Difference between revisions of "Part:BBa K2922021"

Revision as of 21:58, 19 October 2019

T7-RBS-cenA-T7-RBS-bgl1A functioned in Kil secretion cassette with promoter J23109

This part contains the sequence for the protein kil regulated by constitutive promoter J23109 and the sequence for the protein endoglucanase A and beta-D-glucosidase regulated by T7 promoter. We used this part to achieve the secretion of exoglucanase and the expression of beta-D -glucosidase with the function of kil secretion cassette.

Usage and Biology

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.

In order to achieve cooperation between two groups of E. coli, CenA and Bgl1A were selected and expressed in one group of the E. coli, while Cex and Bgl1A in the other. As cellulose cannot be transported into cells, and enzymes selected cannot be released to extracellular spontaneously, the enzymes should be secreted into the medium to degrade cellulose to achieve cooperation by certain means. Then two different secretion mechanisms of enzymes were designed: YebF-cellulase fusion protein and Kil secretion cassette.No part name specified with partinfo tag. were constructed by several basic parts with the expression vectors of T7 and RBS (BBa_K525998). 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.

Fig. 5 Agarose Gel Electrophoresis of BBa_K2922021. (M: Marker. The target gene showed a signal band at about 3100 bp)

2. Cultivation and growth curve determination

We transformed the constructed plasmid into E. coli BL21 (DE3). The positive clones were cultivated in restricted medium where cellobiose is the only carbon source and induced by IPTG.

BBa_K2922021 and BBa_K2922022 were cocultured as experimental group, the two independent non-cocultured groups and blank were set to be ctrl group.

We expected to see the growing tendency of experimental group is greater than ctrl group. But the first try showed that the non-cocultured ctrl group of BBa_K2922021 grew greater than all groups. We thought that was due to the error of OD detection, which is caused by the insoluble microcrystalline cellulose in the medium. We decided to insert a mRFP part to detect the flurescence to avoid the OD detection error. So we constructed BBa_K2922023.(Click to get more information)

Reference

Sequence and Features