"

From 2012.igem.org

---

Contents 1 Methods 1.1 Molecular Biology Methods 1.1.1 plasmid DNA isolation from E.coli (miniprep) 1.1.2 genomic DNA isolation from S.cerevisiae 1.1.3 measurement of DNA concentration 1.1.4 sequencing of plasmid DNA 1.1.5 Agarose gel-electrophoesis 1.1.6 in vitro modification of DNA 1.1.6.1 Site-Directed Mutagenesis 1.1.6.2 polymerase chain reaction (PCR) 1.1.6.2.1 colony PCR 1.1.6.2.2 genomic PCR 1.1.6.3 purification of PCR products 1.1.6.4 Dephosphorylation of DNA 1.1.6.5 DNA restriction enzyme digest 1.1.6.6 ligation / cycled ligation 1.1.6.7 oligohybridization of single-stranded DNA 1.1.6.8 gene synthesis 1.2 Protein biochemical methods 1.2.1 Protein expression in S.cerevisiae 1.2.2 Crude protein extraction from S.cerevisiae 1.2.3 SDS-Polyacrylamid-Gelelektrophoresis (SDS-PAGE) 1.2.4 Western Blot 1.3 Microbiological Methods 1.3.1 cultivation of E.coli 1.3.2 cultivation of S.cerevisiae 1.3.3 heat shock transformation of E.coli with plasmid DNA 1.3.4 transformation of S.cerevisiae 1.3.5 genome integration 1.4 Chemical Methods 1.4.1 Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder 2 Materials 2.1 Bacteria and yeast strains 2.2 used Plasmids 2.3 Reagents 2.4 Buffer and Solutions 2.5 Microbial Media 2.6 References:

Methods

Molecular Biology Methods

plasmid DNA isolation from E.coli (miniprep)

genomic DNA isolation from S.cerevisiae

measurement of DNA concentration

sequencing of plasmid DNA

Agarose gel-electrophoesis

in vitro modification of DNA

Site-Directed Mutagenesis

polymerase chain reaction (PCR)

colony PCR

genomic PCR

purification of PCR products

Dephosphorylation of DNA

DNA restriction enzyme digest

ligation / cycled ligation

oligohybridization of single-stranded DNA

gene synthesis

Protein biochemical methods

Protein expression in S.cerevisiae

Crude protein extraction from S.cerevisiae

SDS-Polyacrylamid-Gelelektrophoresis (SDS-PAGE)

Western Blot

Microbiological Methods

cultivation of E.coli

cultivation of S.cerevisiae

heat shock transformation of E.coli with plasmid DNA

transformation of S.cerevisiae

genome integration

Chemical Methods

Phycocyanobilin (PCB) extraction from dried Spirulina platensis powder

??? • Introducing new Saccharomyces cerevisiae strain (Y190 strain) from Schwab lab for Y2H o Aim of the experiment: The Y190 Saccharomyces cerevisiae is a special strain for Yeast-two-hybrid. This strain carries a Gal4 and Gal80 deletion to higher the signal/noise-ratio of protein-protein interactions. Reporter for protein-protein interactions are HIS3, lacZ and MEL1 and are encoded in the genomic DNA. Transformation markers are trp1, leu2 and cyhR2 and are encoded on the transformation plasmids. • Inoculation of YPD medium with S. cerevisiae and brewing yeast strain 34/70 (part 2/3) o Aim of the experiment: Discrimination of growing ability in wort medium. • Electroporation of electrocompetent E. coli with P698+699

Materials

Bacteria and yeast strains

used Plasmids

Reagents

Buffer and Solutions

Microbial Media

References:

• [http://www.ncbi.nlm.nih.gov/pubmed/20010584 Pubmed: Prasher, 1995: Using GFP to see the light.(Review)]