Team:UPF CRG Barcelona/Methods
METHODS AND PROTOCOLS
Methods
Cupric Acetate Fatty Acid Quantification
Quantifying fatty acids in the medium is a key part of our project. In order to prove the functionality of our genetic constructs, we needed a way to demonstrate that our system had an increased fatty acid uptake over time. To assess this, indirectly measured the fatty acid concentration that remained in the medium after during the assay.
Different systems have been previously used to quantify long chain fatty acid in the medium. Including chemical or thermodynamic titration as well as other enzymatic methods, such as the introduction of Acyl-CoA synthetase that leads to NADH consumption and it can be detected by fluorimetry. Other options such as HPLC or GC/MS are also widely used in the scientific community. However due to funding limitations, it was not doable to use them in our project.
Taking into account a variety of possibilities, we finally chose a method based in the measurement of metal-fatty acid complexes: the cupric acetate-pyridine method. It is a colorimetric technique based in a two-phase system and the following binding of free fatty acids to copper forming copper soaps. These copper soaps can be detected and quantified with spectrophotometric techniques (OD 715 nm). This method constitutes a simple and rapid method for determination of free fatty acids and it has proof good sensitivity and reproducibility for caproic, caprylic, capric, lauric, myristic, palmitic, stearic and oleic acids [1]. Hence, measuring the OD 715nm for a standard curve and for the different concentrations of the fatty acid have been developed, the OD can be directly correlated to the concentration of free fatty acid.
In order to obtain the optimal medium to work with, an assay was performed to determine a standard curve with different palmitic acid and oleic acid concentrations and working with media with an emulsion of the corresponding fatty acid achieved by two methods: (a) diluting using bovine serum albumin (BSA) method or (b) diluting by using Tergitol NP-40, a detergent. The following were the used media for the assay:
- PA diluted with BSA.
- PA diluted with tergitol NP-40.
- Oleic acid diluted with BSA.
- Oleic acid diluted with tergitol NP-40.
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Protocols
Protocol for Extracting parts from iGEM collection
Protocol for Promega Restriction Enzymes kit
Protocol for Transformation
Procotol for Clonning
Protocol for DNA extraction
Protocol for Verifying Digestion
Procotol for PCR
Protocol for SDS-PAGE
Protocol for Protein Quantification
Protocol for Colony PCR
Protocol for Cupric Acetate Fatty Acid Quantification
Protocol for Multiplex Automated Genomic Engineering
Protocol for Growth Assays
Protocol for Mediums