Difference between revisions of "Part:BBa K4437003"
 
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<partinfo>BBa_K4437004 short</partinfo>
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<partinfo>BBa_K4437003 short</partinfo>
  
 
This part contains the coding sequence for the antimicrobial region of NisQ tagged with a 6X-Histag, GST tag, and NusA tag.  
 
This part contains the coding sequence for the antimicrobial region of NisQ tagged with a 6X-Histag, GST tag, and NusA tag.  
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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>
 
<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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===Sequence and Features===
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===Sequence and Features===
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A novel fusion protein with 6XHis-tagged Glutathione S-transferases (GST), NusA, and a 6XHis-tag fused to the N-terminus of NisQ. GST was incorporated into BBa_K4437002 due to its presence within the Xpress expression vector (BBa_K3945014), which we used to successfully produce NisQ protein. GST is a solubility tag which is useful for expressing difficult proteins. NusA is a solubility factor to help increase production and solubility of a desired peptide in E. coli. Double enterokinase cut sites are included for removal of the 6XHis-tag to isolate NisQ alone. A TEV protease site is included for removal of NusA, revealing the 6XHis-tag for subsequent NisQ purification. Using GST and NusA increases the expected band size from 7kDa (NisQ alone) to 91kDa, which is easier to visualize on SDS-PAGE gels.
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<p>A novel fusion protein with 6XHis-tagged Glutathione S-transferases (GST), NusA, and a 6XHis-tag fused to the N-terminus of NisQ. GST was incorporated into BBa_K4437002 due to its presence within the Xpress expression vector (BBa_K3945014), which we used to successfully produce NisQ protein. GST is a solubility tag which is useful for expressing difficult proteins. NusA is a solubility factor to help increase production and solubility of a desired peptide in E. coli. Double enterokinase cut sites are included for removal of the 6XHis-tag to isolate NisQ alone. A TEV protease site is included for removal of NusA, revealing the 6XHis-tag for subsequent NisQ purification. Using GST and NusA increases the expected band size from 7kDa (NisQ alone) to 91kDa, which is easier to visualize on SDS-PAGE gels.</p> 
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[[Image:gb3.png|400px|thumb|center]]
  
 
===Characterization===
 
===Characterization===
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[[Image:SDS1.png|400px|thumb|center]]  
 
[[Image:SDS1.png|400px|thumb|center]]  
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<p>Figure 2. SDS-PAGE analysis of GST-NusA-NisQ (BBa_K4437003) samples from BL21 (DE3) E.coli strain autoinduced, using Anti-Histag antibodies and the complementary capture antibody. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. Large bands in lanes 2, 4, 5, 6, and 9 at 91kDa correspond to our expected protein size.</p>
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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>
  
 
[[Image:SDS2.png|400px|thumb|center]]  
 
[[Image:SDS2.png|400px|thumb|center]]  
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Figure 4. Purified SDS-PAGE of GST-NusA-NisQ (BBa_K4437003) samples samples from BL21 (DE3) E.coli strain autoinduced, using Anti-Histag antibodies and the complementary capture antibody. The process was visualised using 10% SDS-PAGE in 100V for 20 minutes and 180V for 40 minutes. 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.
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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_K4437003 SequenceAndFeatures</partinfo>
  
 
===References===
 
===References===

Latest revision as of 03:31, 14 October 2022

NisQ tagged with GST, NusA, and a 6XHis-tag

This part contains the coding sequence for the antimicrobial region of NisQ tagged with a 6X-Histag, GST tag, and NusA tag.

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.

Sequence and Features

A novel fusion protein with 6XHis-tagged Glutathione S-transferases (GST), NusA, and a 6XHis-tag fused to the N-terminus of NisQ. GST was incorporated into BBa_K4437002 due to its presence within the Xpress expression vector (BBa_K3945014), which we used to successfully produce NisQ protein. GST is a solubility tag which is useful for expressing difficult proteins. NusA is a solubility factor to help increase production and solubility of a desired peptide in E. coli. Double enterokinase cut sites are included for removal of the 6XHis-tag to isolate NisQ alone. A TEV protease site is included for removal of NusA, revealing the 6XHis-tag for subsequent NisQ purification. Using GST and NusA increases the expected band size from 7kDa (NisQ alone) to 91kDa, which is easier to visualize on SDS-PAGE gels.

Gb3.png

Characterization

We used this part to successfully clone and express NisQ protein.

DiagnosticGel.png

Figure 1. 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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2085
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2369
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 136

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.