Part:BBa_K2235009
Sialidase composite with T7 promoter and RBS
- ==Introduction==
This biobrick is a constitute of T7 promoter and RBS followed by the 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. Figure 1 represents the reaction mechanism of an active enzyme.
Characterization
Important parameter
Table 1: Parameters used for expression and purification of sialidase enzyme.
Purification and identification
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 4). 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).
Rheology testing: Deglycosylated mucin samples show a decrease in viscosity
Removing glycans from mucins could induce protein backbone aggregation and thus decrease the viscosity. Due to time restraints, we chose to chemically deglycosylate PGM sample to simulate the enzymatic activity of sialidase for mucus degradation.Using a non-reducing chemical reagent mixture, we efficiently cleaved the glycans with minimal protein or glycan destruction.
With rheology, we can measure the viscoelastic properties of a given structure. Our measurements showed that viscosity of deglycosylated compared to untreated PGM decreased at a higher rate with increasing shear rate (figure 5). Furthermore, this finding is supported by visually checking the fluidity of the PGM samples (figure 6).
Methods
Cultivation
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. By analysing the number of colony forming units and color of the colonies the ligation efficiency of LigA should be assessable. For the negative control, there were no clones able to grow on chloramphenicol supplemented LB medium. For the positive control, a much bigger number of clones was observed when ligation was performed.
Ligation of sialidase insert into iGEM backbone
We cloned our gblock containing T7 promoter-RBS-sialidase into the iGEM compatible backbone (pSB1C3). Cloning of sialidase gBlock into a pSB1C3 vector was performed with T4 Ligase. To confirm successful cloning, we double digested the plasmid (Figure 7) 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.
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.
References
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 127
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 574
Illegal NgoMIV site found at 649
Illegal NgoMIV site found at 739 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1119
| None |