (DH5α with TAPG-JJ01) 4817 bp 0.7 % Agarose gel electrophoresis.
- Lane 1: 1 kb DNA Ladder
- Lane 2-6: DNA sample 1-5
- Lane 7-11: DNA sample 6-11
Figure 5.Heat shock transformation of plasmid into of cComponent E. coli (DH5α) due to its stabilityle and easy forto transformation. properties andThe cells were streaked on a plate to obtain isolated colonies (determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed). Using used PCR to select the coloniesy., afterward Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size According to, gel analysis, the colonies 1, 3, 5 (plasmid with intein DH5α), and colonies 7, 10 (plasmid with intein DH5α) are roughly verifiedcorrect, so we choose colonies 1 and ,7 for further study. again coated, Their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan.
Fig.6. DNA sequence(JJ01).
Figure 6. DNA Sequencing of target gene (JJ01) to checkout DNA Sequence is correct.
Through the DNA sequence result map, the DNA sequence was compared with the originally designed target gene (JJ01) sequence, and it was found that this sequence was consistent with the sequence we originally designed, and we can know that we have successfully linked the target gene (JJ01) into our plasmid (Pet6) and successfully transformed into the competent cell (DH5α)..
Fig.7. Dish with bacterial (E.coli (C43 with PATG-JJ01)) under the ultraviolet light.
Figure 7. Dish with bacterial (E.coli (C43 with PATG-JJ01)) under the ultraviolet light. And determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed into competent cells (C43).
Using heat shock for second transformation, we chose to transformation into E. coli (C43) because it can efficiently enhance the expression of toxic proteins. The following figure shows the image of bacterial disk under ultraviolet light, which can preliminarily determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed into competent cells (C43) and containing the target gene as well.
Fig.8. Checkout protein 44 kDa (plasmid without sec VMA intine).
- Lane 1: Protein Maker (10-245 kDa)
- Lane 2-3: protein samples collected from cell disruption (one-time centrifugation)
Figure 8. SDS-PAGE.(protein without Intein) indicating that the correct position and there are more products in the soup.
Fig.9. Checkout protein 100 kDa (plasmid with sce VMA intein).
- Lane 1-3: protein samples collected from cell disruption (two-time centrifugation)
- Lane 4-6: protein samples collected from cell disruption (one-time centrifugation)
- Lane 7: Protein Maker (10-245 kDa)
Figure 9. SDS-PAGE. (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.
After pProtein oOverexpression, the cells were harvested by centrifugation and disruptedion process was utilized by a Ultrasonic Cell Disruptor to obtain the cell extract containing for the recombinant protein. The protein expression was and also analyzed by SDS-PAGE.
Fig.8. The checkout protein at 44 kDa (plasmid without sec VMA intine) indicatsing that the correct position and high expression of target protein, and also demonstrating the ration between the supernatant and the precipitation up to 90 percent.We added sec VMA intine protein in order to splice target protein also reduce the amino acid residue. According to Fig.9. checking out protein at 100 kDa (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.We added sec VMA intine protein in order to splice target protein also reduce the amino acid residue. According to Fig.9. checking out protein at 100 kDa (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.
Fig.10. We grew the bacteria on the Petri dishes. Left: with JJ01, right: without JJ01.
Fig.11. Antimicrobial activity of JJ01.
We tested the MIC90 (E. coli) of JJ01 with the purified target peptide and confirmed that the JJ01 has the same antibacterial effect as the patent. So we can believe that we have succeeded in doing the Genetic engineering experiments.
Reference
- 1. Wang SJ, Chen BY, Wang YJ, et al. Application of inteins in building protein affinity purification system. Chin J Biotech, 2016.
- 2. Bailey A. Fong, W.-Y. W, David W. Wood, The potential role of self-cleaving Purification tags in commercial-scale processes. Trends in Biotechnology, 2010.
- 3. Li , Y., Self-cleaving fusion tags for recombinant protein production Biotechnology Letters, 2011.
- 4. DW, W., New trends and affinity tag designs for recombinant protein purification. Current Opinion in Structural Biology, 2014.
- 5. Hassouneh W,C.T.,Chilkoti A, Elastin-like polypeptides as a purification tag for recombinant proteins. Current Protocols in Protein Science, 2010.
- 6. Micklos DA, Freyer GA (2003) DNA Science: A First Course, 2nd ed. Cold Spring Harbor Laboratory Press, New York.
Goal:
- Improved the engineering bacteria provided by iGEM by adding four proteins which could be optimized--- eGFP (enhanced green fluorescent protein), SUMO (small ubiquitin-like modifier), sec VMA intine and 6xHis tag.
- 2. Using eGFP as an identifier to monitor the yield of the proteins.
- Improved the engineering bacteria provided by iGEM by adding four proteins which could be optimized--- eGFP (enhanced green fluorescent protein), SUMO (small ubiquitin-like modifier), sec VMA intine and 6xHis tag.
- 2. Using eGFP as an identifier to monitor the yield of the proteins.
Results:
- Execute the production of antimicrobial peptide JJ01.
- Verify the effect of antimicrobial peptide JJ01.
- Execute the production of antimicrobial peptide JJ01.
- Verify the effect of antimicrobial peptide JJ01.
Results
Design the Vector
Improved the engineered bacteria that provided by iGEM by adding four special proteins with different effects. Adding His 6x which can bind to metal divalent ions, and facilitate the purification of the target protein. The proteins with His tag can be purified by divalent metal ions affinity chromatography column under non-denaturing conditions. Then addition of SUMO is to avoid the possible toxicity of our target proteins to the bacterial host and increase its solubility as well. And using eGFP as a reporter to help us quickly screen for successful transformation of colonies and also allow us to quickly monitor the protein production. In order to obtain the pure target peptide, we have added the Intein. which is a self-cleavable polypeptide chain under specific conditions.
Fig.1. Successful PCR 1% Agarose gel electrophoresis.
- Lane 1: 100 bp DNA Ladder
- Lane 2: sample 1 (SUMO: 297 bp)
- Lane 3: sample 2 (eGFP: 717 bp)
- Lane 4: sample 3 (Intein: 1534 bp)
Figure 1. Three DNA fragments were produced by PCR for the major parts in the recombinant plasmid followed by the agarose gel electrophoresis analysis. We could notice the size of each fragment is very close to our expectation.
We have designed a vector that contains eGFP(enhanced green fluorescent protein), SUMO (small ubiquitin-like modifier), and sec VMA intein, then using PCR to amplify these fragments followed by agarose gel electrophoresis. Fig.1 demonstrates the size of each PCR product is similar to that of our expected.
Fig.2. Successful PCR 0.7 % Agarose gel electrophoresis
- Lane 1: 1kb DNA Ladder
- Lane 2: sample1 (vector with Intein:4784 bp)
- Lane 3: sample2 (vector without Intein:3250 bp)
Figure 2.Two vectors were selected for PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis. The size of each fragment is very close to our expectation and their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan.
Ligation with BsaⅠ-HFv2 enzyme and T7 DNA Ligase, then using PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis. The size of each fragment is very close to our expectation and their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan. Heat shock transformation into chemically component E. coli (DH5α) was performed and cells were streaked on a plate to obtain isolated colonies. The colonies were selected by PCR amplification followed by the agarose gel electrophoresis analysis. And their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan.
Plasmid construction
We have designed the target gene (JJ01) and contracted with commissioned Mission Biotech for JJ01 DNA synthesis as well as sequencing later on. Because protein cleavage and purification are commonly not easy and enzymes for protein cleavage are quit expensive. we therefore constructed two plasmids, one of which contains sec VMA Intine for easy purification. We learned from the Journal of Engineering that the addition of Intein is not always conducive to soup up the expression of the recombinant protein due to the large molecular weight of Intein. This result is consistent with our experimental results, which inferred that the addition of Intein in the vector made the target protein to be at the precipitation. Later on, we will commission it to a Bio company for protein purification with HPLC.
Fig.3. Successful PCR 4% Agarose gel electrophoresis.
- Lane 1:50 bp DNA Ladder
- Lane 2-4: sample 1-3 (target: 33 bp)
Figure 3. target gene (JJ01) were used PCR to carry on a small amount of expansion, afterward, Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size.
JJ01 antibacterial peptide was composited by 11 amino acids. We reverse transcript the JJ01 antibacterial peptide from amino acid according to genetic code. We then can design the primers for PCR to amplify the DNA fragment, followed by the agarose gel electrophoresis analysis.
Fig.4. Checkout plasmid 4817 bp 0.7% Agarose gel electrophoresis.
- Lane 1: sample 1 (plasmid without Extraction)
- Lane 2: sample 2 (plasmid with Extraction)
- Lane 3: 1 kb DNA Ladder
Figure 4. Plasmids were used PCR to carry on a small amount of expansion, afterward, Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size and the content is low but, the relative purity is higher.
The ligation reaction was carried out using the cleavage enzyme BsaI-HFv2 and ligase T7 DNA Ligation, and the ligated product was subjected to DNA extraction using a Gene-Spin TM 1-4-3 DNA Purification Kit, and plasmids were used PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis with the proper size. Although the intensity is weak, the purity is relatively higher.
Fig.5. Checkout E.coli (DH5α with TAPG-JJ01) 4817 bp 0.7 % Agarose gel electrophoresis.
- Lane 1: 1 kb DNA Ladder
- Lane 2-6: DNA sample 1-5
- Lane 7-11: DNA sample 6-11
Figure 5.Heat shock transformation of plasmid into of cComponent E. coli (DH5α) due to its stabilityle and easy forto transformation. properties andThe cells were streaked on a plate to obtain isolated colonies (determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed). Using used PCR to select the coloniesy., afterward Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size According to, gel analysis, the colonies 1, 3, 5 (plasmid with intein DH5α), and colonies 7, 10 (plasmid with intein DH5α) are roughly verifiedcorrect, so we choose colonies 1 and ,7 for further study. again coated, Their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan.
Fig.6. DNA sequence(JJ01).
Figure 6. DNA Sequencing of target gene (JJ01) to checkout DNA Sequence is correct.
Through the DNA sequence result map, the DNA sequence was compared with the originally designed target gene (JJ01) sequence, and it was found that this sequence was consistent with the sequence we originally designed, and we can know that we have successfully linked the target gene (JJ01) into our plasmid (Pet6) and successfully transformed into the competent cell (DH5α)..
Fig.7. Dish with bacterial (E.coli (C43 with PATG-JJ01)) under the ultraviolet light.
Figure 7. Dish with bacterial (E.coli (C43 with PATG-JJ01)) under the ultraviolet light. And determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed into competent cells (C43).
Using heat shock for second transformation, we chose to transformation into E. coli (C43) because it can efficiently enhance the expression of toxic proteins. The following figure shows the image of bacterial disk under ultraviolet light, which can preliminarily determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed into competent cells (C43) and containing the target gene as well.
Fig.8. Checkout protein 44 kDa (plasmid without sec VMA intine).
- Lane 1: Protein Maker (10-245 kDa)
- Lane 2-3: protein samples collected from cell disruption (one-time centrifugation)
Figure 8. SDS-PAGE.(protein without Intein) indicating that the correct position and there are more products in the soup.
Fig.9. Checkout protein 100 kDa (plasmid with sce VMA intein).
- Lane 1-3: protein samples collected from cell disruption (two-time centrifugation)
- Lane 4-6: protein samples collected from cell disruption (one-time centrifugation)
- Lane 7: Protein Maker (10-245 kDa)
Figure 9. SDS-PAGE. (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.
After pProtein oOverexpression, the cells were harvested by centrifugation and disruptedion process was utilized by a Ultrasonic Cell Disruptor to obtain the cell extract containing for the recombinant protein. The protein expression was and also analyzed by SDS-PAGE.
Fig.8. The checkout protein at 44 kDa (plasmid without sec VMA intine) indicatsing that the correct position and high expression of target protein, and also demonstrating the ration between the supernatant and the precipitation up to 90 percent.We added sec VMA intine protein in order to splice target protein also reduce the amino acid residue. According to Fig.9. checking out protein at 100 kDa (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.We added sec VMA intine protein in order to splice target protein also reduce the amino acid residue. According to Fig.9. checking out protein at 100 kDa (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.
Fig.10. We grew the bacteria on the Petri dishes. Left: with JJ01, right: without JJ01.
Fig.11. Antimicrobial activity of JJ01.
We tested the MIC90 (E. coli) of JJ01 with the purified target peptide and confirmed that the JJ01 has the same antibacterial effect as the patent. So we can believe that we have succeeded in doing the Genetic engineering experiments.
Reference
- 1. Wang SJ, Chen BY, Wang YJ, et al. Application of inteins in building protein affinity purification system. Chin J Biotech, 2016.
- 2. Bailey A. Fong, W.-Y. W, David W. Wood, The potential role of self-cleaving Purification tags in commercial-scale processes. Trends in Biotechnology, 2010.
- 3. Li , Y., Self-cleaving fusion tags for recombinant protein production Biotechnology Letters, 2011.
- 4. DW, W., New trends and affinity tag designs for recombinant protein purification. Current Opinion in Structural Biology, 2014.
- 5. Hassouneh W,C.T.,Chilkoti A, Elastin-like polypeptides as a purification tag for recombinant proteins. Current Protocols in Protein Science, 2010.
- 6. Micklos DA, Freyer GA (2003) DNA Science: A First Course, 2nd ed. Cold Spring Harbor Laboratory Press, New York.
Ligation with BsaⅠ-HFv2 enzyme and T7 DNA Ligase, then using PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis. The size of each fragment is very close to our expectation and their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan. Heat shock transformation into chemically component E. coli (DH5α) was performed and cells were streaked on a plate to obtain isolated colonies. The colonies were selected by PCR amplification followed by the agarose gel electrophoresis analysis. And their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan.
Plasmid construction
We have designed the target gene (JJ01) and contracted with commissioned Mission Biotech for JJ01 DNA synthesis as well as sequencing later on. Because protein cleavage and purification are commonly not easy and enzymes for protein cleavage are quit expensive. we therefore constructed two plasmids, one of which contains sec VMA Intine for easy purification. We learned from the Journal of Engineering that the addition of Intein is not always conducive to soup up the expression of the recombinant protein due to the large molecular weight of Intein. This result is consistent with our experimental results, which inferred that the addition of Intein in the vector made the target protein to be at the precipitation. Later on, we will commission it to a Bio company for protein purification with HPLC.
Fig.3. Successful PCR 4% Agarose gel electrophoresis.
- Lane 1:50 bp DNA Ladder
- Lane 2-4: sample 1-3 (target: 33 bp)
- Lane 1:50 bp DNA Ladder
- Lane 2-4: sample 1-3 (target: 33 bp)
Figure 3. target gene (JJ01) were used PCR to carry on a small amount of expansion, afterward, Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size.
JJ01 antibacterial peptide was composited by 11 amino acids. We reverse transcript the JJ01 antibacterial peptide from amino acid according to genetic code. We then can design the primers for PCR to amplify the DNA fragment, followed by the agarose gel electrophoresis analysis.
Fig.4. Checkout plasmid 4817 bp 0.7% Agarose gel electrophoresis.
- Lane 1: sample 1 (plasmid without Extraction)
- Lane 2: sample 2 (plasmid with Extraction)
- Lane 3: 1 kb DNA Ladder
Figure 4. Plasmids were used PCR to carry on a small amount of expansion, afterward, Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size and the content is low but, the relative purity is higher.
The ligation reaction was carried out using the cleavage enzyme BsaI-HFv2 and ligase T7 DNA Ligation, and the ligated product was subjected to DNA extraction using a Gene-Spin TM 1-4-3 DNA Purification Kit, and plasmids were used PCR to carry on a small amount of amplification followed by the agarose gel electrophoresis analysis with the proper size. Although the intensity is weak, the purity is relatively higher.
Fig.5. Checkout E.coli (DH5α with TAPG-JJ01) 4817 bp 0.7 % Agarose gel electrophoresis.
- Lane 1: 1 kb DNA Ladder
- Lane 2-6: DNA sample 1-5
- Lane 7-11: DNA sample 6-11
Figure 5.Heat shock transformation of plasmid into of cComponent E. coli (DH5α) due to its stabilityle and easy forto transformation. properties andThe cells were streaked on a plate to obtain isolated colonies (determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed). Using used PCR to select the coloniesy., afterward Run the gel to do initial analysis, we could know from Agarose gel electrophoresis the results of expansion are mostly fit the size According to, gel analysis, the colonies 1, 3, 5 (plasmid with intein DH5α), and colonies 7, 10 (plasmid with intein DH5α) are roughly verifiedcorrect, so we choose colonies 1 and ,7 for further study. again coated, Their sequences were confirmed with DNA sequencing by Mission Biotech Company in Taiwan.
Fig.6. DNA sequence(JJ01).
Figure 6. DNA Sequencing of target gene (JJ01) to checkout DNA Sequence is correct.
Through the DNA sequence result map, the DNA sequence was compared with the originally designed target gene (JJ01) sequence, and it was found that this sequence was consistent with the sequence we originally designed, and we can know that we have successfully linked the target gene (JJ01) into our plasmid (Pet6) and successfully transformed into the competent cell (DH5α)..
Fig.7. Dish with bacterial (E.coli (C43 with PATG-JJ01)) under the ultraviolet light.
Figure 7. Dish with bacterial (E.coli (C43 with PATG-JJ01)) under the ultraviolet light. And determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed into competent cells (C43).
Using heat shock for second transformation, we chose to transformation into E. coli (C43) because it can efficiently enhance the expression of toxic proteins. The following figure shows the image of bacterial disk under ultraviolet light, which can preliminarily determine the colony that emits green fluorescent light, and is a colony that is more likely to be successfully transformed into competent cells (C43) and containing the target gene as well.
Fig.8. Checkout protein 44 kDa (plasmid without sec VMA intine).
- Lane 1: Protein Maker (10-245 kDa)
- Lane 2-3: protein samples collected from cell disruption (one-time centrifugation)
Figure 8. SDS-PAGE.(protein without Intein) indicating that the correct position and there are more products in the soup.
Fig.9. Checkout protein 100 kDa (plasmid with sce VMA intein).
- Lane 1-3: protein samples collected from cell disruption (two-time centrifugation)
- Lane 4-6: protein samples collected from cell disruption (one-time centrifugation)
- Lane 7: Protein Maker (10-245 kDa)
- Lane 1-3: protein samples collected from cell disruption (two-time centrifugation)
- Lane 4-6: protein samples collected from cell disruption (one-time centrifugation)
- Lane 7: Protein Maker (10-245 kDa)
Figure 9. SDS-PAGE. (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.
After pProtein oOverexpression, the cells were harvested by centrifugation and disruptedion process was utilized by a Ultrasonic Cell Disruptor to obtain the cell extract containing for the recombinant protein. The protein expression was and also analyzed by SDS-PAGE.
Fig.8. The checkout protein at 44 kDa (plasmid without sec VMA intine) indicatsing that the correct position and high expression of target protein, and also demonstrating the ration between the supernatant and the precipitation up to 90 percent.We added sec VMA intine protein in order to splice target protein also reduce the amino acid residue. According to Fig.9. checking out protein at 100 kDa (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.We added sec VMA intine protein in order to splice target protein also reduce the amino acid residue. According to Fig.9. checking out protein at 100 kDa (plasmid with sec VMA intine) can make sure the target protein position and most of its are at the part of precipitation.
Fig.10. We grew the bacteria on the Petri dishes. Left: with JJ01, right: without JJ01.
Fig.11. Antimicrobial activity of JJ01.
We tested the MIC90 (E. coli) of JJ01 with the purified target peptide and confirmed that the JJ01 has the same antibacterial effect as the patent. So we can believe that we have succeeded in doing the Genetic engineering experiments.
Reference
- 1. Wang SJ, Chen BY, Wang YJ, et al. Application of inteins in building protein affinity purification system. Chin J Biotech, 2016.
- 2. Bailey A. Fong, W.-Y. W, David W. Wood, The potential role of self-cleaving Purification tags in commercial-scale processes. Trends in Biotechnology, 2010.
- 3. Li , Y., Self-cleaving fusion tags for recombinant protein production Biotechnology Letters, 2011.
- 4. DW, W., New trends and affinity tag designs for recombinant protein purification. Current Opinion in Structural Biology, 2014.
- 5. Hassouneh W,C.T.,Chilkoti A, Elastin-like polypeptides as a purification tag for recombinant proteins. Current Protocols in Protein Science, 2010.
- 6. Micklos DA, Freyer GA (2003) DNA Science: A First Course, 2nd ed. Cold Spring Harbor Laboratory Press, New York.