生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
4期
460-466
,共7页
曲国龙%邵妤%谭俊杰%陈章%金晶%凌焱%李玉霞%刘刚%陈惠鹏
麯國龍%邵妤%譚俊傑%陳章%金晶%凌焱%李玉霞%劉剛%陳惠鵬
곡국룡%소여%담준걸%진장%금정%릉염%리옥하%류강%진혜붕
合成生物学%基因回路%尿酸%稳态控制%共表达
閤成生物學%基因迴路%尿痠%穩態控製%共錶達
합성생물학%기인회로%뇨산%은태공제%공표체
synthetic biology%genetic circuit%uric acid%homeostatic control%co-expression
目的：利用合成生物学方法构建尿酸介导的基因回路，在细胞水平上研究回路对尿酸稳态的调控作用。方法：以耐辐射异常球菌R1基因组中转录抑制物基因hucR及其结合位点基因hucO为基础，化学合成具有转录抑制功能的基因mUTs及其结合位点的8串联结构hucO8，构建基因回路；转染HeLa细胞，通过检测分泌型碱性磷酸酶（SEAP）的表达量来验证回路的作用原理和对尿酸的感应作用；在此基础上，用优化的黄曲霉菌尿酸氧化酶（Uox）基因smUox替换SEAP基因，转染HeLa细胞，通过检测转染前后培养基中尿酸浓度的变化，验证回路对尿酸的调节作用。结果：分别构建了优化的转录抑制物表达载体pcDNA3.1/V5-mUTs、报告基因表达载体pSEAP-hucO8、优化的黄曲霉菌Uox表达载体phucO8-smUox、pBudCE4.1-smUox，双向共表达载体pBudCE4.1-SEAP-mUTs、pBudCE4.1-mUTs-smUox；单独转染pBudCE4.1-SEAP-mUTs或共转染pSEAP-hucO8和pcDNA3.1/V5-mUTs，通过检测培养基中SEAP的表达量，证明双载体及单载体回路对尿酸的感应作用；用smUox替换SAEP基因后，通过检测转染48 h后培养基中尿酸含量的变化，证明双载体及单载体基因回路均具有一定的尿酸调节能力。结论：在细胞水平上，构建的双载体基因回路（phucO8-smUox、pcDNA3.1/V5-mUTs）和单载体基因回路（pBudCE4.1-mUTs-smUox）均可实现对尿酸的感应及调控作用，在一定范围内通过增加mUTs与hucO8的摩尔比，可以改变回路对尿酸的调控范围及调节程度。
目的：利用閤成生物學方法構建尿痠介導的基因迴路，在細胞水平上研究迴路對尿痠穩態的調控作用。方法：以耐輻射異常毬菌R1基因組中轉錄抑製物基因hucR及其結閤位點基因hucO為基礎，化學閤成具有轉錄抑製功能的基因mUTs及其結閤位點的8串聯結構hucO8，構建基因迴路；轉染HeLa細胞，通過檢測分泌型堿性燐痠酶（SEAP）的錶達量來驗證迴路的作用原理和對尿痠的感應作用；在此基礎上，用優化的黃麯黴菌尿痠氧化酶（Uox）基因smUox替換SEAP基因，轉染HeLa細胞，通過檢測轉染前後培養基中尿痠濃度的變化，驗證迴路對尿痠的調節作用。結果：分彆構建瞭優化的轉錄抑製物錶達載體pcDNA3.1/V5-mUTs、報告基因錶達載體pSEAP-hucO8、優化的黃麯黴菌Uox錶達載體phucO8-smUox、pBudCE4.1-smUox，雙嚮共錶達載體pBudCE4.1-SEAP-mUTs、pBudCE4.1-mUTs-smUox；單獨轉染pBudCE4.1-SEAP-mUTs或共轉染pSEAP-hucO8和pcDNA3.1/V5-mUTs，通過檢測培養基中SEAP的錶達量，證明雙載體及單載體迴路對尿痠的感應作用；用smUox替換SAEP基因後，通過檢測轉染48 h後培養基中尿痠含量的變化，證明雙載體及單載體基因迴路均具有一定的尿痠調節能力。結論：在細胞水平上，構建的雙載體基因迴路（phucO8-smUox、pcDNA3.1/V5-mUTs）和單載體基因迴路（pBudCE4.1-mUTs-smUox）均可實現對尿痠的感應及調控作用，在一定範圍內通過增加mUTs與hucO8的摩爾比，可以改變迴路對尿痠的調控範圍及調節程度。
목적：이용합성생물학방법구건뇨산개도적기인회로，재세포수평상연구회로대뇨산은태적조공작용。방법：이내복사이상구균R1기인조중전록억제물기인hucR급기결합위점기인hucO위기출，화학합성구유전록억제공능적기인mUTs급기결합위점적8천련결구hucO8，구건기인회로；전염HeLa세포，통과검측분비형감성린산매（SEAP）적표체량래험증회로적작용원리화대뇨산적감응작용；재차기출상，용우화적황곡매균뇨산양화매（Uox）기인smUox체환SEAP기인，전염HeLa세포，통과검측전염전후배양기중뇨산농도적변화，험증회로대뇨산적조절작용。결과：분별구건료우화적전록억제물표체재체pcDNA3.1/V5-mUTs、보고기인표체재체pSEAP-hucO8、우화적황곡매균Uox표체재체phucO8-smUox、pBudCE4.1-smUox，쌍향공표체재체pBudCE4.1-SEAP-mUTs、pBudCE4.1-mUTs-smUox；단독전염pBudCE4.1-SEAP-mUTs혹공전염pSEAP-hucO8화pcDNA3.1/V5-mUTs，통과검측배양기중SEAP적표체량，증명쌍재체급단재체회로대뇨산적감응작용；용smUox체환SAEP기인후，통과검측전염48 h후배양기중뇨산함량적변화，증명쌍재체급단재체기인회로균구유일정적뇨산조절능력。결론：재세포수평상，구건적쌍재체기인회로（phucO8-smUox、pcDNA3.1/V5-mUTs）화단재체기인회로（pBudCE4.1-mUTs-smUox）균가실현대뇨산적감응급조공작용，재일정범위내통과증가mUTs여hucO8적마이비，가이개변회로대뇨산적조공범위급조절정도。
Objective: To study the regulation of uric acid homeostasis at cellular level introduced by uric acid-mediated gene circuit that was constructed with synthetic biology approach. Methods: Based on the transcriptional inhibitor hucR and its binding site hucO in the genome of Deinococcus radiodurans R1, synthesize optimized tran-scriptional inhibitor gene mUTs and its binding site 8-series structure(hucO8) chemically to construct the circuit;transfect HeLa cells, verifying the mechanisms of the circuit and its reaction to uric acid by assaying the expres-sion of secreted alkaline phosphatase(SEAP); based on these, use optimized Aspergillus flavus urate oxidase gene smUox to replace SEAP gene, transfect HeLa cells, and verify the ability of circuit to regulate the uric acid by as-saying the uric acid concentration change in the culture medium before and after the transfection. Results: The transcriptional inhibitor expression vector pcDNA3.1/V5-mUTs, reporter gene expression vector pSEAP-hucO8, smUox expression vector phucO8-smUox, pBudCE4.1-smUox, the co-direction co-expression vector pBudCE4.1-SEAP-mUTs, pBudCE4.1-mUTs-smUox were constructed; the single transfection with pBudCE4.1-SEAP-mUTs or the co-transfection with pSEAP-hucO8 and pcDNA3.1/V5-mUTs, by assaying SEAP expression level in the culture medium, verifies the impact of the double and single vector circuit to uric acid; replacing SAEP gene with smUox,the ability of double and single vector circuits to mediate uric acid is demonstrated by assaying the concentration change of uric acid concentration in the medium within 48 hours. Conclusion: At the cellular level, the construct-ed double vector circuit(phucO8-smUox、pcDNA3.1/V5-mUTs) and the single vector circuit(pBudCE4.1-mUTs-smUox) could both sense and regulate the urid acid. By increasing the mole ration between mUTs and hucO8 in a certain extent, the level and the extent in which the circuit regulates the uric acid could be changed.
