Part:BBa_K2922015

T7-RBS-cenA (Endoglucanase A) functions in Kil secretion cassette with constitutive promoter J23109

This part contains the sequence for the protein Kil regulated by constitutive promoter J23109 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 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.

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. 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 J23109-kil (BBa_K2922009) and T7-RBS-cenA (linked by BBa_K525998 and BBa_K118023) 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 clonies 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 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-RBS-kil-T7-RBS (linked by BBa_K2922009 and BBa_K525998).

2. Congo Red Assay

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 . 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-RBS-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. Besides, what the results showed was in accordance with the results of SDS-PAGE (Fig. 2) as well.

References

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
  Illegal NotI site found at 328
  Illegal NotI site found at 1472
  
• 21
  INCOMPATIBLE 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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 663
  Illegal NgoMIV site found at 1588
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 567