Difference between revisions of "Part:BBa K2235010"

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Endo beta galactosidase with T7 promoter, RBS and functional unit

Introduction

BBa_K2235010 biobrick is a composite of a T7 promoter and RBS followed by endo-β-galactosidase enzyme coding site which is N-terminally attached to a His-tag. This enzyme has been shown to release saccharide chains from glycans expressed in gastric mucus. It performs this action by hydrolysing the bonds next to galactose saccharides in the polysaccharide chains of mucins. The sequence originates from the species Clostridium Perfringens. The basic part BBa_K2235008 consists of the endo beta galactosidase enzyme coding sequence with a His-tag N-terminally attached.

Usage and Biology

The endo-β-galactosidase (Endo-β-GalGnGa) was originally expressed from Clostridium perfringens. This bacteria strain is capable of releasing GlcNAcα1→4Gal from glycans expressed in the gastric mucous cell-type mucin [1]. This enzyme specifically releases the disaccharide GlcNAcR1f 4Gal from O-glycans expressed in the gastric gland mucous cell-type mucin. This enzyme has been shown to hydrolyze the endo-â-galactosyl linkage not only in the GlcNAcR1f 4Galâ1f4GlcNAc sequence but also in GlcNAcR1f 4Galâ1f3GalNAcR1fSer/Thr. Endo-â-GalGnGa is distinct from the hitherto known endo-â-galactosidases because of its strict specificity for releasing the disaccharide GlcNAcR1f 4Gal. To characterize Endo-â-GalGnGa, we have carried out the molecular cloning of this endoglycosidase. Here we describe the cloning, characterization, and overexpression of the gene encoding Endo-â-GalGnGa and the hypothesis testing of degrading mucus.

Important parameters

Results

After confirming that the cloning worked, the plasmid was transformed into E. coli BL21(DE3) and expression was induced at multiple combinations of OD600 and IPTG concentrations. The expression of EBG, a 47 kDa protein, from one of the successful expressions (at OD600 of 0.4 and an IPTG concentration of 0.5 mM) is shown in figure 2.

Figure 2 is attached here

By carrying out the experiments above we demonstrated successful cloning and expression of both of our mucus degrading enzymes: sialidase and EBG. The next step was to secrete the enzymes, using an already existing biobrick for HylA E.coli secretion system (BBa_K1166002) from the iGEM 2017 distribution kit. 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. 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. Figure 3 shows no secretion of a protein resembling the correct size (≈ 55 kDa).

Figure 3 is attached here

Reference

1. Ashida, H., Anderson, K., Nakayama, J., Maskos, K., Chou, C.-W., Cole, R. B., Li, S.-C., and Li, Y.-T. (2001) J. Biol. Chem. 276, 28226−28232

Sequence and Features