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In their experimental work with Pseudomonas putida the EmPowerPutida partners have developed SOPs on genome engineering (e.g. MAGE, error-prone PCR), modification of bacterial growth and quantification of the products of enzymatic reactions as well as molecular biology techniques (e.g. RNA extraction, Northern blotting, PCR).

The SOPs all use a standard template to describe the purpose of the experiment, equipment and bacterial strains, media, procedure and any troubleshooting or biosafety issues.

These SOPs are available at the following links:

Analysis of 1-Decene biotransformation samples by Gas Chromatography

Bradford Protein Assay

Design and clone spacers in pSEVA231-CRISPR

Evaluation of kinase type enzymes via ADP-dependent ATP depletion assay via NADH-dependent enzyme cascade

Expression of recombinant oleate hydratase from Elizabethkingia meningoseptica

Extraction of total RNA from P. putida

Making a growth curve of a bacterial or yeast strain

Making electro-competent E. coli cells and transformation of them

Optimization of reaction conditions using MODDE for statistical experiment design

Oxygen gradients for adaptation of bacteria to varying oxygen availability

Performing a batch and chemostat cultivation with P. putida in labscale

Plasmid isolation from E. coli and P. putida using the Thermo Scientific GeneJET Plasmid Miniprep Kit

Polyacrylamide Northern Blot

Preparation and transformation of electrocompetent bacterial cells

Preparation of gene libraries by error-prone PCR

Restriction / digestion of DNA

Shaking Flask cultures of Pseudomonas putida

ssDNA-based recombineering in Pseudomonas putida

Two-phase based whole cell system for bioconversions

 

 

 

 

 

 

 

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This project has received funding from the European Union’s Horizon 2020

research and innovation programme under grant agreement No 635536.

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