Composite

Part:BBa_K2922009

Designed by: Fude Chen   Group: iGEM19_XMU-China   (2019-10-13)


Constitutive promoter J23109, strong RBS and Kil protein combination for high secretion levels

        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 J23109/J23112/J23114 promoters with the kil gene and constructed secretion cassettes, where target genes of interest can be easily integrated.


Characterization

        BBa_K2922009 was characterized with different genes[bgl1A(BBa_K2922012), cenA(BBa_K2922015) and cex(BBa_K2922018)] to prove the applicability.


A. 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. The one with correct sequencing was 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. 1-3)

1. Bgl1A(BBa_K2922012)

Fig. 1 SDS-PAGE analysis of protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. Lane 109-kil-bgl1A: protein of BL21 (DE3) carrying J23109-kil-T7-RBS-bgl1A (BBa_K2922012), target bands can be seen in both cells and the medium at about 53 kDa; Lane control: protein of BL21 (DE3) carrying J23109-kil-T7-RBS (linked by BBa_K2922009 and BBa_K525998).


2. CenA(BBa_K2922015)

Fig. 2 SDS-PAGE analysis of protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. Lane 109-kil-cenA: protein of BL21 (DE3) carrying J23109-kil-T7-RBS-cenA (BBa_K2922021), target bands can be seen in both cells and the medium at about 47 kDa; Lane control: protein of BL21 (DE3) carrying J23109-kil-T7-RBS (linked by BBa_K2922009 and BBa_K525998).


3. Cex(BBa_K2922018)

Fig. 3 SDS-PAGE analysis of protein in E. coli BL21 (DE3) cells and the medium by Coomassie blue staining. Lane 109-kil-cex: protein of BL21 (DE3) carrying J23109-kil-T7-RBS-cex (BBa_K2922018), target bands can be seen in both cells and the medium at about 47 kDa; Lane control: protein of BL21 (DE3) carrying J23109-kil-T7-RBS (linked by BBa_K2922009 and BBa_K525998).


B. HPLC Determination [Characterization of bgl1A(BBa_K2922012)]

        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. 4 SWC which is 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. 5 The results of HPLC. (A): J23109-kil-T7-RBS-bgl1A supernatant; (B): J23109-kil-T7-RBS-bgl1A broken supernatant

        Result of the broken supernatant and supernatant of medium cultures with J23109-kil-T7-RBS-bgl1A part shows that D-cellobiose got consumed with extension of reaction time and more D-glucose obtained. Bgl1A can degrade D-cellobiose into D-glucose.


C. Congo Red Assay [Characterization of cenA(BBa_K2922012)]

        Congo Red assay was utilized to qualitatively test the enzymatic activity of CenA in the form of crude enzyme, and this method was from iGEM18-UESTC-China, who had a nice collaboration with us this year. As Congo Red only binds to long chain polysaccharides but not short chain, the short chain therefore are washed off during staining procedure resulting in halo formation [3]. The results are shown in Fig. 6.

Fig. 6 Activity determination of CenA using Congo Red assay. CenA: broken supernatant and medium supernatant of BL21 (DE3) carrying J23109/J23112/J23114-kil-T7-RBS-cenA (BBa_K2922015 / BBa_K2922016 / BBa_K2922017); Control: broken supernatant and medium supernatant of BL21 (DE3) carrying J23109-kil-T7-RBS (linked by BBa_K2922009 and BBa_K525998).


        Zones with the broken sup and sup of 109-CenA added showed due to the hydrolysis of CMC (carboxymethyl cellulose) whereas the blank control didn't show any clearance zones. The obvious difference showed that the broken sup and sup of 109-CenA had enzymatic activity. This was to say that the enzyme, Endoglucanase (CenA), which was expressed successfully, had a certain level of enzymatic activity to hydrolyze cellulose.


Selection of the most efficient secretion system

        Target gene-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). 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.


MUC Assay[Characterization of cex(BBa_K2922018)]

        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. [4]

Fig.1 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(Strains 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-RBS-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.

        In summary, We fused serveral synthetic DNA regions containing the J23109/J23112/114 promoters with the kil gene and constructed secretion cassettes, where target genes of interest can be easily integrated. (BBa_K2922012, BBa_K2922015 and BBa_K2922018) As a result, J23109-RBS-kil was characterized to become the most efficient secretion system among YebF secretion system(BBa_K2922000), and J23109/J23112/J23114-Kil secretion systems(BBa_K2922009, BBa_K2922010 and BBa_K2922011)


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. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).
  4. ↑ S. S. Lakhundi, Synthetic biology approach to cellulose degradation. University of Edinburgh, (2012).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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