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. The The measured values are presented in the bar chart below together with standard error. From this we conclude that the two spinach sequences have equal functionality.

Construct of strains

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Standard series for DFHBI-1T

A standard series was conducted to connect a DFHBI-1T concentration to a specific flourescence signal. Since we discovered that DFHBI-1T itself cause small fluorescence signals we initially examined that. To find the background fluorescence associated with only DFHBI-1T without RNA added a standard curve was made. We find the slope of the curve to be 0.12 µM-1. And have thereby obtain a value for the fluorescence which DFHBI-1T is responsible for.

To generate the DFHBI-1T standard fluorescence was measured with different concentrations of DFHBI-1T. DFHBI-1T was used with excess of RNA. 5 measurements were made for each concentration.Values were subtracted background and blank. Below is the resulting graph. Here Spinach2.1 was used.
The slope is here determine to be 0.10 µM-1.
We have thereby generated a standard curve for DFHBI-1T. The same experiments was conducted with Spinach2.

Degradation of Spinach2.1

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Fluorescence measurement

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