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Cell Specificity and Transcriptional Regulation Study of a Dps Protein In Cell specificity and transcriptional regulation study of a Dps protein in Nostoc punctiforme ATCC 29133 Xin Li, B.S. Degree project in applied biotechnology, Master of Science (two years), 2012 Examensarbete i tillämpad bioteknologi 30 hp till masterexamen, 2012 Dept of Chemistry - Ångström Laboratory, Photochemistry and Molecular Science, Uppsala University Supervisor: Karin Stensjö. 1 Table of Contents Abstract ..................................................................................................................................... 1 1. Introduction ........................................................................................................................... 2 1.1 Background ..................................................................................................................... 2 1.1.1 Cyanobacteria ........................................................................................................... 2 1.1.2 Nostoc punctiforme ATCC 29133 and Anbaena sp. PCC7210 ................................ 3 1.1.3 Nitrogen metabolism in Cyanobacteria .................................................................... 3 1.1.4 Heterocyst differentiation ......................................................................................... 3 1.1.5 Dps-proteins ................................................................................................................. 5 1.1.6 Homologue ferritin domains in Nostoc and Anabaena ............................................. 6 1.2 Aim and strategy of the project ....................................................................................... 6 2. Materials and methods ........................................................................................................... 8 2.1 Chemicals and reagents ................................................................................................... 8 2.2 5’RACE for detecting Transcription start site (TSS) ...................................................... 8 2.2.1 Cyanobacterial strains and growth conditions .............................................................. 8 2.2.2 Heterocysts preparation ................................................................................................ 8 2.2.3 RNA extraction for cyanobacteria ................................................................................ 9 2.2.4 RNA quality check ....................................................................................................... 9 2.2.5 Invitrogen 5’ RACE ................................................................................................... 10 2.3 PCR amplification ......................................................................................................... 11 2.4 Gel electrophoresis ........................................................................................................ 12 2.5 DNA Purification .......................................................................................................... 12 2.6 Plasmid preparation ....................................................................................................... 12 2.7 Digestion and ligation.................................................................................................... 13 2.7.1 Fast Digestion ............................................................................................................. 13 2.7.2 Quick ligation ............................................................................................................. 14 2.8 Transformation .............................................................................................................. 14 2.8.1 Competent cell preparation ..................................................................................... 14 2.8.2 Transformation ....................................................................................................... 14 2.9 Confirmation of constructs ............................................................................................ 15 2 2.9.1 Colony PCR ............................................................................................................ 15 2.9.2 Restriction test ........................................................................................................ 15 2.9.3 Sequencing ............................................................................................................. 16 2.10 Electroporation into cyanobacterial cells .................................................................... 16 2.10.1 Cell preparation for electroporation ......................................................................... 16 2.10.2 Electroptation into Nostoc punctiforme 29133 and Anabaena sp. PCC 7120 .......... 16 2.11 Nitrogen step down...................................................................................................... 17 2.12 Confocal microscopy for determination of GFP presence and location ...................... 17 3. Results ................................................................................................................................. 19 3.1 Homology investigation of the regulatory sites in the promoters .................................. 19 3.2 Identification of TSS by 5’RACE results ...................................................................... 20 3.3 Promoter GFP reporter constructs cloning results ......................................................... 22 3.4 Introduction of shuttle vector to cyanobacteria ............................................................. 26 3.5 Confocal Microscopy study results ............................................................................... 27 4. Discussion ........................................................................................................................... 32 4.1 Different regulation of gene expression of the two homologous .................................. 32 4.2 Constructs with different Dps promoter truncates ......................................................... 32 4.3 Cell localization of the activity of the different truncated promoters ............................ 33 4.4 One step forward base on the previous study ................................................................ 33 4.5 Conclusion ..................................................................................................................... 35 5. Acknowledgement ............................................................................................................... 36 6. References ........................................................................................................................... 37 7. Appendix ............................................................................................................................. 40 7.1 Chemicals and reagents used for this experiment ......................................................... 40 7.2 Recipes of all the medium and buffers used in this study ............................................. 41 7.3 Primers used in 5’ RACE study..................................................................................... 41 3 Abstract DNA-binding proteins from starved cells (Dps) in cyanobacteria perform efficient response to oxidative and nutritional stresses. In order to discover regulatory mechanisms of Dps protein in Nostoc punctiforme ATCC 29133, I focused on the promoter region in Npun_R5799, which is one of the five ferritin-like protein coding genes found in Nostoc punctiforme ATCC 29133 and homologous to alr3808 in Anabaena sp. PCC 7120.A recent differential RNA-sequencing study identified two transcription start sites (TSS) in the promoter region of Anabaena sp. PCC 7120 alr3808, with different properties. One of the TSS appeared to be heterocyst differentiation dependent and could be induced by nitrogen deprivation. In this study I have used 5’RACE to locate the TSS in the Nostoc punctiforme ATCC 29133 Npun_R5799, and promoter truncation-GFP constructs were made to investigate the in vivo regulation of the promoter. The 5’RACE study of N.pun_R5799 Dps promoter identified one TSS located at 44 nt upstream of Dps coding gene, which is just the same location as in alr3808. However the second TSS of alr3808 was not identified in Npun_R5799 upstream region. Six constructs were made which different promoter truncations have been fused to green fluorescent protein (GFP), both on the Nostoc punctiforme ATCC 29133 Npun_R5799 promoters and the homologues region in Anabaena sp. PCC 7120 alr3808, with a GFP reporter on shuttle vector pSUN119. The constructs were transformed into cyanobacteria Nostoc punctiforme via electroporation, and the promoter activity and cell specificity were determined in vivo using Confocal Microscopy. The results indicated that there could be a complex regulatory function within the Anabaena sp. PCC 7120 alr3808 promoter, and the TSS1 region correlated to the initiation of heterocyst differentiation. However, even though the Npun_R5799 and alr3808 are close homologues, our results show that the regulation of the gene expression is different, indicating a different function in Nostoc punctiforme ATCC 29133. 1 1. Introduction The rapid economic and industrial development results in an increase of energy demands. In order to solve the threatening issue of environmental pollution and energy crisis, searching for renewable clean energy approaches seems imminent for human beings in our generation. Hydrogen is a potentially
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