Difference between revisions of "Part:BBa K4437004"

 
 
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This part contains the coding sequence for the antimicrobial region of NisQ, a variant of nisin.  
 
This part contains the coding sequence for the antimicrobial region of NisQ, a variant of nisin.  
  
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__TOC__
 
===Usage and Biology===
 
===Usage and Biology===
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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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[[Image:nisQ.png|400px|thumb|center]]
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4437004 SequenceAndFeatures</partinfo>
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===Sequence and Features===
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This sequence contains only the antimicrobial region of the peptide nisin Q. 
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===Characterization===
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We used the NisQ sequence within 3 composite parts (BBa_K4437001 aka "GB1", BBa_K4437002 aka "GB2", BBa_K4437003) in order to express NisQ protein.
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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 NisQ, 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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[[Image:DiagnosticGel.png|400px|thumb|center]]
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<p>Figure 2. Diagnostic gel for the Xpress expression vector(BBa_K3945014) containing NisQ (within a composite part, BBa_K4437003) miniprepped-samples (samples digested at 37<sup>o</sup>C for 60 minutes with no heat inactivation because of BamHI, therefore we expected the samples to not be fully digested). Lanes 4, 5, 6, and 7 show our expected band sizes. Lane 8 contains a negative control.</p>
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[[Image:SDS1.png|400px|thumb|center]]
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<p>Figure 3. SDS-PAGE analysis of GST-NusA-NisQ (BBa_K4437003) samples from BL21 (DE3) <i>E. coli</i> strain autoinduced, using a Coomassie-blue stain. Large bands in lanes 2, 4, 5, 6, and 9 at 91kDa correspond to our expected protein size.</p>
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[[Image:SDS2.png|400px|thumb|center]]
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<p>Figure 4. His-tag purified SDS-PAGE of GST-NusA-NisQ (BBa_K4437003) samples samples from BL21 (DE3) <i>E. coli</i> strain autoinduced, using a Coomassie-blue stain. Bands in lanes 3, 4, and 5 at 91kDa correspond to our expected protein size. Samples labelled "W-1" indicate wash 1, samples labelled "E-1" indicate elution 1.</p>
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[[Image:westernnisin.png|400px|thumb|center]]
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<p>Figure 5. Western blot of the whole cell lysate of GST+NusA+NisQ auto-induced in overnight express. A his-tagged positive control was also included. The protein ladder used was the Novex sharp pre-stained protein standard. The antibodies used were Mouse Anti-HIS-tag mAb (Abcam) for the primary antibody and Goat Anti-Mouse:HRP (Abcam) for the secondary antibody.</p>
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<partinfo>BBa_K4437004 SequenceAndFeatures</partinfo>
  
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===References===
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===Functional Parameters===
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<ol>
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<partinfo>BBa_K4437004 parameters</partinfo>
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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 03:27, 14 October 2022


Antimicrobial region of nisin Q

This part contains the coding sequence for the antimicrobial region of NisQ, a variant of nisin.

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.

NisQ.png

Sequence and Features

This sequence contains only the antimicrobial region of the peptide nisin Q.

Characterization

We used the NisQ sequence within 3 composite parts (BBa_K4437001 aka "GB1", BBa_K4437002 aka "GB2", BBa_K4437003) in order to express NisQ protein.

PCR amplification of GB1.png

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

DiagnosticGel.png

Figure 2. Diagnostic gel for the Xpress expression vector(BBa_K3945014) containing NisQ (within a composite part, BBa_K4437003) miniprepped-samples (samples digested at 37oC for 60 minutes with no heat inactivation because of BamHI, therefore we expected the samples to not be fully digested). Lanes 4, 5, 6, and 7 show our expected band sizes. Lane 8 contains a negative control.

SDS1.png

Figure 3. SDS-PAGE analysis of GST-NusA-NisQ (BBa_K4437003) samples from BL21 (DE3) E. coli strain autoinduced, using a Coomassie-blue stain. Large bands in lanes 2, 4, 5, 6, and 9 at 91kDa correspond to our expected protein size.

SDS2.png

Figure 4. His-tag purified SDS-PAGE of GST-NusA-NisQ (BBa_K4437003) samples samples from BL21 (DE3) E. coli strain autoinduced, using a Coomassie-blue stain. Bands in lanes 3, 4, and 5 at 91kDa correspond to our expected protein size. Samples labelled "W-1" indicate wash 1, samples labelled "E-1" indicate elution 1.

Westernnisin.png

Figure 5. Western blot of the whole cell lysate of GST+NusA+NisQ auto-induced in overnight express. A his-tagged positive control was also included. The protein ladder used was the Novex sharp pre-stained protein standard. The antibodies used were Mouse Anti-HIS-tag mAb (Abcam) for the primary antibody and Goat Anti-Mouse:HRP (Abcam) for the secondary antibody.


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 37
  • 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.