Part:BBa_K2235009

Sialidase composite with T7 promoter and RBS

Introduction

This biobrick is a constitute of T7 promoter and RBS followed by sialidase enzyme coding site. Sialidase enzyme has the potential to digest terminal sialic acids in a glycoprotein. The sequence originates from the species Arthrobacter Ureafaciens.

Usage and Biology

Sialidase enzyme can hydrolyze glycosidic linkages of terminal sialic acid residues in glycoproteins. Following image represents the reaction mechanism of an active enzyme.

Figure 1: Schematic representation of Sialidase enzyme reaction mechanism.

Characterization

Important parameter

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

Molecular cloning

Ligation of sialidase composite insert into pSB1C3

The gblock containing T7 promoter-RBS-sialidase was ligated into the Chloramphenicol plasmid backbone (pSB1C3). Preliminary confirmation of cloning was done by double digest Ecor1 and Pst1. Figure 1 represents the double digestion result: bands were visible at ~1600 bp and ~2000 bp which represents the insert and plasmid backbone respectively.

Figure 1:From left to right: M DNA ladder, next 2 lanes are plasmids digested with Ecor1 and Pst1.

Purification and identification

Figure 2: SDS-PAGE gel and a protein ladder. From left to right: protein ladder, the remaining three are IMAC purification fractions of sialidase.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  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
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1119