Research On Control Of Uric Acid Homeostasis At Cellular Level By A Synthetic Gene Circuit

Being a burgeoning comprehensive interdiscipline in the post-genomicera, synthetic biology is becoming a research hotspot in modern life science. Based onthe study of traditional biology, combined with engineering, materialogy andcomputer science, synthetic biology studies technologies of artificially synthesizingneotype biology networks or systems, therefore making them perform new functionsas expected. In the past twenty years, synthetic biology has made considerabledevelopments, and its research relates to biomedicine, energy, materials, biologicalcomputing, military application and so on, that brings new hope to solve some of themajor issues faced by human development.Uric acid, the end product of purine metabolism, which is slightly dissoluble inwater and easy to form crystals, is one of the main metabolites in human body.Changes in uric acid concentration of human body fluid can be regarded as reflectionsof physical conditions of metabolism and immune function. Hyperuricemia, referringthe uric acid concentration exceeding the normal physiological range in human body,will cause gout, tumor lysis syndrome if lasts long. Gout is a foreign bodyinflammatory reaction, which is caused by the depositon of urate crystal, on tissueslike joint synovium and cartilage. At present, gout is mainly treated with drugs,including traditional allopurinol and new biological uricase preparations. Traditionaldrugs show the advantage of satisfactory safety, however perform no significanttherapeutic efficacy, and easily result in serious side effects. By contrast, neotypebiological uricase preparations effect quickly and dramatically, but easily induceattacks of acute gout, hypersensitivity and drug resistance. The development ofsynthetic biology brings new hopes on realizing regulation of uric acid as well asother metabolites at the genetic and cellular level. It is reported that hucR inDeinococcus radiodurans can be translated into transcriptional inhibitor protein HucR,which can combine with or depart from hucO mediated by uric acid, and then affectthe expression of a downstream gene of hucO. Based on the transcriptional inhibitor gene hucR and its binding site hucO in thegenome of Deinococcus radiodurans, this study aims to artificially synthesizetranscriptional inhibitor gene mUTs and its binding site8-series structure(hucO8), takemUTs and hucO8as standard biological modules, construct a genetic circuit which iscapable of sensing and regulating the concentration of uric acid, therefore study theregulatory effect of the circuit on the concentration of uric acid at the cellular level.The main contents and results of this study are summarized as below:(1) Fetch the sequences of optimized transcriptional inhibitor gene mUTs and itsbinding site8-series gene hucO8, as well as the sequences of optimized Spergillusflavus uricase gene smUox from NCBI database, chemically synthesize, name thevectors with mUTs, hucO8and smUox as pMD18T-mUTs, pMD18T-hucO8,pGH-smUox separately.(2) Based on pcDNA3.1/V5-his(C) and pMD18T-mUTs, construct optimizedtranscriptional inhibitor expression vector pcDNA3.1/V5-mUTs. Based onpSEAP2-control, which is the expression vector of secreted alkaline phosphatase(SEAP), and pMD18T-hucO8, construct the reporter gene expression vectorpSEAP-hucO8. Co-transfact dual vectors circuit, which consists ofpcDNA3.1/V5-mUTs and pSEAP-hucO8, into HeLa cells, by assaying the expressionof SEAP within48h after transfection, we have verified the basic theory of the dualvectors circuit, and proved the induction effect of the circuit on uric acid.(3) Based on co-expression vector pBudCE4.1, construct the co-directionco-expression single vector circuit pBudCE4.1-SEAP-mUTs by integrating mUTs andSEAP-hucO8into pBudCE4.1. Transfect pBudCE4.1-SEAP-mUTs into HeLa cells, byassaying the expression of SEAP within48h after transfection, we have verified thebasic theory of the single vector circuit, and proved the induction effect of the singlecircuit on uric acid.(4) Transfect pcDNA3.1/V5-mUTs and pSEAP-hucO8, or pcDNA3.1/V5-mUTs andpBudCE4.1-SEAP-mUTs into HeLa cells in different mole ratios. By analyzing thedifferences in SEAP expression concentrations, study the effect of the ratio betweenmUTs and huc08on the circuits. As the results indicate, within certain ranges(1:1-4:1in our study), as the ratio between muTs and hucO8increases, the inhibiting effect ofmUTs on the expression of downstream gene of hucO8increase accordingly, withincertain ranges(1:1-4:1in our study), and when the ratio comes to4:1, the inhibitingeffect reaches its peak. (5) Based on pSEAP-hucO8and pGH-smUox, construct optimized Aspergillus flavusuricase expression vector phucO8-smUox, and then based on phucO8-smUox andpBudCE4.1, construct and transfect optimized Aspergillus flavus uricase expressionvector pBudCE4.1-smUox into HeLa cells. By assaying the concentration of uricasein the medium48h after transfection, we have verified that smUox can expresscorrectly in HeLa cell. Finally based on pBud4.1-smUox andpBudCE4.1-SEAP-mUTs, construct the single vector uric acid regulation circuitpBudCE4.1-mUTs-smUox.(6) Co-transfect dual vectors uric acid regulation circuit(phucO8-smUox,pcDNA3.1/V5-mUTs) or singly transfect single vector uric acid regulationcircuit(pBudCE4.1-mUTs-smUox). By assaying the concentration of uric acid in themedium48h after transfection and contrasting the concentration of uric acid aftertransfection with that before the transfection, we have proved that both single anddual vectors circuits are capable of regulating uric acid.This study has constructed the dual vectors uric acid induction circuit, verifiedthe theory of mUTs and hucO8, as well as the induction effect of the circuits on uricacid.On this basis, we have imported optimized Aspergillus flavus uricase genesmUox, constructed dual vectors and single vector uric acid regulation circuitsseparately. Other than single vector circuit, dual vectors circuit shows its advantage offreely regulating the ratio between mUTs and hucO8at the time oftransfecting, whereas disadvantages as below:1, Owe to uncertain transfectionefficiency, dual vectors circuit is not capable of accurately regulating the ratiobetween mUTs and hucO8transfected into HeLa cells;2, The experimentalcomplexity of co-transfection raises the experimental costs;3, When selecting stabletransfection cell lines, the dual vectors circuit requires2different selection markers,thus raising the difficulty and complexity of the process of selection. On thecontrast, single vector circuit occupies several advantages as below:1, Replacingco-transfection with single transfection, reduces the complexity and costs ofexperiment;2, Able to ensure the mole ratio between mUTs and hucO8when singlytransfecting pBudCE4.1-mUTs-smUox, avoid the problem of uncertain ratio betweenmUTs and hucO8caused by uncertain transfection efficiency;3, Single transfectionrequires only1selection marker, making it much more convenient to stabilize theselection of trasfection HeLa cell line in further study.In conclusion, this study has set the mUTs and hucO8as standard biological modules, using synthetic biological methods, successfully constructed dual vectorsand single vector uric acid regulation circuits, verified the theory of thecircuits, proved the induction and regulation effect of the circuits on uric acid, reachedthe goal of regulating uric acid at the cellular level. The study also shows that withincertain ranges, the ration between mUTs and hucO8will affect the expression of thedownstream gene of hucO8, further affect the regulation effect of the circuits on uricacid. This study lays a foundation for further achieving the control of uric acidhomeostasis at cellular level.