Using Synthetic Biology Method To Construct A Heme Response Dynamic Element For Regulating Gene Transcription

Recent years, Synthetic biology is a kind of important and frontier research method in biology studies which combines multi-subject disciplines in research.This subject is based on the artificial design construction and optimization of the core components to form the engineered, standardized and replaceable modules. It can synthesize new elements such as enzymes, genetic circuit and even new life. Given the modular nature of engineering, the element in synthetic biology can be designed in larger combination to achieve a larger biological engineering innovation. At present, synthetic biology has achieved important progress in many aspects such as biological medicine, biological energy and synthetic life.Heme is an important form of porphyrin compoundand plays an important function in the process of normal metabolism. It is a protoporphyrin IX and ferrous chelate compound. In the metabolic process of Escherichia coli, heme is indispensable for growth and transformed by the intermediate product 5-aminolevulinic acid (ALA). ALA is the precursor of almost all kind tetrapyrrole compounds and has broad application prospect in agricultural and medical field. Biological method has been widely used in the production of this important intermediate compound.Accroding to previous research, we found that the production of ALA was a complex metabolic process, which was influenced by many genes upstream or downstream. The simple method of overexpress genes upstream or weakened genes downstream may not work and heme is the end product in ALA metabolic process, it is prone to accumulate causing excessive waste in the process of metaolic regulation, dynamically regulating the intracellular heme is particularly important. Based on this problem, our research use the way and ideas of synthetic biology and construct a dynamic regulator contained three parts: IRRr1-Pice, CI-Pciand the reporter gene gfp-ssrA to reduce the energy waste and regulate gene transcription.This research selected the IRRr1 protein from Rhizobium leguminosarum as the sensor protein and inserted negative regulatory element CI-Pci from λ phage to display the function of negative regulation, degradation lable ssrA was added at the end of C-terminus of the reporter gene gfp-ssrA to reduce the matabolic stress. The IRRr1 protein inhitit the promoter Pice, thus stop accumulation of the CI protein. Promoter Pci start transcription of the reporter gene gfp-ssrA. When the heme level is high, IRRr1 protein was occupied by heme thus can’t bind the promoter Pice. The accumulated CI protein can stop the Pci promoter and inhibit the tranccription of the reporter gene gfp-ssrA. If the reporter gene was changed into other genes upstream of heme, the element can dynamically regulate heme level intracellularly and it is conductive to ALA accumulation.To study the effection of this element, we verified the function of the promoters, the repressor elements and the series element successively. Firstly, we chosed DH5a strains for the verification of the single promoter. Promoter Pice and Pci was assembled with gfp-ssrA respectively to construct plasmids pGFPI and pGFPII. The fluorescence values and F/OD was continiusly rise in 16h. Secondly, single element was inserted into plasmid pUC19 to construct plasmids pGFPIII and pGFPIV. From the fermentation curve we found that the expressed protein can’t inhibit gene transcription. Adding certain concentration of glucose or replace the strains with TOP 10 from MC1061 could reduce the promoter leakage effectively. Next, we constructed plasmids pGFPIII2 and pGFPIV2, from the fermentation curve we found that the promoters was inhibited after 5h. Finally, we combined the two elements in series for constructting the plasmid pGFPV. We then transformed the plasmid into DHGAL strain and the element stop the transcription of gfp-ssrA.This work successfully constructed a dynamic element, which can response to heme level and regulate gene transcription. These results will provide research direction and new idea for metabolic pathway reconstruction, and have a broad application value.