Difference between revisions of "Part:BBa K1362101"

 
 
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__NOTOC__
 
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<partinfo>BBa_K1362101 short</partinfo>
 
<partinfo>BBa_K1362101 short</partinfo>
  
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This intein assembly construct is part of the iGEM team Heidelberg 2014's strategy for cloning with split inteins. Inteins are naturally occuring peptide sequences that splice out of a precursor protein and attach the remaining ends together to form a new protein. When splitting those intein sequence into an N-terminal and a C-terminal split intein one is left with a powerful tool to post-translationally modify whole proteins on the amino-acid sequence level. This construct was designed to express any protein of interest fused to the <i>Nostoc punctiforme</i> DnaE N-terminal split intein. The corresponding C-terminal construct is <partinfo>BBa_K1362101</partinfo>.
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This intein assembly construct is part of the iGEM team Heidelberg 2014's strategy for cloning with split inteins. Inteins are naturally occuring peptide sequences that splice out of a precursor protein and attach the remaining ends together to form a new protein. When splitting this intein sequence into an N-terminal and a C-terminal split intein one is left with a powerful tool to post-translationally modify whole proteins on the amino-acid sequence level. This construct was designed to express any protein of interest fused to the <i>Nostoc punctiforme</i> [[Part:BBa_K1362401|DnaE C-terminal]] split intein. The corresponding N-terminal construct is <partinfo>BBa_K1362100</partinfo>.
 
Upon coexpression or mixture of the N- and C-constructs protein splicing takes place and the N- and C-terminal proteins of interest are irreversibly assembled via a newly formed peptide bond.
 
Upon coexpression or mixture of the N- and C-constructs protein splicing takes place and the N- and C-terminal proteins of interest are irreversibly assembled via a newly formed peptide bond.
  
 
This mechanism can be applied for a variety of different uses such as the activation of a protein through reconstitution of individually expressed split halves. See our [http://2014.igem.org/Team:Heidelberg split sfGFP experiment] and the respective [[Part:BBa_K1362170|parts]] in the registry for more information. Protein splicing offers many new possibilities and we hope to have set a foundation that you guys can build on!
 
This mechanism can be applied for a variety of different uses such as the activation of a protein through reconstitution of individually expressed split halves. See our [http://2014.igem.org/Team:Heidelberg split sfGFP experiment] and the respective [[Part:BBa_K1362170|parts]] in the registry for more information. Protein splicing offers many new possibilities and we hope to have set a foundation that you guys can build on!
  
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===<i>Nostoc punctiforme</i> DnaE split intein (<i>Npu</i>DnaE)===
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<h3> Improvement </h3>
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Inteins can be found in all kingdoms of life, however their use for the organism remains yet unknown. In the past years many intein sequences were found in the genomes of cyanobacteria.
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<i>Npu</i>DnaE is a naturally split intein from the alpha subunit of Dna Polymerase III in Nostoc punctiforme bacteria which shows [[#References|[2]]] extraordinarly high splicing activity and therefore is on of the gold-standards for inteins since many years.
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This part was improved upon by the UCL 2018 iGEM team by building [https://parts.igem.org/Part:BBa_K2842669 BBa_K2842669] . This improved construct used a brighter fluorescent protein that achived higher levels of fluoresence of the reporter driven purly by the leaky expression of the T& promoter in BL21* cells(fig.1).
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[[File: UCLImprovedPartComparison.png |400px|thumb|left|
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<center>'''Figure 1: Comparisons between BBa K1362101 and the improved part BBa_K2842669: '''</center>
  
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<pre>
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By measuring fluorescence and OD600 over time with a plate reader we compared the fluorescence of the two constructs. 
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Amino Acid Sequence:
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]]
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[[File: BBa_K1362101_vs_controll.png |400px|thumb|right|
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<center>'''Figure 2: The fluorescence of BBa_K1362101 over time compared to a control cell culture.'''</center>
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BL21* cells expressing BBa K1362101 were cultured then diluted to 0.2 OD600 and measured overnight (20 hours) in a plate reader.
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]]
  
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N-Intein: AEY | CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN
 
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C-Intein: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN | CFN
 
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</pre>
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 22:27, 16 October 2018

RBS+NpuDnaE C-intein RFC[105] assembly construct (with His6)

This intein assembly construct is part of the iGEM team Heidelberg 2014's strategy for cloning with split inteins. Inteins are naturally occuring peptide sequences that splice out of a precursor protein and attach the remaining ends together to form a new protein. When splitting this intein sequence into an N-terminal and a C-terminal split intein one is left with a powerful tool to post-translationally modify whole proteins on the amino-acid sequence level. This construct was designed to express any protein of interest fused to the Nostoc punctiforme DnaE C-terminal split intein. The corresponding N-terminal construct is BBa_K1362100. Upon coexpression or mixture of the N- and C-constructs protein splicing takes place and the N- and C-terminal proteins of interest are irreversibly assembled via a newly formed peptide bond.

This mechanism can be applied for a variety of different uses such as the activation of a protein through reconstitution of individually expressed split halves. See our [http://2014.igem.org/Team:Heidelberg split sfGFP experiment] and the respective parts in the registry for more information. Protein splicing offers many new possibilities and we hope to have set a foundation that you guys can build on!

Improvement

This part was improved upon by the UCL 2018 iGEM team by building BBa_K2842669 . This improved construct used a brighter fluorescent protein that achived higher levels of fluoresence of the reporter driven purly by the leaky expression of the T& promoter in BL21* cells(fig.1).

Figure 1: Comparisons between BBa K1362101 and the improved part BBa_K2842669:
By measuring fluorescence and OD600 over time with a plate reader we compared the fluorescence of the two constructs.
Figure 2: The fluorescence of BBa_K1362101 over time compared to a control cell culture.
BL21* cells expressing BBa K1362101 were cultured then diluted to 0.2 OD600 and measured overnight (20 hours) in a plate reader.


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 958
    Illegal AgeI site found at 1070
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1247
    Illegal BsaI.rc site found at 172