Difference between revisions of "Part:BBa K2922000"

Revision as of 13:10, 19 October 2019

Beta-D-glucosidase fused at N-terminal with YebF secretion protein

This part contains the sequence for the protein beta-glucosidase with protein yebF fused to its N-terminus by GS linker. It can achieve the secretion of Beta-glucosidase with the function of yebF protein.

Usage and Biology

Biology

BBa_K2922000 is a composite of bgl1A (BBa_K2564000) with yebF ( BBa_K1659003), a protein reported to be naturally secreted into the extracellular medium by E.coli BL21:

1. bgl1A

Cellulose is a polymer composed of beta-1,4-linked glucosyl residues. Cellulases (endoglucanases), cellobiosidases (exoglucanases), and beta-glucosidases are required by organisms (some fungi, bacteria) that can consume it. These enzymes are powerful tools for degradation of plant cell walls by pathogens and other organisms consuming plant biomass.

Beta-glucosidase is an 53 kDa enzyme that catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in beta-D-glucosides and oligosaccharides, with release of glucose.^[1]

Depending on the organism cellobiose may be cleaved extracellularly by β-glucosidases (cellobiases) and imported as glucose, or imported directly and cleaved in the cytoplasm. Import generally occurs through phosphotransferase transport systems.^[2]

2. YebF

YebF is a 13 kDa protein of unknown function that is perhaps the only protein that has been conclusively documented to be secreted into the extracellular medium by a laboratory E. coli strain. At the N-terminus, YebF has a 2.2 kDa sec-leader sequence which mediates its translocation through the bacterial inner membrane via the Sec pathway, and is cleaved upon translocation into the periplasm to give the 10.8 kDa "mature" form.

Export from periplasm into the extracellular space takes places via the Omp pathway, whereby the electropositive dynamic region of YebF electrostatically helps load YebF onto the OmpF/OmpC porins at their electronegative periplasmic face, and after which the disordered N-terminal region of YebF gets threaded through the OmpF lumen. YebF has been used successfully to mediate the secretion of recombinant proteins.^[3]

Usage

In order to let yebF help secrete our cellulase out of the E. coli membrane, we fused the cellulase gene fragment with yebF gene fragment at the N-terminal by Overlap Extension Polymerase Chain Reaction(OE-PCR), and inserted a flexible GS Linker (GGGGS). PCR product was identified by agarose gel electrophoresis (Fig.1)

Characterization

These parts were insert into the expression vectors with T7 and RBS (BBa_K525998) by restriction sites EcoRI and PstI. Then transformed the expression vectors into E. coli DH5α, and the correct construction of this recombinant plasmid was confirmed by chloramphenicol, colony PCR and plasmid sequencing.

We transformed the constructed plasmid into E. coli BL21 (DE3). After confirmed by the same method, the positive clones were cultivated and induced to express by IPTG. The supernatant of culture medium was obtained by centrifugation. And we gain the total protein by ultrasonic crushing. The lysate was then centrifuged and the supernatant was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/ol) polyacrylamide gel, followed by Coomassie blue staining. (Fig. 2)

Reference

1. ↑ M. Cox, D. Nelson, Lehninger Principles of Biochemistry. (2000), vol. 5. New York: Worth Publishers. pp. 306–308.
2. ↑ R. M. Weiner et al., Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T. 4, e1000087 (2008).
3. ↑ https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659003#Biology

Sequence and Features