Difference between revisions of "Part:BBa K1763000"

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(→‎IMPROVEMENT REFERENCE: GDSYZX 2020)
 
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<partinfo>BBa_K1763000 short</partinfo>
 
<partinfo>BBa_K1763000 short</partinfo>
  
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This is the coding region for the honeybee silk protein #3. Previous literature has shown that it is possible to generate honeybee fibers by expressing this protein recombinantly in ''e. coli ''and spinning the protein into fibers ''in vitro'' It does not contain any of the regulatory elements necessary for protein expression.
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This is the coding region for the honeybee silk protein #3. Previous literature has shown that it is possible to generate honeybee fibers by expressing this protein recombinantly in ''e. coli ''and spinning the protein into fibers ''in vitro'' (Sutherland) This part does not contain any of the regulatory elements necessary for protein expression.
  
  
 
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===Usage and Biology===
 
===Usage and Biology===
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Because this is just the coding region for the honeybee silk sequence, it is not meant to be expressed on its own. For an expressible honey bee silk biobrick part, please see [https://parts.igem.org/Part:BBa_K1763007 BBa_K1763007 ].
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'''Because this is just the coding region for the honeybee silk sequence, it is not meant to be expressed on its own. For an expressible honey bee silk biobrick part, please see [https://parts.igem.org/Part:BBa_K1763007 BBa_K1763007 ].''' Silk from ''Apis Mellifera'' represents an intriguing alternative to silks from spiders or silkworms. Although it is not quite as strong as these other types of silks, working with honey bee silk has certain advantages over spider and silkworm silk (Weisman). The size of the honey bee silk protein gene is considerably smaller than the silk genes of spiders or silkworms. More importantly, the gene sequence is non repetitive, which allows us to synthesize and make modifications to the gene without the complications that are inherent to repetitive DNA sequences. Honey bee silk also has a very different secondary and tertiary structure than spider and silkworm silks. It forms primary alpha helices, and four silk proteins come together to form a coiled coil structure these coiled coils are formed from four similar, yet unique proteins, Amelf 1-4 (Sutherland 2007). However, a study has shown that using one of these proteins, (Amelf3) is sufficient to reproduce the physical properties of the wild type fibers (Sutherland).
  
 
===Characterization===
 
===Characterization===
 
#Protein Expression
 
#Protein Expression
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*Here is the SDS gel of our first attempt at expression of the honeybee silk protein. The purified protein should be in the lane labeled "inclusion body." It was purified using [http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf this inclusion body protocol]
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*Here is the SDS gel of our expression of the honeybee silk protein. It was purified using [http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf this inclusion body protocol]
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*[http://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/28_July_2015 Here] is the link to the protocol that we used for expressing the protein starting from a colony. (We were using Kanamycin as our antibiotic, but chloramphenicol should be used if using this biobrick.)
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[[File:UCLA Honeybee silk SDS PAGE9.18.jpg|none|thumb|650px|'''Fig. 1''' Expected size of product is 40.0 kDA]]
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*There are contaminating bands present on the SDS PAGE gel, indicating that the purification is not completely effective.
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*We also ran BCA protein concentration assays on our purified honeybee proteins. Based on this assay we were able to get around 12 mg of protein from a 300 ml cell culture.
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**However, this 12 mg might not be composed entirely of honeybee protein because based on the SDS PAGE results, the purification was not 100% complete.
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===References===
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*Weisman, S., Haritos, V., Church, J., Huson, M., Mudie, S., Rodgers, A., Dumsday, G., Sutherland, T. Honeybee silk: Recombinant protin production, assembly, and fiber spinning. Elsevier Ltd. 2009.
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*Sutherland, T., Church, J., Hu, X., Huson, M., Kaplan, D., Weisman, S. Single Honeybee Silk Protein Mimics Properties fo Multi-Protein Silk. PLoS ONE 2011. e16489
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*Sutherland, T. Conservation of Essential Design Features in Coiled Coil Silks. Mol Biol Evol 2007;24:2424-2432
  
 
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<partinfo>BBa_K1763000 parameters</partinfo>
 
<partinfo>BBa_K1763000 parameters</partinfo>
 
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==IMPROVEMENT REFERENCE: BNU-China 2017 ==
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The part was linked with a blue chromoprotein amilCP to make it visualized. The new part is : <html><a href='https://parts.igem.org/Part:BBa_K2220028'>BBa_K2220028</a></html>(Group: iGEM17_BNU-China), Designed by: Jiawei Xing.
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==IMPROVEMENT REFERENCE: GDSYZX 2020 ==
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According to the codon preference of Arabidopsis thaliana, our team optimized the codon of this part to make it more suitable for expression in plants, providing a basis for subsequent research on this part in plants. The new part is : <html><a href='https://parts.igem.org/Part:BBa_K3458005'>BBa_K3458005</a></html> (Group: iGEM20_GDSYZX), Designed by: Jiahua Zou.

Latest revision as of 09:12, 25 October 2020

Apis mellifera (honeybee) silk fibroin 3

This is the coding region for the honeybee silk protein #3. Previous literature has shown that it is possible to generate honeybee fibers by expressing this protein recombinantly in e. coli and spinning the protein into fibers in vitro (Sutherland) This part does not contain any of the regulatory elements necessary for protein expression.


Usage and Biology

Because this is just the coding region for the honeybee silk sequence, it is not meant to be expressed on its own. For an expressible honey bee silk biobrick part, please see BBa_K1763007 . Silk from Apis Mellifera represents an intriguing alternative to silks from spiders or silkworms. Although it is not quite as strong as these other types of silks, working with honey bee silk has certain advantages over spider and silkworm silk (Weisman). The size of the honey bee silk protein gene is considerably smaller than the silk genes of spiders or silkworms. More importantly, the gene sequence is non repetitive, which allows us to synthesize and make modifications to the gene without the complications that are inherent to repetitive DNA sequences. Honey bee silk also has a very different secondary and tertiary structure than spider and silkworm silks. It forms primary alpha helices, and four silk proteins come together to form a coiled coil structure these coiled coils are formed from four similar, yet unique proteins, Amelf 1-4 (Sutherland 2007). However, a study has shown that using one of these proteins, (Amelf3) is sufficient to reproduce the physical properties of the wild type fibers (Sutherland).

Characterization

  1. Protein Expression
  • Here is the SDS gel of our expression of the honeybee silk protein. It was purified using [http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf this inclusion body protocol]
  • [http://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/28_July_2015 Here] is the link to the protocol that we used for expressing the protein starting from a colony. (We were using Kanamycin as our antibiotic, but chloramphenicol should be used if using this biobrick.)
Fig. 1 Expected size of product is 40.0 kDA
  • There are contaminating bands present on the SDS PAGE gel, indicating that the purification is not completely effective.
  • We also ran BCA protein concentration assays on our purified honeybee proteins. Based on this assay we were able to get around 12 mg of protein from a 300 ml cell culture.
    • However, this 12 mg might not be composed entirely of honeybee protein because based on the SDS PAGE results, the purification was not 100% complete.

References

  • Weisman, S., Haritos, V., Church, J., Huson, M., Mudie, S., Rodgers, A., Dumsday, G., Sutherland, T. Honeybee silk: Recombinant protin production, assembly, and fiber spinning. Elsevier Ltd. 2009.
  • Sutherland, T., Church, J., Hu, X., Huson, M., Kaplan, D., Weisman, S. Single Honeybee Silk Protein Mimics Properties fo Multi-Protein Silk. PLoS ONE 2011. e16489
  • Sutherland, T. Conservation of Essential Design Features in Coiled Coil Silks. Mol Biol Evol 2007;24:2424-2432

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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IMPROVEMENT REFERENCE: BNU-China 2017

The part was linked with a blue chromoprotein amilCP to make it visualized. The new part is : BBa_K2220028(Group: iGEM17_BNU-China), Designed by: Jiawei Xing.


IMPROVEMENT REFERENCE: GDSYZX 2020

According to the codon preference of Arabidopsis thaliana, our team optimized the codon of this part to make it more suitable for expression in plants, providing a basis for subsequent research on this part in plants. The new part is : BBa_K3458005 (Group: iGEM20_GDSYZX), Designed by: Jiahua Zou.