Difference between revisions of "Part:BBa K4907027"

 
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<partinfo>BBa_K4907027 short</partinfo>
 
<partinfo>BBa_K4907027 short</partinfo>
  
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===Biology===
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<i>cbm</i> encodes the cellulose-binding domain (CBD) of an exoglucanase from <i>Cellulomonas fimi</i>., which can bind to cellulose irreversibly. At the same time, it performs excellent at low temperatures and in a wide pH range (1), which is suitable for our project.
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===Usage and design ===
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To avoid irrigation water or rainwater washing away our engineered bacteria, we plan to use a <b>Cellulose-binding module (CBM)</b>  to make them adhere to the surface of crop roots. It was subcloned and modified with his-tag for protein purification and further biochemical characterization.
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===Characterization===
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====Agarose gel electrophoresis (AGE)====
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This part was constructed into a pET-28a(+) vector using seamless cloning and the correct plasmid was transformed into <i>E. coli</i> BL21(DE3). Colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (506 bp) can be observed at the position around 500 bp (Fig. 1).
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/ctt/t7promotor-cbm-histag-t7t-pet28a-ctt111.png"width="400px"></html></center>
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<center><b>Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4907027_pET-28a(+).</b></center>
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====SDS-PAGE====
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The plasmid verified by sequencing was successfully transformed into <i>E. coli </i>BL21(DE3). After being cultivated and induced by 0.75 mM IPTG, the GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. CBM  was verified by sodium dodecyl sulfate (SDS) - 12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image (Fig. 2), the bands of the target protein (11.9 kDa) could be observed at the position around 10 kDa on the purified protein lanes (FR).
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/ctt/cbm-1-2.png"width="400px"></html></center>
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<center><b>Fig. 2  SDS-PAGE analysis of CBM protein.  </b></center>
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====The binding ability to cellulose====
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We conducted an experiment to compare the adsorption capacity of CBM and BSA on cellulose. In this experiment, we filtered 4 mL of 10 &micro;M CBM and BSA three times using cellulose filter paper and washed it three times with 1xPBS. We diluted the final filtrate to the same volume, measured the amount of protein in the filtrate using the Bradford method, and repeated the experiment three times. As shown in Fig. 3, the difference between CBM and BSA can be observed significantly through OD<sub>595</sub>.
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<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/ctt/tt-cbmbsa.png" width="400px"></html></center>
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<center><b>Fig. 3 Comparison of the CBM and BSA in terms of cellulose-binding ability. (<i>p</i>=0.0003)</b></center>
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===Reference===
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1.E. Ong, N. R. Gilkes, R. C. Miller Jr, R. A. J. Warren, D. G. Kilburn, The cellulose‐binding domain (CBDCex) of an exoglucanase from <i>Cellulomonas fimi</i>: Production in <i>Escherichia coli</i> and characterization of the polypeptide. <i>Biotechnol. Bioeng</i>. <b>42</b>, 401-409 (1993).
  
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
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===<span class='h3bb'>Sequence and Features</span>===
 
<partinfo>BBa_K4907027 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4907027 SequenceAndFeatures</partinfo>
  

Latest revision as of 12:00, 12 October 2023


cbm-his tag

Biology

cbm encodes the cellulose-binding domain (CBD) of an exoglucanase from Cellulomonas fimi., which can bind to cellulose irreversibly. At the same time, it performs excellent at low temperatures and in a wide pH range (1), which is suitable for our project.

Usage and design

To avoid irrigation water or rainwater washing away our engineered bacteria, we plan to use a Cellulose-binding module (CBM) to make them adhere to the surface of crop roots. It was subcloned and modified with his-tag for protein purification and further biochemical characterization.

Characterization

Agarose gel electrophoresis (AGE)

This part was constructed into a pET-28a(+) vector using seamless cloning and the correct plasmid was transformed into E. coli BL21(DE3). Colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (506 bp) can be observed at the position around 500 bp (Fig. 1).

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4907027_pET-28a(+).

SDS-PAGE

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by 0.75 mM IPTG, the GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. CBM was verified by sodium dodecyl sulfate (SDS) - 12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image (Fig. 2), the bands of the target protein (11.9 kDa) could be observed at the position around 10 kDa on the purified protein lanes (FR).

Fig. 2 SDS-PAGE analysis of CBM protein.

The binding ability to cellulose

We conducted an experiment to compare the adsorption capacity of CBM and BSA on cellulose. In this experiment, we filtered 4 mL of 10 µM CBM and BSA three times using cellulose filter paper and washed it three times with 1xPBS. We diluted the final filtrate to the same volume, measured the amount of protein in the filtrate using the Bradford method, and repeated the experiment three times. As shown in Fig. 3, the difference between CBM and BSA can be observed significantly through OD595.

Fig. 3 Comparison of the CBM and BSA in terms of cellulose-binding ability. (p=0.0003)

Reference

1.E. Ong, N. R. Gilkes, R. C. Miller Jr, R. A. J. Warren, D. G. Kilburn, The cellulose‐binding domain (CBDCex) of an exoglucanase from Cellulomonas fimi: Production in Escherichia coli and characterization of the polypeptide. Biotechnol. Bioeng. 42, 401-409 (1993).


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 268
  • 1000
    COMPATIBLE WITH RFC[1000]