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 comparably to Spinach2, by performing fluorescence measurements on RNA produced in vitro. We encountered some complications when generating the spinach RNA by in vitro transcription. This can be due to different parameters e.g. the instability of RNA and presence of RNases. We therefore had to work with what small amounts of RNA we could produce, and we were not able to perform as many replicates as we would have liked.

(and chose to use all the generated RNA to have the best foundation for measuring fluorescence. This is why Spinach2 and Spinach2.1 are not used in equal concentrations.)

To test the ability of Spinach2.1 to activate DFHBI-1T fluorescence compared to Spinach2, we added DFHBI-1T to excess amounts of RNA and calculated to slope of the linear relationship between DFHBI-1T concentration and fluorescence

(GRAFER AF DE TO STANDARDKURVER)) As seen in FIGURE the slopes are very similar, implying that the two Spinach sequences activate DFHBI-1T fluorescence equally well. <math>\alpha^2+\beta^2=1</math> We also compared the fluorescence of Spinach2 and Spinach2.1 with excess DFHBI, to determine if Spinach2.1 folds as well as Spinach2.

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 9 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. As mentioned in our experimental design we started out with two suggestions on durable mutations. Since we had most confidence in the second modified spinach, now referred to as Spinach2.1, and this was now proved working we decided to continue with this.

Construct of strains

We constructed a library of DH5α strains with promoters of different strength in front of the Spinach2.1 flanked by the tRNA scaffold. We intended to use the 15 of the 20 Anderson Promoters available on the standard pSB1C3 backbone with chloramphenicol resistance available in the iGEM distribution kit. Thereby creating a library of applicable for measuring promoter strength. The following promoters was sucessfully inserted in front of Spinach2.1 and have been verified by sequencing:

• BBa_J23101
• BBa_J23102
• BBa_J23103
• BBa_J23106
• BBa_J23113
• BBa_J23114
• BBa_J23114
• BBa_J23118
• BBa_J23119

As the list indicates 9 constructs out of the 15 possible succeeded. These constructs were used for further measurement in the promoter strength.

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 the DFHBI-1T Spinach2.1 complex.

The same experiments was conducted with the original Spinach2. These values are not as linear as for the Spinach2.1. This can be due to the low concentration of RNA, and the fact that we figured out on a later stage how to mix the solution properly. However a clear correlation between DFHBI-1T concentrations and fluorescence signal is observed.

Degradation of Spinach2.1

bla
bla

Fluorescence measurement

We selected 5 of the 9 constructed strains with different promoters. We selected 5 strains containing promoters with pronounced difference in strength to demonstrate our developed method for measuring promoter strength. The measured fluorescence signals were divided by the measured OD600. For each sample fluorescence was measured 5 times. However we are aware of that an ideal experiment would have included triplicate each strain. An average value of these was used and illustrated in the bar chart below.


The strength of the promoter BBa_J23119 is not yet characterised other that stated as a very strong promoter. The strength of the of the other promoters is given as a relative number relative to BBa_J23100. The expected activities are included in the bar chart. In this case the reference is J23101. According to our data J23119 should have a relative activity of 238 percent. We conclude that the fluorescence measurements of ours are in accordance with the expected order of the promoter activities.

bla
bla