SOPs

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.

EmPowerPutida is collaborating with IBISBA sharing ideas on key elements to include in experimental SOPs to help inform how SOPs can be improved.

The final SOPs are accessible in the categories (Molecular Biology, Microbiology, Cultivation and Production, Analytics) through the following links: 

Molecular Biology

• Cloning
  • Golden gate cloning
  • Design and clone spacers in pSEVA231-CRISPR
  • Design of spacers and cloning of them in pSEVA231-CRISPR
  • Expression of recombinant oleate hydratase from Elizabethkingia meningoseptica
  • Identification of target alcohol dehydrogenases enzymes (ADHs) involved in small alcohols and terpene alcohols catabolism.
  • Marker-less multiple gene deletions in P. putida KT2440
  • Multiplex automated genome engineering (MAGE) for Pseudomonas putida
  • Plasmid isolation from E. coli and P. putida using the Thermo Scientific GeneJET Plasmid Miniprep Kit
  • ssDNA-based recombineering in Pseudomonas putida
  • CRISPR/Cas9-enhanced ssDNA recombineering for Pseudomonas putida
• PCR
  • MASC-PCR
  • Colony PCR of S. cerevisiae, P. putida, E. coli
  • Preparation of gene libraries by error-prone PCR
• Protein assay
  • Acetate kinase protein activity assay
  • Bradford Protein Assay
  • Enzymatic acetyl-CoA determination
  • Evaluation of kinase type enzymes via ADP-dependent ATP depletion assay via NADH-dependent enzyme cascade
• Transformation
  • Making electro-competent E. coli cells and transformation of them
  • Mix & Go competent cells and transformation of E. coli
  • Preparation and transformation of electrocompetent bacterial cells
  • Preparation of P. putida electrocompetent cells and transformation
  • Selection of suitable Pseudomonas putida genetic backgrounds for small alcohols biotransformations
• Nucleic acids
  • Agarose gel electrophoresis of DNA fragments
  • cDNA synthesis
  • DNA and RNA concentration measurement
  • Extraction of total RNA from P. putida
  • Genomic DNA isolation
  • Polyacrylamide Northern Blot
  • Restriction / digestion of DNA

Cultivation and Production

• Bacterial cultivation
  • Cultivation of P. putida (and E. coli) in rich-, minimal-, and selective medium
  • Inoculating and sampling anaerobic cultures and CFU determination
  • Making a growth curve of a bacterial or yeast strain
  • 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
  • Sampling for nucleotide analysis from chemostat cultivation with P.putida
  • Shaking Flask cultures of Pseudomonas putida

Microbiology

• Bacterial staining
  • Gram staining of bacteria
  • Determination of cell dry weight
  • Live/dead staining of bacteria
• Electroporation
  • Cleaning of electroporation cuvettes

Analytics

• Gas chromatography
  • Analysis of alcohols in culture supernatant by gas chromatography
  • Analysis of 1-Decene biotransformation samples by Gas Chromatography
  • Downstream processing and analysis of samples of degradation experiments 1:short chain alcohols
  • Downstream processing and analysis of samples of degradation experiments 2:terpenoids
  • Two-phase based whole cell system for bioconversions
• RNA-Seq analysis
  • Differential RNA-Seq data analysis