Difference between revisions of "Part:BBa K2922008"

Revision as of 16:37, 19 October 2019

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 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).

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 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.

1. SDS-PAGE

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/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

In order to determine the enzyme activity of cex, we carried out the qualitative 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 when and after interval time since reaction started. Dilute reaction sample 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 the ratio of fluorescence and OD600 to determine the activity of exoglucanase in test samples. 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.

Assay for supernatant shows that NO fluorescence intensity can be detected, which means no enzymatic activity. But broken supernatant of culture with YebF linked Cex protein can be detected fluorescence intensity.

References

1. ↑ https://parts.igem.org/wiki/index.php?title=Part:BBa_K1659003#Biology

Sequence and Features