Team:TU-Munich/Notebook/Methods

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Molecular-biological Methods

Preparation of Nucleotide fragments

Digestion, Dephosphorylation

Hybridization of Oligonucleotides

...using complementary oligo-nucleotides that contained the desired sequence with specific overhangs for cloning. For oligohybridization, 25 ml of 100 mM of forward and reverse oligos were put together in one tube and heated to 90 °C for 5 min. The samples were slowly cooled to room temperature in a styrofoam box overnight.

Polymerase Chain Reaction

Polymerase chain reaction (PCR) was used for the selective amplification of desired DNA fragments (for example from a plasmid). Primers were designed for the desired target sequences. The PCR reaction was divided in to three steps which were repeated up to 30 times. Firstly, the DNA template strand was heat-denatured at 95 °C to produce single-stranded DNA. Secondly, the temperature of the reaction batch was lowered to 55 – 60 °C to allow the primers to bind. Thirdly, the temperature was raised to 72 °C. This enabled the DNA polymerase to synthesize the other DNA strand. Special PCR methods that were used include colony and genomic PCR.

Site-directed Mutagenesis

Site-Directed Mutagenesis was used to mutate specific bases of DNA sequences. Therefore, specific primers, which bind at the same site and contain a mismatch at the specific base, were required. The original base pair that had to be replaced was replaced by the mismatch. The method works just as PCR by amplifying the desired product that contains the mismatch. Afterwards, the product was digested with the restriction enzyme DpnI to destroy the plasmids strands which do not contain the desired base pair exchange. The QuikChange Site-Directed Mutagenesis Kit by Agilent Technologies was used.

Agarose Gel Electrophoresis

Agarose gel-electrophoresis was used to separate double-stranded DNA fragments by length. Ethidium bromide was applied as a nucleic acid stain (Sambrook et al., 1989). This method was used for the restriction analysis of plasmids (analytical gel-electrophoresis) as well as for the isolation of DNA fragments (preparative gel-electrophoresis). After preparative gel-electrophoresis, the bands were cut out and purified using a Qiagen Gel extraction kit.

TAE-Buffer

Reagent Amount

Ligation of DNA-Fragments

After digestion with an restriction enzyme, plasmid fragments were inserted into vectors (which were cut with matching restriction enzymes) by ligation. The enzyme T4 ligase connected complementary overhangs of fragments by catalyzing the formation of the bond between the 5'phosphoryl group and the 3' hydroxyl group.

Microbiological Methods

Preparation of LB-Media

LB-Media

Reagent Amount

Transformation of E. coli

Cultivation of E. coli

Protein-biochemical Methods

Protein production in E. coli

Cell disruption

Streptavidin-Affinity chromatography (SAC)

Konzentration of proteins using a centrifugal filter unit

Size-exclusion chromatography (SEC)

Physcomitrella Methods

The techniques used for the cultivation and manipulation of Physcomitrella patens are based on the knowledge of the chair for Plantbiotechnology of Prof. Dr. Reski at Freiburg University which are availible at [http://www.plant-biotech.net Plant-Biotech.net].

Preparation of Knob-Media

Knob-Media

Reagent Amount

Cultivation of P. patens

Homogenization

Bioinformatical Methods

Generation of Annimated GIFs

  • A homology search was performed for the amino acid sequece using [http://toolkit.tuebingen.mpg.de/hhpred HHpred]
  • Entry for the best matching structure(> 50%)in PDB-database was opened
  • PDB-file was was saved
  • The structure was opened with Pymol
  • The approach was adapted from a PyMol-Tutorial http://ihome.cuhk.edu.hk/~b102142/pymol/pymol_tutorial.html Wong
  • The following commands were entered into the command line:

PyMOL> orient
PyMOL> hide everything, all
PyMOL> show cartoon, all
PyMol> bg_color white
click> Settings>Rendering>Shadows>Occlusion
PyMOL> color purple, ss h; color yellow, ss s; color green, ss ""
PyMOL> show surface, all
PyMOL> set transparency, 0.75
PyMOL> set surface_quality, 1 (command will prolongue the process significantly, you may skip it)
PyMOL> set ray_trace_frames=1 (command will prolongue the process significantly, you may skip it)
PyMOL> mset 1 x180
PyMOL> util.mroll 1,180
PyMOL> mpng frame

  • Afterward the *.png files were automatically converted to *.tif files using xxx
  • The program xxx was used to generate the annimated GIFs
    • A new working folder was generated and the *.gif files were imported
    • The background was set to white
    • The range of colors was set to 256
    • Finally the annimated Gif was computed

The result is the following:

File:TUM13 Annimated Gif test.gif
Description

References:

http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984

  1. http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984 Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. Cell, 37(2):629–33.
  2. http://ihome.cuhk.edu.hk/~b102142/pymol/pymol_tutorial.html Wong A tutorial on using PyMOL to generate publication quality figures. http://ihome.cuhk.edu.hk/~b102142/pymol/pymol_tutorial.html]]


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