Shanghai_city
Description
1. Background
The emergence of antibiotics has provided a powerful tool for human beings to fight against various bacterial
infections and has saved hundreds of millions of lives in the past hundred years. Improved livestock health, and
significantly increased meat and dairy production and hygiene. However, the abuse of antibiotics has led to the
emergence of widespread drug resistance since only a small amount of antibiotics can be effectively used, most of
them stay in water or soil as a pollutant. The remaining constantly increase the occurrence of resistance genes in
bacteria. In 2011, livestock excretion in China reaches 2.2 billion, and most contains resistance gene in it. These
genes can extend into harmful microorganisms through the biological membrane and becomes ‘Super bacteria’, which
reduces the effectiveness of antibiotics.
Fig.1. Abuse of antibiotics led to the birth of ‘Super bacteria’.
Antibacterial peptides (AMPs) were first found by G.Boman in 1980 named Cecropins. AMPs
are natural active substances, mostly composed of 20-60 amino acids, and have different effects including
antibacterial, antifungal, antiviral, etc., and are ideal substitutes with a little side effects for antibiotics.
These days, it got attention in agriculture, animal husbandry, fish farming, medicine and health, and so forth.
Fig.2.Main procedure of AMPs destroying biological membrane.
In the process of fish farming, whether it is large-scale farming for the purpose of fishery or family farming for
the purpose of ornamental purposes, maintaining a clean growth environment is of great significance to the growth of
fish. In particular, the lack of a perfect water circulation system for domestic ornamental fish can easily lead to
poor water quality in the fish tank, which will directly affect the growth of ornamental fish. Usually, fish tanks
use a very simple and crude method, namely activated carbon filtration and water changes, to maintain excellent
water quality. However, for large-scale fish farming enterprises and aquariums, filtering with activated carbon is
obviously not enough, and frequent water changes will consume a lot of water resources.
To help solve these problems, our project plans to launch a fish tank cleaner made of antimicrobial peptides, which
can effectively improve the water environment of fish. The use of antimicrobial peptide products can not only
effectively avoid the generation of drug resistance, but also reduce the frequency of water changes in the fish tank
and save water resources.
2.Experiment Design
Our team aims to create AMPs for cleaning aquarium. We will synthesize five types of AMPs which have potential value
in inhibiting the common bacteria in fish tank. Further tests are done for different scenarios to produce the
finalized product.
AMPs (Antimicrobial Peptides)
AMPs kill cells by disrupting membrane integrity by targeting the lipopolysaccharide
layer of the cell membrane,
thus inhibiting bacterial growth or killing bacteria. Compared to antibiotics that target a particular site of
action, AMPs act on a wider range and thus reduce the possibility of drug resistance. Besides this advantage, AMPs
also have a rapid killing effect and can enhance the activity of antibiotics. It is usually effective against one
class of microorganisms such as bacteria and fungi, but exceptions do occur.
Fig.3. Distribution of different types of antimicrobial peptides.
In this project, five types of AMPs are produced: Hydramacin-1, Spheniscin-2, Sparamosin26-54, LL-37, and Fusion.
The characteristics of each will be introduced separately:
1. Hydramacin-1
Hydramacin-1 is an antimicrobial protein discovered while investigating the epithelial defense of the ancient
metazoan Hydra. It is active both against Gram-positive and Gram-negative bacteria including multi-resistant human
pathogenic strains.
2. Spheniscin-2
Speniscin-2 can be antibacterial and antifungal against Gram-positive and Gram-negative bacteria, acting as the
beta-defensin of king penguin (Aptenodytes patagonicus)’s Stomach.
3. Sparamosin26-54
Sparamosin was identified in the mud crab Scylla paramamosain, being
active against Gram-negative bacteria, Gram-positive bacteria and fungi. It is particularly effective in fungicidal
functions but has limited effects on mammalian cells.
4. LL-37
LL-37 is expressed widely in human body cells, such as epithelial skin
cell，bone marrow cells, and neutrophils. It is responsible for immune defense against bacterial invasion, with study
showing its effect of significant reduction in attachment of bacteria and biofilm production
5. Fusion
In the fusion AMP, the functional gene for LL-37 and Sparamosin26-54 are
combined, intended to express a new AMP with both proficiencies of the two AMPs in bacterial and fungal inhibition.
This is a technical innovation of the project as a new attempt.
General Experiment Procedure
First, we downloaded the gene sequence of Hydramacin-1, Spheniscin-2, Sparamosin26-54, and LL-37 from the NCBI
database, and designed the fusion gene sequence of Sparamosin26-54 and LL-37, after codon optimization of the gene
sequence, it was synthesized into the pUC57 plasmid by a gene synthesis company.
Then, the five AMPs’gene were put into PCR apparatus for mass replication, and then the products were DNA gel electrophoresis to obtain our target genes.
Next, we extracted plasmids pET28a(+)-vector from E. coli DH5-alpha, linked the target gene and the vector into complete plasmids with homologous recombinant enzymes, and transferred the recombinant plasmids into BL21 by opening the membrane pores with a hot water bath, and then put them into ice to shrink the receptive state membrane pores.
Then, kanamycin was used to select whether the plasmid was inserted into the competent bacteria E. coli BL21(DE3) and screened, and the screened bacteria were coated with a culture dish for selection by PCR machine and gel electrophoresis. If it presents positive results, we remove it into the conical flask for shaking.
After that, when our bacteria grew, we used IPTG to induce it to express much protein, and then wash buffer and elution buffer were used to purify the protein we wanted..
Finally, we divided the protein into 11 gradients to conduct bacteriostatic experiments. We judged the survival rate of bacteria by the light transmittance of bacteria.
Then, the five AMPs’gene were put into PCR apparatus for mass replication, and then the products were DNA gel electrophoresis to obtain our target genes.
Next, we extracted plasmids pET28a(+)-vector from E. coli DH5-alpha, linked the target gene and the vector into complete plasmids with homologous recombinant enzymes, and transferred the recombinant plasmids into BL21 by opening the membrane pores with a hot water bath, and then put them into ice to shrink the receptive state membrane pores.
Then, kanamycin was used to select whether the plasmid was inserted into the competent bacteria E. coli BL21(DE3) and screened, and the screened bacteria were coated with a culture dish for selection by PCR machine and gel electrophoresis. If it presents positive results, we remove it into the conical flask for shaking.
After that, when our bacteria grew, we used IPTG to induce it to express much protein, and then wash buffer and elution buffer were used to purify the protein we wanted..
Finally, we divided the protein into 11 gradients to conduct bacteriostatic experiments. We judged the survival rate of bacteria by the light transmittance of bacteria.
3.Expected Results
1. Successfully construct Hydramacin-1, Spheniscin-2, Sparamosin26-54, LL-37, and fusion by removing from plasmid
pUC57 into pET-28a(+).
2. Expressed and purified antimicrobial peptide protein.
3. The expressed peptide can restrain the growth of bacteria compared with control group.
2. Expressed and purified antimicrobial peptide protein.
3. The expressed peptide can restrain the growth of bacteria compared with control group.
4.Reference
1. Bahar, A., & Ren, D. (2013). Antimicrobial peptides. Pharmaceuticals, 6(12), 1543–1575.
https://doi.org/10.3390/ph6121543
2. Chen, Y.-C., Yang, Y., Zhang, C., Chen, H.-Y., Chen, F., & Wang, K.-J. (2021). A novel antimicrobial peptide SPARAMOSIN26–54 from the mud crab scylla paramamosain showing potent antifungal activity against Cryptococcus neoformans. Frontiers in Microbiology, 12. https://doi.org/10.3389/fmicb.2021.746006
3. Hannah. (2022, February 20). LL-37 provides immune defense against bacterial, viral and fungal invasion.PeptideSciences.com. Retrieved July 10, 2022, from https://www.peptidesciences.com/blog/what-is-ll-37
4. Jung, S., Dingley, A. J., Augustin, R., Anton-Erxleben, F., Stanisak, M., Gelhaus, C., Gutsmann, T., Hammer, M. U., Podschun, R., Bonvin, A. M. J. J., Leippe, M., Bosch, T. C. G., & Grötzinger, J. (2009). Hydramacin-1, structure and antibacterial activity of a protein from the basal metazoan hydra. Journal of Biological Chemistry, 284(3), 1896–1905. https://doi.org/10.1074/jbc.m804713200
5. Lei, J., Sun, L., Huang, S., Zhu, C., Li, P., He, J., Mackey, V., Coy, D. H., & He, Q. (2019). The antimicrobial peptides and their potential clinical applications. American journal of translational research, 11(7), 3919–3931.
6. Madej, T., Lanczycki, C. J., Zhang, D., Thiessen, P. A., Geer, R. C., Marchler-Bauer, A., & Bryant, S. H. (2013). MMDB and vast+: Tracking structural similarities between macromolecular complexes. Nucleic Acids Research, 42(D1). https://doi.org/10.1093/nar/gkt1208
7. uniprot. (n.d.). P83430 · SPHE2_APTPA. Retrieved July 10, 2022, from https://www.uniprot.org/uniprotkb/P83430/entry#subcellular_location
8. Zhou M.,Xu Y.B.*,Ouyang P.Q.,Ling J.Y.,Cai Q.J.,Du Q.P.,Zheng L.,2020.Spread of resistance genes from duck manure to fish intestine in simulated fish-duck pond and the promotion of cefotaxime and As.Science of the Total Environment 731,138693.
2. Chen, Y.-C., Yang, Y., Zhang, C., Chen, H.-Y., Chen, F., & Wang, K.-J. (2021). A novel antimicrobial peptide SPARAMOSIN26–54 from the mud crab scylla paramamosain showing potent antifungal activity against Cryptococcus neoformans. Frontiers in Microbiology, 12. https://doi.org/10.3389/fmicb.2021.746006
3. Hannah. (2022, February 20). LL-37 provides immune defense against bacterial, viral and fungal invasion.PeptideSciences.com. Retrieved July 10, 2022, from https://www.peptidesciences.com/blog/what-is-ll-37
4. Jung, S., Dingley, A. J., Augustin, R., Anton-Erxleben, F., Stanisak, M., Gelhaus, C., Gutsmann, T., Hammer, M. U., Podschun, R., Bonvin, A. M. J. J., Leippe, M., Bosch, T. C. G., & Grötzinger, J. (2009). Hydramacin-1, structure and antibacterial activity of a protein from the basal metazoan hydra. Journal of Biological Chemistry, 284(3), 1896–1905. https://doi.org/10.1074/jbc.m804713200
5. Lei, J., Sun, L., Huang, S., Zhu, C., Li, P., He, J., Mackey, V., Coy, D. H., & He, Q. (2019). The antimicrobial peptides and their potential clinical applications. American journal of translational research, 11(7), 3919–3931.
6. Madej, T., Lanczycki, C. J., Zhang, D., Thiessen, P. A., Geer, R. C., Marchler-Bauer, A., & Bryant, S. H. (2013). MMDB and vast+: Tracking structural similarities between macromolecular complexes. Nucleic Acids Research, 42(D1). https://doi.org/10.1093/nar/gkt1208
7. uniprot. (n.d.). P83430 · SPHE2_APTPA. Retrieved July 10, 2022, from https://www.uniprot.org/uniprotkb/P83430/entry#subcellular_location
8. Zhou M.,Xu Y.B.*,Ouyang P.Q.,Ling J.Y.,Cai Q.J.,Du Q.P.,Zheng L.,2020.Spread of resistance genes from duck manure to fish intestine in simulated fish-duck pond and the promotion of cefotaxime and As.Science of the Total Environment 731,138693.