Difference between revisions of "Part:BBa K3802000"

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(→‎Sequence and Features)
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===Sequence and Features===
 
===Sequence and Features===
  
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This fusion protein aims to anchor MlrA, or microcystinase, an enzyme capable of degrading freshwater toxin microcystin-LR (MC-LR) to the outer membrane of E. coli. This part  puts together a codon-optimized  subunit of  PgsA (poly-γ-glutamic acid synthetase) [https://parts.igem.org/Part:BBa_K2963020 (BBa_K2963020)], an anchoring motif to MlrA with 6x His [https://parts.igem.org/Part:BBa_K1907002 (BBa_K1907002)], with a 2 amino acid Gly-Ser linker [https://parts.igem.org/Part:BBa_J18920 (BBa_J18920)].  The addition of the His tag to MlrA gives this part the ability to be purified using Nickel affinity chromatography and detected using Western blotting with anti-His antibodies.  
+
This fusion protein aims to anchor MlrA, or microcystinase, an enzyme capable of degrading freshwater toxin microcystin-LR (MC-LR) to the outer membrane of <i> E. coli </i>. This part  puts together a codon-optimized  subunit of  PgsA (poly-γ-glutamic acid synthetase) [https://parts.igem.org/Part:BBa_K2963020 (BBa_K2963020)], an anchoring motif to MlrA with 6x His [https://parts.igem.org/Part:BBa_K1907002 (BBa_K1907002)], with a 2 amino acid Gly-Ser linker [https://parts.igem.org/Part:BBa_J18920 (BBa_J18920)].  The addition of the His tag to MlrA gives this part the ability to be purified using Nickel affinity chromatography and detected using Western blotting with anti-His antibodies.  
 
This BioBrick was put under the control of an IPTG inducible ptac promoter [https://parts.igem.org/Part:BBa_K3802001 (BBa_K3802001)], as using a constitutive promoter could potentially lead to the depletion of resources for the bacteria.
 
This BioBrick was put under the control of an IPTG inducible ptac promoter [https://parts.igem.org/Part:BBa_K3802001 (BBa_K3802001)], as using a constitutive promoter could potentially lead to the depletion of resources for the bacteria.
  
 
<partinfo>K3802000 SequenceAndFeatures</partinfo>
 
<partinfo>K3802000 SequenceAndFeatures</partinfo>
 +
 +
===Usage and Biology===
 +
 +
Our project aims to develop a novel MC-LR degradation solution by utilizing recombinant E. coli that heterologously over-express MlrA, an enzyme known to degrade MC-LR in the native <i> Sphingomonas sp </i>[1]. This composite part aimed to improve upon previous systems of MC-LR detection by creating a method where this toxin could be degraded in the outer membrane of <i> E.coli </i>. The advantage of this system is that MC-LR does not have to be transported into the cytosol of <i>E. coli</i> for degradation, but rather can be directly broken down if MC-LR is within proximity of <i>E. coli</i>.
 +
 +
This fusion protein links MlrA with  the gene for PgsA (poly-γ-glutamate synthetase A), a protein natively found in Bacillus subtilis [2] Though past iGEM teams such as JNU- China from 2019 have genetically engineered  <i> Corynebacterium glutamicium </i> to produce PgsA for various applications such as food additives, anti-freeze, protective antigen coupling, PgsA can also act as an anchoring motif. This protein has been used to successfully and heterologously express enzymes and proteins such as α-amylase, lipase B, Laccase COTA, and VP2--an antigen for IBDV virus in chickens [5][3][4].
 +
 +
===References===
 +
[1] Dziga, D., Wladyka, B., Zielińska, G., Meriluoto, J., & Wasylewski, M. (2012).
 +
Heterologous expression and characterisation Of microcystinase. Toxicon, 59(5), 578–586. https://doi.org/10.1016/j.toxicon.2012.01.001
 +
 +
[2] Narita, J., Okano, K., Tateno, T., Tanino, T., Sewaki, T., Sung, M.-H., Fukuda, H., &
 +
Kondo, A. (2005). Display of active enzymes on the cell surface of Escherichia COLI using PgsA ANCHOR protein and their application to bioconversion. Applied Microbiology and Biotechnology, 70(5), 564–572. https://doi.org/10.1007/s00253-005-0111-x
 +
 +
[3] Zhang, Y., Dong, W., Lv, Z., Liu, J., Zhang, W., Zhou, J., Xin, F., Ma, J., & Jiang, M.
 +
(2018). Surface display of BACTERIAL Laccase COTA on Escherichia COLI cells and its application in Industrial Dye Decolorization. Molecular Biotechnology, 60(9), 681–689. https://doi.org/10.1007/s12033-018-0103-6
 +
 +
[4] Maqsood, I., Shi, W., Wang, L., Wang, X., Han, B., Zhao, H., Nadeem, A. M., Moshin, B.
 +
S., Saima, K., Jamal, S. S., Din, M. F., Xu, Y., Tang, L., & Li, Y. (2018). Immunogenicity and Protective efficacy of orally administered RECOMBINANTLACTOBACILLUS Plantarumexpressing VP2 protein against IBDV in chicken. Journal of Applied Microbiology, 125(6), 1670–1681. https://doi.org/10.1111/jam.14073
 +
 +
[5] MASEDA, HIDEAKI., SHIMIZU, KAZUYA, DOI, YOSHIAKI,
 +
INAMORI, YUHEI, UTSUMI, MOTOO, SUGIURA, NORIO, & KOBAYASHI, MICHIHIKO (2012). MlrA located in the inner membrane is essential for Initial degradation Of microcystin In sphingopyxis sp. C－1. Japanese Journal of Water Treatment Biology, 48(3), 99–107. https://doi.org/10.2521/jswtb.48.99

Revision as of 16:56, 21 October 2021

PgsA-MlrA fusion protein

Sequence and Features

This fusion protein aims to anchor MlrA, or microcystinase, an enzyme capable of degrading freshwater toxin microcystin-LR (MC-LR) to the outer membrane of E. coli . This part puts together a codon-optimized subunit of PgsA (poly-γ-glutamic acid synthetase) (BBa_K2963020), an anchoring motif to MlrA with 6x His (BBa_K1907002), with a 2 amino acid Gly-Ser linker (BBa_J18920). The addition of the His tag to MlrA gives this part the ability to be purified using Nickel affinity chromatography and detected using Western blotting with anti-His antibodies. This BioBrick was put under the control of an IPTG inducible ptac promoter (BBa_K3802001), as using a constitutive promoter could potentially lead to the depletion of resources for the bacteria.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1792
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 827
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 813
    Illegal NgoMIV site found at 2323
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 304
    Illegal BsaI.rc site found at 2160

Usage and Biology

Our project aims to develop a novel MC-LR degradation solution by utilizing recombinant E. coli that heterologously over-express MlrA, an enzyme known to degrade MC-LR in the native Sphingomonas sp [1]. This composite part aimed to improve upon previous systems of MC-LR detection by creating a method where this toxin could be degraded in the outer membrane of E.coli . The advantage of this system is that MC-LR does not have to be transported into the cytosol of E. coli for degradation, but rather can be directly broken down if MC-LR is within proximity of E. coli.

This fusion protein links MlrA with the gene for PgsA (poly-γ-glutamate synthetase A), a protein natively found in Bacillus subtilis [2] Though past iGEM teams such as JNU- China from 2019 have genetically engineered Corynebacterium glutamicium to produce PgsA for various applications such as food additives, anti-freeze, protective antigen coupling, PgsA can also act as an anchoring motif. This protein has been used to successfully and heterologously express enzymes and proteins such as α-amylase, lipase B, Laccase COTA, and VP2--an antigen for IBDV virus in chickens [5][3][4].

References

[1] Dziga, D., Wladyka, B., Zielińska, G., Meriluoto, J., & Wasylewski, M. (2012). Heterologous expression and characterisation Of microcystinase. Toxicon, 59(5), 578–586. https://doi.org/10.1016/j.toxicon.2012.01.001

[2] Narita, J., Okano, K., Tateno, T., Tanino, T., Sewaki, T., Sung, M.-H., Fukuda, H., & Kondo, A. (2005). Display of active enzymes on the cell surface of Escherichia COLI using PgsA ANCHOR protein and their application to bioconversion. Applied Microbiology and Biotechnology, 70(5), 564–572. https://doi.org/10.1007/s00253-005-0111-x

[3] Zhang, Y., Dong, W., Lv, Z., Liu, J., Zhang, W., Zhou, J., Xin, F., Ma, J., & Jiang, M. (2018). Surface display of BACTERIAL Laccase COTA on Escherichia COLI cells and its application in Industrial Dye Decolorization. Molecular Biotechnology, 60(9), 681–689. https://doi.org/10.1007/s12033-018-0103-6

[4] Maqsood, I., Shi, W., Wang, L., Wang, X., Han, B., Zhao, H., Nadeem, A. M., Moshin, B. S., Saima, K., Jamal, S. S., Din, M. F., Xu, Y., Tang, L., & Li, Y. (2018). Immunogenicity and Protective efficacy of orally administered RECOMBINANTLACTOBACILLUS Plantarumexpressing VP2 protein against IBDV in chicken. Journal of Applied Microbiology, 125(6), 1670–1681. https://doi.org/10.1111/jam.14073

[5] MASEDA, HIDEAKI., SHIMIZU, KAZUYA, DOI, YOSHIAKI, INAMORI, YUHEI, UTSUMI, MOTOO, SUGIURA, NORIO, & KOBAYASHI, MICHIHIKO (2012). MlrA located in the inner membrane is essential for Initial degradation Of microcystin In sphingopyxis sp. C－1. Japanese Journal of Water Treatment Biology, 48(3), 99–107. https://doi.org/10.2521/jswtb.48.99