Part:BBa_K2235011

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

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

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.

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).

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.

References

1. Egebjerg, J. and Christensen, S. (2005). Cloning, expression and characterization of a sialidase gene from Arthrobacter ureafaciens. Biotechnology and Applied Biochemistry, 41(3), p.225.

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

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