Difference between revisions of "Part:BBa K2922008"

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Expression system of YebF-Cex coding YebF-Exoglucanase with T7 promoter

This part contains the sequence for the protein exoglucanase with protein YebF fused to its N-terminus by GS Linker. We use T7 promoter and RBS related to achieve the high level secretion of exoglucanase with the function of YebF protein.

Biology

BBa_K2922008 is a composite of cex (BBa_K118022) with yebF ( BBa_K1659003)expressed in T7-RBS (BBa_K525998) system with the related protein reported to be naturally secreted into the extracellular medium by E.coli BL21:

1. Exoglucanase

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.

The cellulolytic bacterium Cellulomonas fimi uses an exoglucanase (from cex, accession M15824) along with 3 endoglucanases in the degradation of cellulose into cellobiose, before use B-glucosidase to catalyse the conversion of cellobiose to D-glucose.

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 ^[1].

Usage

In order to secrete our cellulases out of the E. coli membrane, we fused the cellulase gene fragments with yebF gene fragment at the N-terminal using a flexible GS linker (GGGGS) by overlap extension polymerase chain reaction (OE-PCR). PCR product was purified by agarose gel electrophoresis (Fig. 1).

Fig.1 Agarose Gel Electrophoresis of yebF-cex OE-PCR product. Lane M: Marker.

Characterization

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

1. SDS-PAGE

The constructed plasmid was transformed into E. coli BL21 (DE3). Positive clones that were selected by chloramphenicol preliminarily and then by colony PCR, while finally confirmed by sequencing were cultivated and induced by IPTG to express cellulases. The supernatant of culture, namely sup, was obtained by centrifugation. And the total protein was gained by ultrasonication. The lysate underwent centrifugation and its supernatant, namely broken sup, was electrophoresed on a sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining (Fig. 2)

Fig.2 SDS-PAGE analysis of E. coli BL21 (DE3) by Coomassie blue staining. YebF-cex: protein of E. coli BL21 (DE3) carrying T7-RBS-yebF-cex (BBa_K2922008), target bands can be seen in both cells and the medium at 62 kDa; Control: protein of E. coli BL21 (DE3) carrying T7 and RBS(BBa_K525998).

2. MUC Assay

Methylumbelliferyl cellobioside (MUC) in the presence of Exoglucanase is broken down into methylumbelliferone and cellobiose. Methylumbelliferone fluoresces under long wave length (λ=366 nm) ultra-violet light. Add 200 μL MUC working solution (5×) into 800 μL culture supernatant / crushed cell supernatant as reaction system. Add 200 μL MUC working solution (5×) into 800 μL LB Broth / PBS Buffer as background group. Incubate under the condition of 37 °C, 200 rpm using a shaking incubator for reaction. Take out one tube of reaction system into boiling water bath for 8 minutes to stop the reaction after interval time since reaction started. Dilute reaction samples for 100 times and pipet 200 μL diluent into Black opaque 96-well plate, measure fluorescence (Excitation 364 nm, Emission 640 nm) with TECAN^® infinite M200 PRO. Using fluorescence intesity to determine the activity of Exoglucanase in test samples^[2]. Fig. 3 shows the results from the qualitative MUC assay.

Fig.3 Assay for Quantitative Determination of cex Activity using MUC. (A) Supernatant and control. (B) Broken supernatant and control.

All supernatant show greater fluorescence intensity than control group(Strain with T7-RBS or T7-RBS-Cex), which means all of the secretion systems have enzymatic activity. We also cultivated the strain with J23109/J23112/J23114-kil-T7-RBS-yebF-cex (BBa_K2922018/BBa_K2922019/BBa_K2922020) to compare YebF secretion system with Kil secretion system. Among these curves, fluorescence intensity J23109-RBS-kil-PT7-Cex (BBa_K2922018)increased fastest, the enzymatic activity is highest, and efficiency of secretion is strongest.

References

1. ↑ https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659003#Biology
2. ↑ S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).

Sequence and Features