1. The glycerol kinase gene (glpK) of M. pneumoniae contains 10 TGA codons Idea: changing simultaneously all the TGA codons into TGG codons (TGG = tryptophan in E. coli) to prevent premature translation termination in E. coli The Glycerol Kinase of Mycoplasma pneumoniae

2. First step: amplification of glpK from chromosomal DNA of M. pneumoniae External reverse primer changed the TGA at the last position into a TGG during the PCR Primer fwPrimer rev MMR: Multiple Mutation Reaction

3. Difference between external and mutation primer: External primer ( ): much lower melting temperature (about 4°C) no phosphorylation Mutation primer ( ): have a slightly increasing melting temperature towards the downstream positioned primer 5´phosphorylated PPPPPPPPP P MMR: Multiple Mutation Reaction

4. Out of 5 independent MMRs, 5 different MMR products were sequenced: 3 contained all 9 desired mutations 1 contained 7 of 9 desired mutations 1 contained 9 desired mutations and 1 additional mutation Evaluation of the MMR

5. overexpression purification Purification of Glycerol Kinase