Difference between revisions of "Part:BBa K2235011"
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<partinfo>BBa_K2235011 short</partinfo>
 
<partinfo>BBa_K2235011 short</partinfo>
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==Introduction==
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Among all of the type I secretion systems, the α-hemolysin(HlyA) secretion system is the best characterized, studied and most used. Also, the type I secretion system is able to transport various molecules, from ions, drugs, to proteins of various sizes (20-900 kDa) (1). Because of these two reasons, we chose to use the α-hemolysin(HlyA) secretion system for the secretion of our enzymes (≈50 kDa).
  
 
==Usage and Biology==
 
==Usage and Biology==
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We used an already existing biobrick for HylA E.coli secretion system (BBa_K1166002) to ligate our functional protein from the iGEM 2017 distribution kit in order to secrete the sialidase.
  
 
==Characterization==
 
==Characterization==
  
 
===Important Parameter===
 
===Important Parameter===
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[[File:SiaSec_Parameter.png|400px|thumb|left|Table 1: Parameters used for expression and purification of sialidase enzyme in supernatent.]]
  
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===Purification and identification===
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<p align="justify">
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</p>
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<br style="clear: both" />
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===Secretion of Sialidase===
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The newly cloned plasmid was transformed into E.coli and expression in flask was induced with 0.5 mM IPTG. The enzyme was extracted from the medium using IMAC purification. SDS-PAGE results (figure 1) shows no secretion of a protein resembling the correct size (≈ 55 kDa).
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[[File:SiaSec_SDSPAGE.png|400px|thumb|left|Figure 1: SDS-PAGE gel and a protein ladder. From left to right: protein ladder, following four are IMAC purification fractions of a control that wasn’t induced with IPTG, the last four show the IPTG induced sample fractions.]]
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<p align="justify">
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</p>
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<br style="clear: both" />
  
 
===Ligation of sialidase insert into secretion device===
 
===Ligation of sialidase insert into secretion device===
  
  
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Firstly, we removed the stop codon at the end of the sialidase gblock sequence using PCR and thereafter cloned sialidase without the stop codon upstream of the secretion system (BBa_K2235011). To confirm successful cloning, we double digested the plasmid (figure 2). Two bands were observed, one at ~7000 bp, corresponding to the size of T7 promoter-RBS-Sialidase-HylA E.coli secretion system, and one at ~2000 bp, corresponding to the size of the plasmid backbone.
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[[File:SiaSec_ligation.png|400px|thumb|left|Figure 2: Agarose gel. From left to right: DNA ladder, the next five are digested BBa_K2235011 plasmids.]]
  
  
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==References==
 
  
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</p>
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<br style="clear: both" />
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<p>
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<br>More details to the characterization of Sialidase can be found in the composite part BBa_K2235009.
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<br>Following parts constitute the sialidase enzyme:
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<br>Basic part BBa_K2235005 consists of the sialidase enzyme coding sequence with a His tag C-terminally attached.
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<br>BBa_K2235006 biobrick has the RBS functional unit attached to sialidase part (BBa_K2235005) that can be used to test on various promoters.
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<br>BBa_K2235007 biobrick constitutes OmpR fused to sialidase enzyme with RBS (BBa_K2235006).
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</p>
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==References==
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1. Green, E. and Mecsas, J. (n.d.). Bacterial Secretion Systems: An Overview. Virulence Mechanisms of Bacterial Pathogens, Fifth Edition, pp.215-239.
  
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2. Gentschev I, e. (2017). The E. coli alpha-hemolysin secretion system and its use in vaccine development. - PubMed - NCBI. [online] Ncbi.nlm.nih.gov. Available at: https://www.ncbi.nlm.nih.gov/pubmed/11755084 [Accessed 1 Nov. 2017].
  
  

Latest revision as of 14:37, 1 November 2017


Sialidase enzyme coding composite N terminally attached to secretion system type 1

Introduction

Among all of the type I secretion systems, the α-hemolysin(HlyA) secretion system is the best characterized, studied and most used. Also, the type I secretion system is able to transport various molecules, from ions, drugs, to proteins of various sizes (20-900 kDa) (1). Because of these two reasons, we chose to use the α-hemolysin(HlyA) secretion system for the secretion of our enzymes (≈50 kDa).

Usage and Biology

We used an already existing biobrick for HylA E.coli secretion system (BBa_K1166002) to ligate our functional protein from the iGEM 2017 distribution kit in order to secrete the sialidase.

Characterization

Important Parameter

Table 1: Parameters used for expression and purification of sialidase enzyme in supernatent.



Secretion of Sialidase

The newly cloned plasmid was transformed into E.coli and expression in flask was induced with 0.5 mM IPTG. The enzyme was extracted from the medium using IMAC purification. SDS-PAGE results (figure 1) shows no secretion of a protein resembling the correct size (≈ 55 kDa).

Figure 1: SDS-PAGE gel and a protein ladder. From left to right: protein ladder, following four are IMAC purification fractions of a control that wasn’t induced with IPTG, the last four show the IPTG induced sample fractions.



Ligation of sialidase insert into secretion device

Firstly, we removed the stop codon at the end of the sialidase gblock sequence using PCR and thereafter cloned sialidase without the stop codon upstream of the secretion system (BBa_K2235011). To confirm successful cloning, we double digested the plasmid (figure 2). Two bands were observed, one at ~7000 bp, corresponding to the size of T7 promoter-RBS-Sialidase-HylA E.coli secretion system, and one at ~2000 bp, corresponding to the size of the plasmid backbone.

Figure 2: Agarose gel. From left to right: DNA ladder, the next five are digested BBa_K2235011 plasmids.





More details to the characterization of Sialidase can be found in the composite part BBa_K2235009.
Following parts constitute the sialidase enzyme:
Basic part BBa_K2235005 consists of the sialidase enzyme coding sequence with a His tag C-terminally attached.
BBa_K2235006 biobrick has the RBS functional unit attached to sialidase part (BBa_K2235005) that can be used to test on various promoters.
BBa_K2235007 biobrick constitutes OmpR fused to sialidase enzyme with RBS (BBa_K2235006).

References

1. Green, E. and Mecsas, J. (n.d.). Bacterial Secretion Systems: An Overview. Virulence Mechanisms of Bacterial Pathogens, Fifth Edition, pp.215-239.

2. Gentschev I, e. (2017). The E. coli alpha-hemolysin secretion system and its use in vaccine development. - PubMed - NCBI. [online] Ncbi.nlm.nih.gov. Available at: https://www.ncbi.nlm.nih.gov/pubmed/11755084 [Accessed 1 Nov. 2017].




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3178
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3117
    Illegal XhoI site found at 127
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 574
    Illegal NgoMIV site found at 649
    Illegal NgoMIV site found at 739
    Illegal AgeI site found at 2952
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1119
    Illegal SapI site found at 2934