Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies

• 1 Summary

• 2 Introduction

  • 2.1 Properties of phototropin

  • 2.2 Phototropin in Chlamydomonas reinhardtii

  • 2.3  Chlamydomonas reinhardtii as host for recombinant protein expression

  • 2.4 RNA interference and gene silencing

  • 2.5 The goal of C. reinhardtii mating assay

• 3 Results

  • 3.1 Localization and expression profile of Phototropin

    • 3.1.1 Distribution and localization of phototropin

    • 3.1.2 Distributions of phototropin in different growth stages

    • 3.1.3 The degradation of phototropin is induced by light.

  • 3.2 Reduction of the phototropin level in C. reinhardtii by RNAi

    • 3.2.1 The Phot1- RNAi construct

    • 3.2.2 Transformation of C. reinhardtii strain cw15 arg- A with the RNAi construct

    • 3.2.3 Transformation of C. reinhardtii strain CC32pab1mt(+) with phototropin RNAi construct

  • 3.3 Mating Assay

    • 3.3.1 The design of mating assay

    • 3.3.2 Optimization of growth conditions for precultures before gametogenesis

    • 3.3.3 Mating assay results

  • 3.4 Expression and purification of phototropin in different expression systems

    • 3.4.1 Expression of Phototropin in Oocytes

    • 3.4.2 Fusion expression of Ble-Phot1 in C. reinhardtii

    • 3.4.3 Analysis of pLY2-A transformant No.21

      • 3.4.3.1 Distribution of the expressed product and the endogenous phototropin

      • 3.4.3.2 Light induced degradation of the fusion protein

      • 3.4.3.3 Purification of the fusion protein

    • 3.4.4 Expression of phototropin in diatom

    • 3.4.5 Establishment of a tandem affinity purification system in C. reinhardtii

      • 3.4.5.1 Use of a directly combined Strep tag II at the N-terminus of the fusion products

      • 3.4.5.2 Final construct for tandem affinity purification and purification of fusion expression product

• 4 Discussion

  • 4.1 Locaization and distribution of phototropin in Chlamydomonas reinhardtii

  • 4.2 Silencing of phototropin in C.reinhardtii

  • 4.3 Mating assay

  • 4.4 Expression and purification of phototropin

• 5 Materials and methods

  • 5.1 Methods about handling with C. reinhardtii (buffer recipes included)

    • 5.1.1 Growing C. reinhardtii in liquid culture

    • 5.1.2 Growing Chlamydomonas reinhardtii on Agar

    • 5.1.3 Gametogenesis

    • 5.1.4 Isolation of Flagella (pH Shock Protocol)

    • 5.1.5 Flagella fractionation

    • 5.1.6 Preparation of Gamete Autolysin

    • 5.1.7 Transformation of Chlamydomonas

      • 5.1.7.1 Transformation of cell-wall-deficient strain (Glass bead method)

      • 5.1.7.2 Transformation of wild type cell

    • 5.1.8 Mating Assay

    • 5.1.9 Fractionation of Chlamydomonas cells

  • 5.2 Methods about handling with Diatom Cylindrotheca fusiformis (buffer recipes included)

    • 5.2.1 Growing C. fusiformis

    • 5.2.2 Transformation of diatom cells

      • 5.2.2.1 Adsorption of plasmid-DNA on tungsten particles (M17)

      • 5.2.2.2 Preparation of diatom cells

      • 5.2.2.3 Shooting of cells

      • 5.2.2.4 Selection of resistant C. fusiformis Cells

  • 5.3 Methods about handling with Escherichia coli(buffer recipes included)

    • 5.3.1 Growing E. coli

    • 5.3.2 Making competent cells of E. coli

    • 5.3.3 Transformation of E. coli cells

    • 5.3.4 Storing and retrieving E. coli in frozen at -80°C

      • 5.3.4.1 Preparing frozen stocks of E. coli

      • 5.3.4.2 Retrieving E. coli from -80°C frozen stocks

  • 5.4 Methods about handling with X. laevis oocyte (buffer recipes included)

    • 5.4.1 Microinjection of X. laevis oocyte and detection of target protein expression

  • 5.5 Methods about handling with DNA (buffer recipes included)

    • 5.5.1 Isolation of genomic DNA from C. reinhardtii

    • 5.5.2 Isolation plasmid-DNA from E. coli

    • 5.5.3 Agarose gel electrophoresis of DNA

    • 5.5.4 Purification of DNA from agarose gel

    • 5.5.5 Amplification of DNA fragment by Polymerase Chain Reaction(PCR)

    • 5.5.6 Digestion of DNA

    • 5.5.7 Ligation of DNA

    • 5.5.8  in vitro synthesis of mRNA from DNA

  • 5.6 Method about handling with Protein (buffer recipes included)

    • 5.6.1 Protein amount detection by BC Assay

    • 5.6.2 SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis)

    • 5.6.3 Staining of SDS-PAG

      • 5.6.3.1 Commassie Brilliant Blue staining

      • 5.6.3.2 Silver staining

    • 5.6.4 Western blot

    • 5.6.5 In gel tryptic digestion

    • 5.6.6 Purification with Ni-NTA column

    • 5.6.7 Purification with Strep-tactin column

  • 5.7 Materials

    • 5.7.1 Strains

    • 5.7.2 Plasmids

    • 5.7.3 Chemicals, Enzymes and Kits

      • 5.7.3.1 Chemicals and Kits

      • 5.7.3.2 Enzymes and Proteins

    • 5.7.4 Oligonucleotides

      • 5.7.4.1 Primers for making RNAi construct

      • 5.7.4.2 Primers for oocyte expressions

      • 5.7.4.3 Primers used for pLY2-phot1

      • 5.7.4.4 Primers for diatom expressions

      • 5.7.4.5 Primers for pLY2-A

      • 5.7.4.6 Primers for pLY2-B

      • 5.7.4.7 Primers for pLY2-C

      • 5.7.4.8 Primers for pLY2-D, pLY2-E

      • 5.7.4.9 Primers for Chapter 3.4.5.1

      • 5.7.4.10 Primers for pLY2-OV

      • 5.7.4.11 Sequencing primers

• Acknowledgments

• Abbreviations

• References

• Lebenslauf

• Erklärung

• Form 3.4.3.1.1 Distribution of phototropin and the fusion expression product.

• Figure 2.1.1 Domain features of plant phototropin

• Figure 2.1.2 Photocycle of LOV1 domain from C. reinhardtii phototropin (Kottke et al 2003)

• Figure 2.2.1 Amino acid sequence of Chlamydomonas phototropin gene (Huang et al., 2002)

• Figure 2.2.2 Life cycle of Chlamydomonas reinhardtii (Huang et al., 2003)

• Figure 2.4.1 The RNAi pathway. (Dykxhoorn et al 2003)

• Figure 2.4.2 Self-amplifying process of RNAi and transitive RNA siliencing (Ahlquist, 2002).

• Figure 2.4.3 Methods to generate RNAs that silence gene expression (Dykxhoorn et al., 2003).

• Figure 3.1.1.1 Localization of phototropin

• Figure 3.1.1.2 Phototropin level and distribution in different strains grown under same conditions

• Figure 3.1.2.1 Phototropin distributions in pellet, membrane and soluable fraction in different cell stages and in different light conditions.

• Figure 3.1.3.1 Light induced degradation of phototropin.

• Figure 3.2.1.1 Construction of RNAi construct of Chlamydomonas Phototropin gene

• Figure 3.2.2.1 Relative amount of phototropin in the transformants and recipient strain cw15 arg- A

• Figure 3.2.3.1 Relative phototropin amount in transformant, recipient and control strain.

• Figure 3.3.1.1 Description of mating assay

• Figure 3.3.2.1 Difference in phototropin levels in transformant C4 and recipient strain CC32pab1mt(+) under different growth conditions

• Figure 3.3.3.1 Result of mating assay

• Figure 3.4.1.1 Schematic drawing of the two plasmids used for expression of Phot1 and Phot1 (C57S, C250S) in Xenopus laevis oocytes.

• Figure 3.4.1.2 Expression of Phot1 and Phot1 mutant (C57S, C250S) in Xenopus Laevis oocytes.

• Figure 3.4.1.3 Distribution and solubilization of Phot1 expressed in oocyte and light induced degradation experiment.

• Figure 3.4.2.1 Different constructs tested for expressing Phototropin in C. reinhardtii

• Figure 3.4.2.2 Overexpression of fusion protein Ble-TEV-Phot1-12 His in C. reinhardtii

• Figure 3.4.2.3 Western blot of pLY2-A transformants.

• Figure 3.4.2.4 Western blot result from screening of pLY2-B transformants

• Figure 3.4.2.5 Western blot result from screening of pLY2-C transformants

• Figure 3.4.3.1.1 Distribution of fusion expression product and phototropin

• Figure 3.4.3.2.1 Light gradient test for overexpression product and phototropin

• Figure 3.4.3.3.1 Ni-NTA spin column purification of fusion product.

• Figure 3.4.3.3.2 Mass Spectroscopy analysis of the expressed product

• Figure 3.4.4.1 Plasmid construct for fusion expression of Ble-Phot1in Diatom

• Figure 3.4.4.2 Screening of fusion expression transformants and localization of the expressed product in diatom.

• Figure 3.4.5.1.1 Schematic drawing and restriction sites of parts of the five plasmids used for expressing Ble fusion protein.

• Figure 3.4.5.1.2 Comparison of transformation efficiency and products of the different fusion expression constructs with or without Strep tag II in the N-terminus.

• Figure 3.4.5.1.3 Western blot of screening transformants with pLY2-SBP

• Figure 3.4.5.2.1 Final construct for fusion expression of Ble and Phototropin for tandem affinity purification.

• Figure 3.4.5.2.2 Screening of transformants with pLY2-OV

• Figure 3.4.5.2.3 Purification of expressed fusion product (Fb) from strain No. 9.

• Figure 3.4.5.2.4 Purification of full-length fusion product from transformant No.24

• Figure 3.4.5.2.5 MS result: Sequence coverage of transformant No.9. The high-lighted sequences were found in MS analysis.

• Figure 3.4.5.2.6 MS results: Sequence coverage of transformant No.24. The high-lighted sequences were found by MS analysis.

• Figure 4.1.1 Phosphorylation prediction of C. r. phototropin (NetPhos 2.0 Server).

• Figure 4.4.1 Schematic illustration of the roles of LOV1 and LOV2 domains in light regulation of substrate phosphorylation by Kinase domain in A.t. Phot2. P: phosphate; S, substrate; LL, low light conditions; HH, high light conditions ( Mastuoka and Tokutomi, 2005).