Difference between revisions of "Part:BBa K4437001"

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<partinfo>BBa_K4437001 short</partinfo>
 
<partinfo>BBa_K4437001 short</partinfo>
  
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===Usage and Biology===
  
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<!-- Nisin is an antimicrobial protein derived from Lactococcus lactis bacteria strain. Amongst all the derivatives of nisin, nisin Q has the highest microbial activity and loses its activity the slowest by inhibiting oxidation. The mechanisms of nisin works by it binding to lipid II present on the cell membrane. This binding results in the creation of a pore and prevents the further synthesis of peptidoglycan - a protein found in the cell membrane- which leads to death. Nisin targets both gram positive and gram negative bacteria, however, is more effective towards gram positive bacteria as they have a higher quantity of peptidoglycan present. Nisin is pH and thermostable.
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<p>Derived from <I>Lactococcus lactis</I>, nisin is a food-safe, antimicrobial peptide (AMP) that targets a wide range of Gram-positive bacteria by binding to lipid II on the pathogens membrane, creating a pore, and causing cell death [1]. Literature suggests that nisin Q (NisQ) demonstrates greater antimicrobial and antioxidant activity against pathogens compared to other variants of nisin, such as NisA (BBa_K1365000) [2]. Nisin’s optimal pH stability is between 2 and 7 but can maintain its antibacterial activity up to a pH of 12, and can also retain its antimicrobial activity at temperatures of 120<sup>o</sup>C [2]. Unlike other AMPs, nisin is non-toxic to Gram-negative bacteria, meaning that successful recombinant expression in <I>E. coli</I> can be achieved without an inhibitory protein.</p>
  
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Our project focused on developing a fruit packaging which has been integrated with our antimicrobial peptide-nisin. Nisin has been shown to be effective against many fungi but there is scare research done on its effectiveness on bacteria. We designed a gene fragment containing nisin in order to test its effective against bacteria commonly found on fruit. We characterized its properties using Kirby Bauer tests as well as minimum inhibitory concentration tests.  
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===Design===
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<p> Nisin GB1 (BBa_K4437001) includes, a T7 promoter, a lac operon, RBS, TEV protease site, 6XHis-tag, two enterokinase cut sites, nisQ, and T7 terminator. The two enterokinase sites serves to make sure the 6XHis-tag is cleaved off and surely leaves us with nisQ. The lac operon functions to be able to use different protein induction media such as autoinduction medias when expression protein. The presence of the lac operon will allow the bacteria to switch from a glucose sugar source to a lactose sugar source. The rest if the elements are regulatory elements necessary to express our protein.</p>
  
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===Usage and Biology===
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[[Image:gb1.png|400px|thumb|center]]
  
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===Characterization===
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<span class='h3bb'>Sequence and Features</span>
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<p>We were able to insert our Nisin GB1 (BBa_K4437001) into a pSB1A3 plasmid. Once it was PCR amplified, it was run on a gel in which its indicated band size 331 was seen.</p>
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[[Image:PCR amplification of GB1.png|400px|thumb|center]]
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<p>Figure 1. PCR amplification of pSB1A3 plasmid containing GB1 (BBa_K4437001), using T7 promoter and terminator-specific primers, indicating successful cloning. Lane 2 contains our expected band size at 331 bp</p>
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===Sequence and Features===
 
<partinfo>BBa_K4437001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4437001 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K4437001 parameters</partinfo>
 
<partinfo>BBa_K4437001 parameters</partinfo>
 
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===References===
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<ol>
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<li>Zhou H, Fang J, Tian Y, Lu XY. Mechanisms of nisin resistance in Gram-positive bacteria. Annals of microbiology. 2014 Jun;64(2):413-20.</li>
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<li>Mai HT, Van Hau N, Nghia NH, Thao DT. Expression and Purification of Nisin in Escherichia coli. Int. J. Life. Sci. Scienti. Res. eISSN. 2018 Jul;2455(1716):1716.</li>
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</ol>

Latest revision as of 03:19, 14 October 2022


NisQ with a N-terminus 6x His-tag (NisQ-His)

Usage and Biology

Derived from Lactococcus lactis, nisin is a food-safe, antimicrobial peptide (AMP) that targets a wide range of Gram-positive bacteria by binding to lipid II on the pathogens membrane, creating a pore, and causing cell death [1]. Literature suggests that nisin Q (NisQ) demonstrates greater antimicrobial and antioxidant activity against pathogens compared to other variants of nisin, such as NisA (BBa_K1365000) [2]. Nisin’s optimal pH stability is between 2 and 7 but can maintain its antibacterial activity up to a pH of 12, and can also retain its antimicrobial activity at temperatures of 120oC [2]. Unlike other AMPs, nisin is non-toxic to Gram-negative bacteria, meaning that successful recombinant expression in E. coli can be achieved without an inhibitory protein.

Design

Nisin GB1 (BBa_K4437001) includes, a T7 promoter, a lac operon, RBS, TEV protease site, 6XHis-tag, two enterokinase cut sites, nisQ, and T7 terminator. The two enterokinase sites serves to make sure the 6XHis-tag is cleaved off and surely leaves us with nisQ. The lac operon functions to be able to use different protein induction media such as autoinduction medias when expression protein. The presence of the lac operon will allow the bacteria to switch from a glucose sugar source to a lactose sugar source. The rest if the elements are regulatory elements necessary to express our protein.

Gb1.png

Characterization

We were able to insert our Nisin GB1 (BBa_K4437001) into a pSB1A3 plasmid. Once it was PCR amplified, it was run on a gel in which its indicated band size 331 was seen.

PCR amplification of GB1.png

Figure 1. PCR amplification of pSB1A3 plasmid containing GB1 (BBa_K4437001), using T7 promoter and terminator-specific primers, indicating successful cloning. Lane 2 contains our expected band size at 331 bp

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 220
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. Zhou H, Fang J, Tian Y, Lu XY. Mechanisms of nisin resistance in Gram-positive bacteria. Annals of microbiology. 2014 Jun;64(2):413-20.
  2. Mai HT, Van Hau N, Nghia NH, Thao DT. Expression and Purification of Nisin in Escherichia coli. Int. J. Life. Sci. Scienti. Res. eISSN. 2018 Jul;2455(1716):1716.