Part:BBa_K2922017
T7-RBS-cenA (Endoglucanase A) functions in Kil secretion cassette with constitutive promoter J23114
This part contains the sequence for the protein kil regulated by constitutive promoter J23114 and the sequence for the protein endoglucanase A regulated by T7 promoter. We used this part to achieve the secretion of endoglucanase with the function of kil secretion cassette.
Biology
1. Kil Secretion Cassette
In wild-type E.coli exist 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 BBa_K118022 of interest can be easily integrated.
2. Endoglucanase A
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.
Bacterium Cellulomonas fimi uses 3 endoglucanases (including CenA, accession M15823) and an exoglucanase in the degradation of cellulose into cellobiose, before using beta-glucosidase to catalyse the conversion of cellobiose to D-glucose.
Usage
To construct this part, we moved J23114-kil (BBa_K2922011) and T7-RBS-cenA (linked by BBa_K525998 and BBa_K118023) into the expression vector pSB1C3 by standard assembly.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.
Characterization
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 protein in E. coli BL21 (DE3) cells and the medium by Silver staining. 114-kil-cenA: protein of BL21 (DE3) carrying J23114-kil-PT7-RBS-cenA (BBa_K2922017), target bands can be seen in the medium at about 47 kDa after dimming the screen; Control: protein of BL21 (DE3) carrying J23114-kil-T7-RBS (linked by BBa_K2922011 and BBa_K525998).
2. Congo Red Assay
We used Congo Red assay to verify the enzyme activity of YebF-CenA, and this method is from [http://2018.igem.org/Team:UESTC-China iGEM18-UESTC-China]. Luria agar plates with 0.2% CMC are addd with the crude enzyme. Then the agar is flooded with 1 mg/mL Congo Red solution for 1 h. Congo Red solution is poured off into a toxic waste bottle and 1 M NaCl is added and left for another 1 h. Then pour off NaCl solution. Cellulases can cut CMC-Na into short chains. As Congo Red only binds to long chain polysaccharides but not short chain which therefore are washed off during staining procedure resulting in halo formation[2]. The results are shown in Fig. 3.
- Fig.3 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).
Zone added with 114-CenA boken supernatant and cluture supernatant were shown clearance zones produced by hydrolysis of CMC. The empty control didn't show any zone of clearance. The results showed that both intracellular and extracellular 114-CenA had enzyme activity.
References
- ↑ 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).
- ↑ S. S. J. U. o. E. Lakhundi, Synthetic biology approach to cellulose degradation. (2012).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NotI site found at 328
Illegal NotI site found at 1472 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1385
Illegal BamHI site found at 521
Illegal XhoI site found at 883
Illegal XhoI site found at 1132 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 663
Illegal NgoMIV site found at 1588 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 567
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