Composite

Part:BBa_K2922006

Designed by: Tianyu Feng   Group: iGEM19_XMU-China   (2019-10-13)


Expression system of YebF-bgl1A coding YebF-Beta-D-glucosidase with T7 promoter

This part contains the sequence for the protein beta-glucosidase 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 Beta-glucosidase with the function of YebF protein.

Biology

BBa_K2922006 is a composite of Bgl1A (BBa_K2564000) with YebF (BBa_K1659003) expressed in T7-RBS (BBa_K525998) system with 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 a 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 Beta-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 place 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 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-bgl1A (BBa_K2922000)OE-PCR product. Lane M: Marker. The PCR product in the second step, it showed a signal band at about 1800 bp.


Characterization

These parts were inserted into the expression vectors with T7 and RBS (BBa_K525998). 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). 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 Silver staining. (Fig. 2)


Fig. 2 SDS-PAGE analysis of E. coli BL21 (DE3) by Sliver staining. Lane yebF-bgl1A(R): protein of E. coli BL21 (DE3) carrying T7-RBS-yebF-bgl1A (BBa_K2922006), target bands can be seen in both cells and the medium at 66 kDa; Lane control: protein of E. coli BL21 (DE3) carrying T7 and RBS (BBa_K525998).


2. HPLC

We use HPLC to verify the activity of Bgl1A. First of all, we used the different concentrations of glucose solution and cellobiose solution to make SWC (Standard Working Curve) of HPLC.


Fig. 3 SWC for T7-RBS-yebF-bgl1A, made through the relationship between peak area and concentration.


Then mix the crude enzyme solution with cellobiose, 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. And then carry out HPLC on the sample.


Fig. 4 Quantitative analysis for the enzymatic activity of YebF-Bgl1A (T7-RBS-yebF-bgl1A) was supported through HPLC. (A) The concentration of cellobiose and glucose of sup at different times. (B) The concentration of cellobiose and glucose of broken sup at different times.

Result of the broken supernatant and supernatant of medium cultures with T7-RBS-yebF-bgl1A part shows that D-cellobiose got consumed with extension of reaction time and more D-glucose obtained. Bgl1A could degrade D-cellobiose into D-glucose.


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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE WITH RFC[25]
    Unknown
  • 1000
    COMPATIBLE WITH RFC[1000]


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