Investigations
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Handling and manipulation of S. pneumoniae using molecular, cell biological and genetic tools.
Submitter: Jan-Willem Veening
Studies: Automated time-lapse microscopy, Chromosome segregation in S. pneumoniae, Investigation of bacterial transcription fidelity and processivity, The role of transcription factor GreA in transcription fidelity and proc...
Assays: Kinetics of misincorporation and proofreading by bacterial RNA polymerase, Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae usin..., ParB-GFP ChIP on chip, RNA-Seq
An experimental workflow to provide detailed information of the molecular mechanisms of enzymes is described. This workflow will help in the application of enzymes in technical processes by providing crucial parameters needed to plan, model and implement biocatalytic processes more efficiently. These parameters are homogeneity of the enzyme sample (HES), kinetic and thermodynamic parameters of enzyme kinetics and binding of reactants to enzymes. The techniques used to measure these properties are ...
Submitter: Gudrun Gygli
Studies: DLS measurements (homogeneity of an enzyme sample), ITC binding experiments, ITC kinetic experiments (enzyme activity), Spectrophotometric Activity Measurements
Assays: Analysis of data from ITC experiments (binding), Analysis of data from ITC experiments (kinetics), Binding of HK to Gre2p (ITC-BIND), Binding of NADP+ to Gre2p in HEPES Buffer (ITC-BIND), Binding of NADP+ to Gre2p in KPi Buffer (ITC-BIND), Binding of NADP+ to Gre2p in PBS Buffer (ITC-BIND), Binding of NADPH to Gre2p in HEPES Buffer (ITC-BIND), Binding of NADPH to Gre2p in KPi Buffer (ITC-BIND), Binding of NADPH to Gre2p in PBS Buffer (ITC-BIND), Binding of NADPH to Gre2p in Tween-KPi Buffer (ITC-BIND), Binding of NDK to Gre2p (ITC-BIND), DLS measurements in 2 buffers, DLS measurements in KPi Buffer and in KPi buffer with Tween added, DLS measurements in KPi buffer with BSA added, Kinetic parameters of Gre2p, Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-MIM) in HEPES ..., Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-MIM) in KPi bu..., Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-MIM) in PBS bu..., Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-MIM) in Tween-..., Kinetics of the reaction of NDK and NADPH with Gre2p (ITC-rSIM) in 3 buf..., Selwyn test of Gre2p, Specific activity of Gre2p
The objectives of WP1 within the colosys project are:
- Produce resources of data and information that will be used for the identification of CC driver genes (WP2) and for the construction and configuration of computational models of cancer-related biological processes (WP2 and WP3)
- Integrate information from public patient data and cancer cell line data repositories with information from project specific clinical and model data
- Convert information from public cellular signalling databases ...
Objectives: Protocol identification and establishment for the synthesis of high-performing catalytic GO in the desired oxidation reactions under mild conditions. Characterization of the most prominent materials synthesized and comparison to commercially available material.
Description of Work: Several established chemical methods will be used for the synthesis of GO. Each batch will be characterized, for instance for its C/O ratio, surface area and conductivity. The catalytic profile of the ...
Objectives: Identification of amine transaminases (ATAs) able to catalyze efficiently the amination of a desired set of ketones and aldehydes (expected products of GO oxidation). Optimization of the most potent biocatalysts, in terms of catalytic efficiency, stability, availability of functional groups for covalent immobilization.
Description of Work: ATAs from our construct selection (>30 wild-type and variants of both (R)- and (S)-enantioselectivity) with different substrate selectivity will ...
Objectives: Development of efficient ATA immobilization approach. Production of a hybrid catalyst of high catalytic efficiency.
Description of Work: Τhe ATAs selected in WP2 will be expressed, purified and covalently and/or non-covalently immobilized on the GO selected in WP1. The catalytic behavior (in terms of catalytic activity, stability and reusability) and the structural implications of the immobilization will be investigated. For comparison purposes, the non-optimized ATAs (prior to the ...
Objectives: Establishment of the chemoenzymatic process with the best GO-ATA hybrid catalysts. Highlighting of the potential of the process in semi-preparative scale.
Description of Work: The best hybrid catalysts identified in WP3 will be investigated in coupled one-pot reactions selected in WP1, in batch and continuous flow reactors. The productivity of the system will be optimized with response surface methodology (RSM), for parameters such as temperature, duration, substrate concentration ...
Objectives: Empowering smooth implementation and fruitful completion of all WPs and tasks. Implementation of a data management plan for efficient dissemination under F.A.I.R. principles.
Description of Work: The PI of the project with the heads of the collaborating groups will closely monitor the progress of the technical and administrative tasks and it will implement actions to correct any deviation from the established work-plan. The whole group will meet regularly every six months or more ...
