We have constructed many parts successfully in microcin regulation project, mucosal healing project and in metabolic balancing part.
Microcin Regulation Project
To construct a huge plasmid containing nine genes, we put them into four polycistrons: mcmK-mcmL, mchI-mchB-mchX, mchC-mchD, mchE-mchF. We had constructed them successfully.(Fig.1) For polycistrons containing the regulatory genes(mcmK-mcmL, mchC-mchD, mchE-mchF), we placed them under a constitutive expression promoter -- J23119. For mchI-mchB-mchX, we used Ptac-lacO to represent a regulatory promoter. Unfortunately, we failed to construct the final version plasmid, as the mcmKL failed to merge with other polycistrons.(Fig.1)
Fig. 1 The identification of four polycistrons in DNA level.
Mucosal healing project
In order to heal the intestinal tract damaged by IBD, we adopted a special therapy expressing the therapeutic proteins by E.coli Nissle 1917 (EcN) in situ. The design is based on a ternary system: sensor - secretion peptide - therapeutic proteins. Thus, EcN can be induced by inflammatory signals to secrete therapeutic proteins in the damaged intestine, promoting the intestinal mucosal healing.
For each of the three sections, we can replace with different parts freely for a more comprehensive treatment system. Therefore, there are various of combinations, making the whole method more flexible, so that we can provide the treatment with multi-aspects.
Fig.2 The design of sensor-secretion peptide-therapeutic proteins.
CsgA and TFF1/2/3 were chosen separately as secretion peptide and therapeutic proteins. To ensure the existence of the plasmid in the nissle 1917, we perform clony PCR and agarose gel electrophoresis. The results are shown in Fig.2.
Fig.3 Identification of TFF1/2/3 in DNA level
Metabolic balancing part
We had constructed BSH1 successfully. Fig.4 show the extraction of assembled BSH1 DNA. Lane 4,5,6,8 represented the plasmids containing N tagged BSH1 gene which was later sequenced.
Fig4.The identification of BSH1 in DNA level.