Part:BBa_K2922022

T7-RBS-cex-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 exoglucanase 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.BBa_K2922009 was tested to be the most strongest secretion among those.

After cellulose in culture medium was degraded into cellobiose by CenA and Cex, both of the two groups of E. coli could use cellobiose as substrate since they had exogenous gene bgl1A. However, cellobiose still needed to be transported into cell for usage. We found that cellobiose can be transported into cells by the Beta-galactoside permease encoded by the lacY gene in the lac operon, which could be achieved by induction of lactose or its analogues.

Fig. 1 Cellobiose could be transported into cell due to the function of beta-galactoside permease coded by lacY.

Once cellobiose is transported into cell, Beta-glucosidase can function at the final step of cellulose degradation, and glucose is generated for living finally.

Fig. 2 The complete schematic of "cooperative" in our project.

Thus, the cellulose will be degraded due to the cooperation of two groups of E. coli, and they both survive (Fig. 2). In summary, according to the thoughts we depicted above, combining the results we obtained, we constructed the gene circuits (Fig. 3) to achieve the cooperation.

Fig. 3 The gene circuits to achieve cooperation between two groups of E. coli. (A) The gene circuits (BBa_K2922021) was constructed according to the function of Kil, CenA and Bgl1A in one group of E. coli. (B) The gene circuits (BBa_K2922023) was constructed according to the function of Kil, Cex and Bgl1A in the other group of E. coli.

Characterization

1. Restriction digestion

Fig. 4 One of the gene circuits to achieve cooperation. (BBa_K2922022)

BBa_K2922022 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_K2922022. (M: Marker. The target gene showed a signal band at about 3200 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 control group. But the first try showed that the non-cocultured control 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

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 818
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 451
  Illegal NgoMIV site found at 824
  Illegal NgoMIV site found at 1326
  Illegal NgoMIV site found at 3014
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 871
  Illegal SapI.rc site found at 954