Synthetic Control of Transcription: From Hybrid Promoters to Promoter Engineering to Synthetic ... PDF
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Engineering Conferences International ECI Digital Archives Metabolic Engineering IX Proceedings Summer 6-6-2012 Synthetic Control of Transcription: From Hybrid Promoters to Promoter Engineering to Synthetic Operon Design Hal Alper The University of Texas at Austin Follow this and additional works at:http://dc.engconfintl.org/metabolic_ix Part of theBiomedical Engineering and Bioengineering Commons Recommended Citation Hal Alper, "Synthetic Control of Transcription: From Hybrid Promoters to Promoter Engineering to Synthetic Operon Design" in "Metabolic Engineering IX", E. Heinzle, Saarland Univ.; P. Soucaille, INSA; G. Whited, Danisco Eds, ECI Symposium Series, (2013). http://dc.engconfintl.org/metabolic_ix/15 This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Metabolic Engineering IX by an authorized administrator of ECI Digital Archives. For more information, please [email protected]. Synthetic control of transcription : From hybrid promoters to promoter engineering to synthetic operon design Hal Alper Department of Chemical Engineering The University of Texas at Austin h1 [email protected] September 7, 2005 http://www.che.utexas.edu/alper_group/ Metabolic Engineering has opened up possibilities… • Metabolic engineering advances have expanded the breath of chemicals pprroodduucceedd bbyy cceellllss – Pharmaceuticals and Nutraceuticals – Fuels – Commodity/Specialty Chemicals – Polymers and Precursors Curran & Alper, Metabolic Engineering, 2012 Laboratory for Cellular June 6, 2012 Hal Alper and Metabolic Engineering Slide # 2 …but, requires synthetic control elements... Pathway Control Circuit Design A B C D Heterologous Expression Probing via Graded Expression HAT A promoter HAT* promoter B D promoter C promoter Laboratory for Cellular June 6, 2012 Hal Alper and Metabolic Engineering Slide # 3 …with a range of expression capacities. Native range of mRNA levels promoter Require a promoter series(s) that span above the highest promoter native expression down to essentially zero expression: promoter Affords both amplification and knockdowns. promoter Holstege, et al. (1998), Cell 95:717-728. Laboratory for Cellular June 6, 2012 Hal Alper and Metabolic Engineering Slide # 4 Synthetic control of transcription occurs at many levels Synthetic 5’UTR and MCSs Transcriptional Genetic Context Amplifiers EElleemmeennttss tthhaatt Influence Terminators and Expression 3’UTR Mutant Promoters Selection Markers and Vectors Laboratory for Cellular June 6, 2012 Hal Alper and Metabolic Engineering Slide # 5 Designing novel genetic control elements • Promoter Engineering is an effective method for generating a collection of genetic control elements Prior Promoter engineering example + GFP Range of promoter Promoter Promoter strength to control gene Library expression Alper et al. PNAS, 2005. Laboratory for Cellular June 6, 2012 Hal Alper and Metabolic Engineering Slide # 6 Development of diversified promoters Mutant T CYC1 Promoter yECitrine Nevoigt et al. AEM, 2006. Laboratory for Cellular June 6, 2012 Hal Alper and Metabolic Engineering Slide # 7 Development of novel low-strength promoters Mutant T CYC1 Promoter yECitrine 4 3.5 11 CC FF TT 3 oo tt ee 22..55 vv ii tt aa elel 2 RR ee cc 1.5 nn ee cc ss 1 ee rr oo uu 0.5 ll FF 0 Promoter Laboratory for Cellular June 6, 2012 Hal Alper and Metabolic Engineering Slide # 8 An expanding range of yeast promoters Native range of mRNA levels Holstege, et al. (1998), Cell 95:717-728. Laboratory for Cellular June 6, 2012 Hal Alper and Metabolic Engineering Slide # 9
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