Difference between revisions of "Part:BBa K2922014"

Latest revision as of 03:44, 22 October 2019

T7-RBS-bgl1A (Beta-D-glucosidase) functions in Kil secretion cassette with promoter J23114

This part contains the sequence for the protein Kil regulated by constitutive promoter J23114 and the sequence for the protein Beta-D-glucosidase regulated by T7 promoter. We used this part to achieve the secretion of Beta-D-glucosidase with the function of Kil secretion cassette.

Biology

1. Kil Secretion Cassette

In wild-type E.coli exists a plasmid named colE1, the kil gene of the colE1 plasmid encodes a peptide that, at low levels, causes the release of periplasmic proteins without cell lysis. In contrast, high-level induction results in cell lysis and death. This indicates that the regulation of kil gene expression is critical for utilization in a protein secretion system.

The main problem when using constitutive promoters for kil gene expression is the rapid decrease of the viability of bacterial cells before a sufficient amount of target protein has been produced. Using the kil gene under the control of the weak constitutive promoter enabled viability to be maintained.^[1]

Here, we use BBa_J23109, BBa_J23112 and BBa_J23114 to demonstrate the effect of the kil gene controlled by the J21309 series promoters (BBa_J23109) on the release of periplasmic enzymes into the extracellular medium. We fused a synthetic DNA region containing the promoter of the J23109/J23112/J23114 genes with the kil gene and constructed secretion cassettes, where target genes of interest can be easily integrated.

2. 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.^[2]

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.^[3]

Usage

To construct this part, we moved J23114-kil (BBa_K2922011) and T7-RBS-bgl1A (linked by BBa_K525998 and BBa_K2564000) into the expression vector pSB1C3 by standard assembly. Then the ligation mixture was transformed into E. coli DH5α, and the correct recombinant one was confirmed by chloramphenicol, colony PCR and sequencing.

Characterization

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 protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. Lane 114-kil-bgl1A: protein of BL21 (DE3) carrying J23114-kil-PT7-RBS-bgl1A (BBa_K2922014), target bands can be seen in both cells and the medium at about 53 kDa; Lane control: protein of BL21 (DE3) carrying J23114-kil-T7-RBS (linked by BBa_K2922011 and 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 J23114-kil-T7-RBS-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 when and after interval time since reaction started. And then carry out HPLC on the sample.

Fig. 4 Quantitative analysis for the enzymatic activity of Bgl1A (114-kil) 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 J23114-kil-T7-RBS-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. ↑ G. Miksch, E. Fiedler, P. Dobrowolski, K. J. A. o. M. Friehs, The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase. 167, 143-150 (1997).
2. ↑ M. Cox, D. Nelson, Lehninger Principles of Biochemistry. (2000), vol. 5. New York: Worth Publishers. pp. 306–308.
3. ↑ R. M. Weiner et al., Complete genome sequence of the complex carbohydrate-degrading marine bacterium, Saccharophagus degradans strain 2-40 T. 4, e1000087 (2008).

Sequence and Features