Difference between revisions of "Part:BBa K4607013"

 
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<partinfo>BBa_K4607013 short</partinfo>
  
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===Description===
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===Usage and Biology===
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<p align="justify">
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The biobrick consists of the albumin binding domain (ABD) from streptococcal protein G and is capable of increasing the lifetimes of antibodies, proteins, and enzymes. For this to be possible, the ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. This part consists of an affinity-maturated variant of the streptococcal protein G, which has been used for LysK expression with results of up to 34 hours in increasing the lifetime of the protein in mice [1]. The domain has a length of 29 amino acids and a molecular weight of 3.2388 kDa.
  
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<center>https://static.igem.wiki/teams/4607/wiki/parts/new-parts-e-coli/parts/900-px-composite-part-lysss-biorender-1.jpg</center>
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<span class='h3bb'>Sequence and Features</span>
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<center><b>Figure 1.</b> Expression casstte's PCNP-CecA-LysSS protein diagram.</center>
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===<span class='h3bb'><b>Sequence and Features</b></span>===
 
<partinfo>BBa_K4607013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4607013 SequenceAndFeatures</partinfo>
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===Biology and usage===
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<div style="text-align:justify;">
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As a brief contextualization, bovine mastitis is the result of the infection of the bovine mammary glands caused by pathogenic microorganisms, mainly gram-positive and negative bacteria. This disease reduces milk quality production to a great extent and produces painful damage to the bovine. The main treatment for mastitis is the use of diverse antibiotics, therefore the overuse and misuse of them have caused a real problem in the development of multidrug-resistant pathogens [2]. Our team has conducted an extensive investigation to find an alternative treatment for bovine mastitis without risking the environment.
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The principle behind our proposal is the use of fused proteins based on efficient bacteriophage endolysins. The function of a bacteriophage is to infect bacteria in order to kill them. Once the bacteria are infected and the virions are mature, they release holins, which are enzymes that create pores in the inner cell membrane. Endolysins now have access to the cell wall, so they can degrade it. Endolysins have lytic activity for the purpose of setting free the phage progeny to continue infecting other cells [3]. Endolysins are composed of two main domains: the N-terminal, which represents the catalytic domain, and the C-terminal, which is a cell wall binding domain, which interacts by binding itself to the bacterium's cell wall, activating the catalytic region, and causing cell wall lysis. However, the average endolysin lifetime is 20 minutes [4] [3].
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Tomar como ejemplo:
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The main purpose of the albumin binding domain (ABD) is to counteract the problems related to the brief in vivo time life of the endolysins. These domains have the capacity to increase the lifetime of antibodies, proteins, and enzymes through the incorporation of their sequences into the fusion protein. For this to be possible, the ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. This part consists of an affinity-maturated variant of the streptococcal protein G which has been used  for LysK expression, with results of up to 34 hours in increasing the lifetime of the protein in mice [1]. The best results have been achieved with the following conformation: CHAP domain-ABD-SH3 domain [5]. The domain has a length of 29 amino acids and a molecular weight of 3.2388 kDa. It keeps its stability in a range of 4 to 37 °C and a pH of 7 to 9. The average ABD-endolysin lifetime is about 30 hours [1].
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===References===
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[1] Seijsing, J., Sobieraj, A. M., Keller, N., Shen, Y., Zinkernagel, A. S., Loessner, M. J., & Schmelcher, M. (2018). Improved Biodistribution and Extended Serum Half-Life of a Bacteriophage Endolysin by Albumin Binding Domain Fusion. Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.02927
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[2] World Health Organization. (2021, November 17). Antimicrobial resistance. Who.int; World Health Organization: WHO. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance
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[3] Gutiérrez, D., Fernández, L., Rodríguez, A., & García, P. (2018). Are phage lytic proteins the secret weapon to kill Staphylococcus aureus?. MBio, 9(1), 10-1128. https://doi.org/10.1128/mbio.01923-17
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[4] Fernández, L., González, S., Campelo, A. B., Martínez, B., Rodríguez, A., & García, P. (2017). Downregulation of Autolysin-Encoding Genes by Phage-Derived Lytic Proteins Inhibits Biofilm Formation in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 61(5), e02724-16. https://doi.org/10.1128/AAC.02724-16
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[5] Schmelcher, M., Powell, A. M., Becker, S. C., Camp, M. J., & Donovan, D. M. (2012). Chimeric Phage Lysins Act Synergistically with Lysostaphin To Kill Mastitis-Causing Staphylococcus aureus in Murine Mammary Glands. Applied and Environmental Microbiology, 78(7), 2297–2305. https://doi.org/10.1128/aem.07050-11
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4607011 parameters</partinfo>
 
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Revision as of 19:09, 9 September 2023

Expression cassette for PCNP-CecA-LysSS protein


Description

The biobrick consists of the albumin binding domain (ABD) from streptococcal protein G and is capable of increasing the lifetimes of antibodies, proteins, and enzymes. For this to be possible, the ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. This part consists of an affinity-maturated variant of the streptococcal protein G, which has been used for LysK expression with results of up to 34 hours in increasing the lifetime of the protein in mice [1]. The domain has a length of 29 amino acids and a molecular weight of 3.2388 kDa.

900-px-composite-part-lysss-biorender-1.jpg
Figure 1. Expression casstte's PCNP-CecA-LysSS protein diagram.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 833
    Illegal XbaI site found at 96
    Illegal SpeI site found at 127
    Illegal SpeI site found at 814
    Illegal PstI site found at 505
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 833
    Illegal SpeI site found at 127
    Illegal SpeI site found at 814
    Illegal PstI site found at 505
    Illegal NotI site found at 975
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 833
    Illegal BglII site found at 30
    Illegal BamHI site found at 827
    Illegal XhoI site found at 984
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 833
    Illegal XbaI site found at 96
    Illegal SpeI site found at 127
    Illegal SpeI site found at 814
    Illegal PstI site found at 505
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 833
    Illegal XbaI site found at 96
    Illegal SpeI site found at 127
    Illegal SpeI site found at 814
    Illegal PstI site found at 505
  • 1000
    COMPATIBLE WITH RFC[1000]

Biology and usage

As a brief contextualization, bovine mastitis is the result of the infection of the bovine mammary glands caused by pathogenic microorganisms, mainly gram-positive and negative bacteria. This disease reduces milk quality production to a great extent and produces painful damage to the bovine. The main treatment for mastitis is the use of diverse antibiotics, therefore the overuse and misuse of them have caused a real problem in the development of multidrug-resistant pathogens [2]. Our team has conducted an extensive investigation to find an alternative treatment for bovine mastitis without risking the environment.

The principle behind our proposal is the use of fused proteins based on efficient bacteriophage endolysins. The function of a bacteriophage is to infect bacteria in order to kill them. Once the bacteria are infected and the virions are mature, they release holins, which are enzymes that create pores in the inner cell membrane. Endolysins now have access to the cell wall, so they can degrade it. Endolysins have lytic activity for the purpose of setting free the phage progeny to continue infecting other cells [3]. Endolysins are composed of two main domains: the N-terminal, which represents the catalytic domain, and the C-terminal, which is a cell wall binding domain, which interacts by binding itself to the bacterium's cell wall, activating the catalytic region, and causing cell wall lysis. However, the average endolysin lifetime is 20 minutes [4] [3].



Tomar como ejemplo: The main purpose of the albumin binding domain (ABD) is to counteract the problems related to the brief in vivo time life of the endolysins. These domains have the capacity to increase the lifetime of antibodies, proteins, and enzymes through the incorporation of their sequences into the fusion protein. For this to be possible, the ABD binds with high affinity to serum albumin, creating a large hydrodynamic volume complex that reduces its degradation. This part consists of an affinity-maturated variant of the streptococcal protein G which has been used for LysK expression, with results of up to 34 hours in increasing the lifetime of the protein in mice [1]. The best results have been achieved with the following conformation: CHAP domain-ABD-SH3 domain [5]. The domain has a length of 29 amino acids and a molecular weight of 3.2388 kDa. It keeps its stability in a range of 4 to 37 °C and a pH of 7 to 9. The average ABD-endolysin lifetime is about 30 hours [1].

References

[1] Seijsing, J., Sobieraj, A. M., Keller, N., Shen, Y., Zinkernagel, A. S., Loessner, M. J., & Schmelcher, M. (2018). Improved Biodistribution and Extended Serum Half-Life of a Bacteriophage Endolysin by Albumin Binding Domain Fusion. Frontiers in Microbiology, 9. https://doi.org/10.3389/fmicb.2018.02927

[2] World Health Organization. (2021, November 17). Antimicrobial resistance. Who.int; World Health Organization: WHO. https://www.who.int/news-room/fact-sheets/detail/antimicrobial-resistance

[3] Gutiérrez, D., Fernández, L., Rodríguez, A., & García, P. (2018). Are phage lytic proteins the secret weapon to kill Staphylococcus aureus?. MBio, 9(1), 10-1128. https://doi.org/10.1128/mbio.01923-17

[4] Fernández, L., González, S., Campelo, A. B., Martínez, B., Rodríguez, A., & García, P. (2017). Downregulation of Autolysin-Encoding Genes by Phage-Derived Lytic Proteins Inhibits Biofilm Formation in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 61(5), e02724-16. https://doi.org/10.1128/AAC.02724-16

[5] Schmelcher, M., Powell, A. M., Becker, S. C., Camp, M. J., & Donovan, D. M. (2012). Chimeric Phage Lysins Act Synergistically with Lysostaphin To Kill Mastitis-Causing Staphylococcus aureus in Murine Mammary Glands. Applied and Environmental Microbiology, 78(7), 2297–2305. https://doi.org/10.1128/aem.07050-11