Comparison of protein quantification and extraction methods suitable for E-coli cultures

Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and sonication-BCA_P combinations

Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli

L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity