Difference between revisions of "Part:BBa K2922020"

Latest revision as of 03:00, 22 October 2019

T7-RBS-cex expressing Exoglucanase 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 Exoglucanase regulated by T7 promoter. We used this part to achieve the secretion of Exoglucanase with the function of Kil secretion cassette.

Kil protein (BBa_K1350001)accumulates in the periplasmic space of the bacteria, the periplasmic space was increased and the membrane permeability of bacteria was improved, thereby the protein inside the bacteria can secrete out of bacteria. But only when the intensity of the promoter is proper, the secretion can be achieved and prevent cell lysis and death. Here we use different constitutive promoters to regulate the expression of Kil protein to enhance its secretion ability.^[1]

Biology and Usage

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.^[2]

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-cex BBa_K118022 of interest can be easily integrated.

Target genes-cex was inserted into the expression vectors with T7 and RBS (BBa_K525998), then constructed the final parts(BBa_K2922018, BBa_K2922019 and BBa_K2922020). We transformed the constructed plasmid into E. coli BL21 (DE3). After confirmed by the same method, the positive clones were cultivated and induced by IPTG.

In the meantime, we also cultivated the strain with T7-RBS-yebF-cex (BBa_K2922002) to compare YebF secretion system with Kil secretion system, hoping to find the best secretion system among them.

Characterization

SDS-PAGE

We transformed the constructed plasmid BBa_K2922020 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 gained 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 Silver staining (Fig. 1).

Fig.1 SDS-PAGE analysis of protein in E. coli BL21 (DE3) cells and the medium by Silver staining. 114-kil-cex: protein of BL21 (DE3) carrying J23114-kil-T7-RBS-cex (BBa_K2922020), target bands can't be seen in cells at about 47 kDa; Control: protein of BL21 (DE3) carrying J23114-kil-T7-RBS (linked by BBa_K2922011 and BBa_K525998).

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 460 nm) with TECAN^® infinite M200 PRO. Using fluorescence intesity to determine the activity of Exoglucanase in test samples. Fig. 1 shows the results from the qualitative MUC assay. ^[3]

Fig.2 Assay for Quantitative experiment 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. Among these curves, fluorescence intensity J23109-RBS-kil-T7-cex (BBa_K2922018)increased fastest, the enzymatic activity is highest, and efficiency of secretion is strongest.

Thus, we finally chose Kil secretion cassette with promoter BBa_J23109 for characterization.

Reference

1. ↑ http://2014.igem.org/Team:SZU-China/Project/Kil
2. ↑ 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).
3. ↑ S. S. Lakhundi, Synthetic biology approach to cellulose degradation. University of Edinburgh, (2012).

Sequence and Features