Team:DTU-Denmark/Achievements/Experimental Results

Comparison of Spinach2 and Spinach2.1

Since we introduced a mutation in the Spinach2 sequence to overcome a SpeI restriction site, our first task was to confirm that this modified Spinach2.1 was performing compatible to Spinach2. We met some complications when generating the spinach RNA by in vitro transcription. This can be due to different parameters ie. the instability of RNA and presence of RNAses. We therefore chose to use all the generated RNA to have the best foundation for measuring fluorescence. This is why Spinach2 and Spinach2.1 is not used in identical concentrations.

The RNA concentrations were measured:

• Spinach2: 40 ng/µl
• Spinach2.1: 14 ng/µl

Excess of DFHBI-1T was added and fluorescence was measured in plate reader: Average of measurements

• Spinach2: 306.9
• Spinach2.1: 116.2

When these are normalised with the RNA concentration we conclude that our generated mutant Spinach2.1 is compatible with the existing Spinach2. Spinach2.1 is registered as a BioBrick (link). We actually even observe a higher fluorescence signal from Spinach2.1

• Spinach2: 7.7
• Spinach2.1: 8.3

Since that is the fact we continued working with the mutant, and all experiments from now on is conducted with this. Surely it would have been ideal to make triplicate measurements of fluorescence of both the wildtype and the mutant. However since we as mentioned had complications with amplifying high concentrations of RNA we chose to use all generated RNA in one sample. We conducted multiple measurements on each sample, where the standard error is obtained from.

Construct of strains

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Standard series for DFHBI-1T

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Degradation of Spinach2.1

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Fluorescence measurement

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