Difference between revisions of "Part:BBa K5530001"

 
 
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ChBD3&#65288;CBM2&#65289;
 
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===Usage and Biology===
 
  
 
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<partinfo>BBa_K5530001 SequenceAndFeatures</partinfo>
 
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    <title>ChBD3(CBM2) - Profile</title>
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===Functional Parameters===
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    <h2>Profile</h2>
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<partinfo>BBa_K5530001 parameters</partinfo>
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    <p><strong>Name:</strong> ChBD3(CBM2)</p>
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    <p><strong>Gene length:</strong> 341 bp</p>
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    <p><strong>Source:</strong> <i>Thermococcus kodakarensis</i> KOD1 (5DHE)</p>
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    <p><strong>Properties:</strong></p>
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    <p>ChBD3(CBM2) consists of two catalytic domains and three binding domains (ChBD1, ChBD2, and ChBD3). ChBD2 and ChBD3 can bind to both chitin and cellulose. The spacing of the side chains of the three tryptophan residues on the molecular surface is equivalent to twice the length of the chitin lattice [1]. CBM2-encoded protein has a special affinity for chitin, which is another main component of the fungal cell wall, allowing its binding to fungal cells with the presence of chitin.</p>
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    <h2>Usage and Biology</h2>
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    <p>There are few studies on ChBD3, with most research focusing on its binding domain characteristics. Some studies examine the effect of ChBD3 and cellulose on each other's binding efficiency [2]. We expanded the application of ChBD3 by utilizing its specific binding characteristics with chitin for fungal detection.</p>
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    <h2>Cultivation, Purification, and SDS-PAGE</h2>
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    <p>Both gene sequences were amplified by PCR, confirmed by bands of approximately 300 bp and 500 bp on the electrophoretic gel. The gene length of ChBD3(CBM2) was 341 bp, indicating successful amplification of the target band. Figure 1 shows that the plasmid was successfully linearized.</p>
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        <img src="https://static.igem.wiki/teams/5530/bba-k5530001/1.png" width="40%" alt="Figure 1: The electrophoretic gel map">
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            <caption>Figure 1: The electrophoretic gel map</caption>
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    <p>Figure 2 represents the purified protein. The ChBD3(CBM2) is 35.9 kDa. In Figure 3, the bands corresponding to CBM2-mcherry proteins, ranging from 34 kDa to 43 kDa, are notably intense, confirming successful expression in the supernatant.</p>
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        <img src="https://static.igem.wiki/teams/5530/bba-k5530001/2.png" width="40%" alt="Figure 2: The SDS-PAGE of the CBM2-mcherry">
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            <caption>Figure 2: The SDS-PAGE of the CBM2-mcherry.CBM2-mcherry:35.9kDa</caption>
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    <h3>References</h3>
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    <p>[1] Hanazono Y, Takeda K, Niwa S, Hibi M, Takahashi N, Kanai T, Atomi H, Miki K. Crystal structures of chitin binding domains of chitinase from <i>Thermococcus kodakarensis</i> KOD1. FEBS Lett. 2016 Jan;590(2):298-304. doi: 10.1002/1873-3468.12055. PMID: 26823175.</p>
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    <p>[2] Kikkawa Y, Fukuda M, Kashiwada A, et al. Binding ability of chitinase onto cellulose: an atomic force microscopy study. Polymer Journal, 2011, 43(8):742-744.</p>
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Latest revision as of 07:11, 29 September 2024


ChBD3（CBM2）

ChBD3（CBM2）


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 334
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

ChBD3(CBM2) - Profile

Profile

Name: ChBD3(CBM2)

Gene length: 341 bp

Source: Thermococcus kodakarensis KOD1 (5DHE)

Properties:

ChBD3(CBM2) consists of two catalytic domains and three binding domains (ChBD1, ChBD2, and ChBD3). ChBD2 and ChBD3 can bind to both chitin and cellulose. The spacing of the side chains of the three tryptophan residues on the molecular surface is equivalent to twice the length of the chitin lattice [1]. CBM2-encoded protein has a special affinity for chitin, which is another main component of the fungal cell wall, allowing its binding to fungal cells with the presence of chitin.

Usage and Biology

There are few studies on ChBD3, with most research focusing on its binding domain characteristics. Some studies examine the effect of ChBD3 and cellulose on each other's binding efficiency [2]. We expanded the application of ChBD3 by utilizing its specific binding characteristics with chitin for fungal detection.

Cultivation, Purification, and SDS-PAGE

Both gene sequences were amplified by PCR, confirmed by bands of approximately 300 bp and 500 bp on the electrophoretic gel. The gene length of ChBD3(CBM2) was 341 bp, indicating successful amplification of the target band. Figure 1 shows that the plasmid was successfully linearized.

Figure 1: The electrophoretic gel map
Figure 1: The electrophoretic gel map

Figure 2 represents the purified protein. The ChBD3(CBM2) is 35.9 kDa. In Figure 3, the bands corresponding to CBM2-mcherry proteins, ranging from 34 kDa to 43 kDa, are notably intense, confirming successful expression in the supernatant.

Figure 2: The SDS-PAGE of the CBM2-mcherry
Figure 2: The SDS-PAGE of the CBM2-mcherry.CBM2-mcherry:35.9kDa

References

[1] Hanazono Y, Takeda K, Niwa S, Hibi M, Takahashi N, Kanai T, Atomi H, Miki K. Crystal structures of chitin binding domains of chitinase from Thermococcus kodakarensis KOD1. FEBS Lett. 2016 Jan;590(2):298-304. doi: 10.1002/1873-3468.12055. PMID: 26823175.

[2] Kikkawa Y, Fukuda M, Kashiwada A, et al. Binding ability of chitinase onto cellulose: an atomic force microscopy study. Polymer Journal, 2011, 43(8):742-744.