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

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

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.

Purification and identification

Figure 3: SDS-PAGE gel and a protein ladder. From left to right: protein ladder, the remaining three are IMAC purification fractions of sialidase.

Figure 4: SDS-PAGE gel and a protein ladder.From left to right: first well is a protein ladder, second well is protein expressed from the au54 plasmid, third well contains sialidase expressed from the biobrick plasmid.

The cloned gblock plasmid culture was induced with IPTG for expression. Post expression the cells were sonicated and purified using Immobilized Metal Affinity Chromatography(IMAC). The purified enzymes were tested on SDS PAGE(Figure 2). As control, sialidase enzyme plasmid from literature research lab(see reference) was used. The control plasmid(au54) holds the enzyme coding site on a backbone which is not compatible with iGEM standards. Similar cloning, expression and purification methods were carried out on both, gblock+pSB1C3 plasmid and the control plasmid. A band could be observed at 60 kDa. Note: According to the literature the expected size of the enzyme should be at 55 kDa. However, the size of enzyme purified from the control plasmid(au54) and designed plasmid are consistent. To demonstrate the above, an SDS-PAGE on both samples was carried out. The au54 sialidase and our designed biobrick sialidase, with molecular sizes of 54kDa and 55 kDa respectively. Figure 3 shows that there is no observable difference in size between the two proteins. Sialidase is a protein with a high content of basic amino acids, therefore it was hypothesized that this might affect the travelling speed through the gel.

Hypothesis testing: Successfully expressed sialidase shows enzymatic activity on mucin

With the goal of testing the enzymatic activity of sialidase on mucin, an assay was developed and optimized. The objective was to measure the concentration of sialic acid released after digestion, which was quantified using high performance anion exchange chromatography (HPAEC).

Using industrially purchased sialidase to treat bovine submaxillary mucin (BSM) gave a positive result. Sialic acid was proved to be digested from the mucin. Therefore, the next step was to repeat the experiment with sialidase that we expressed in E. coli. A range of different sialidase concentrations were used and their respective sialic acid digestion quantified (figure 9). We were expecting a linear increase of substrate degraded with increase of enzyme. It is believed that excessive enzyme was used in the experiment. The sialic acid concentration released even at the lowest enzyme concentration is large when compared to the positive control (deglycosylation using sulfuric acid).

Figure 5: Concentrations of sialic acid digested from BSM in comparison to the concentration of sialidase used.

Methods

IMAC purification

To purify the sialidase sample A1-A3, B1-B2 and control using the His-tag. IMAC purification was carried out using nickel column of volume 3.2mL and three cobalt columns, each with a volume of 1.2 mL. The protein samples was eluted to five fractions each.

Ligation of sialidase insert into iGEM backbone

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

We cloned our gblock containing T7 promoter-RBS-sialidase into the iGEM compatible backbone (pSB1C3). To confirm successful cloning, we double digested the plasmid (figure 5) and could see one band at ~1600 bp and one at ~2000 bp. The band at 1600 bp corresponds to the size of sialidase and the band at 2000 bp to the linearized plasmid backbone.

Cultivation Cloning of sialidase gBlock into a pSB1C3 vector was performed with T4 Ligase. Ligation was done with ThermoFisher T4 DNA Ligase Buffer (10X) at 22 °C and the vectors were transformed into E. coli TOP10 and BL21(DE3) by heat shock. After growth overnight at 37 °C on petri dishes the results were documented.

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

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