IGEM:IMPERIAL/2009/M1/Assays

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M1 Assays

Cellulase

Activity of most cellulases is monitored by the fluorescent substrate, Resorufin Cellobioside, contained in the kit. Upon cleavage, the fluorescent compound, Resorufin, is released and fluorescent activity measurements can be taken. The Fluorescent Cellulase Assay Kit allows fast and easy detection of most cellulases in a microtiter plate based assay format.

PAH

This assay allows us to quantify the amount of tyrosine in the sample, thanks to spectrophotometry. The enzyme of interest, PAH, degrades Phenylalanine into Tyrosine. By shining light at the optimal wavelength of Tyrosine, we obtain absorbance readings. Using the Beer-Lambert law, we can then determine the concentration of the amino acid. Subsequently plotting this information (absorbance readings) against time, allows us to determine the activity rate of the enzyme. However, we need to assume that only the PAH enzyme is able to catalyse this reaction.

Promoter characterisation

To characterise the promoters, a promoter measurement kit will be used.[1] The Assembly Instructions describe a method to insert a test promoter into the promoter test construct by combining the test promoter with the GFP reporter device (BBa_E0240) and the backbone plasmid (pSB3K3). The Measurement Instructions describe a method for measuring the activity of a test promoter relative to the reference standard promoter (BBa_J23101) so that a user of the measurement kit can report the activity of their test promoter in Standard Promoter Units (SPUs). The Registry of Standard Biological Parts includes the SPU calculator to convert the raw fluorescence and OD data into SPUs.

References

  1. Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, de Mora K, Glieberman AL, Monie DD, and Endy D. Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng. 2009 Mar 20;3:4. DOI:10.1186/1754-1611-3-4 | PubMed ID:19298678 | HubMed [promoter1]