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 sequence, 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.

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

Figure 1: The plots show the relationship between DFHBI-1T concentration and fluorescence in the presence of excess Spinach2 (top) or Spinach2.1 (bottom) RNA.

The slopes of the curves in Figure 1 are 1.5 μM^-1 for Spinach2 and 1.0 μM^-1 for Spinach2.1. Since these two slopes are of the same order of magnitude we concluded that Spinach2.1 did not have a dramatically reduced ability to activate DFHBI-1T fluorescence, but in order to determine whether it is significantly reduced, experiments with more replicates should be carried out.

We also compared the fluorescence of Spinach2 and Spinach2.1 with excess DFHBI, to determine if Spinach2.1 folds as well as Spinach2.

Spinach2 and Spinach2.1 RNA was produced and the concentrations were measured:

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

Excess of DFHBI-1T was added and fluorescence was measured:

• Spinach2: 306.9
• Spinach2.1: 116.2

Because of the low RNA concentrations we chose to use all the RNA we had produced, instead of using equal concentrations, as we wanted to make sure that fluorescence was detectable. The low RNA concentrations also resulted in a fairly weak signal compared to measurement noise. The above fluorescence signals are means of 9 successive measurements.

FIGURE XX shows the fluorescence intensities divided by the RNA concentrations. The error bars denote the standard error of the mean. The two different Spinach versions show comparable fluorescence per concentration, and we conclude that our generated mutant Spinach2.1 folds as well as the existing Spinach2.

Figure X: …….

It would have been ideal to make triplicate measurements of fluorescence of both Spinach2 and Spinach2.1. However since we had complications with producing high concentrations of RNA in vitro we chose to use all generated RNA in one sample. We conducted multiple measurements on each sample, where the standard error is obtained from.
We conclude that the two spinach sequences function equally well. Spinach2.1 has been submitted as a BioBrick (link to PARTS) and we have only used Spinach2.1 in the rest of the project. After learning that Spinach2.1 worked as we had hoped we decided not to investigate the function of the other mutant, mentioned in experimental design further.

Note: We observed that fluorescence from Spinach was strongly dependent on temperature, with lower temperatures giving higher fluorescence. Therefore all samples have been chilled on ice before being measured, to ensure high signals and to minimize variation.

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 in the iGEM distribution kit, in standard pSB1C3 backbone with chloramphenicol resistance. This would create a library of strains applicable for measuring promoter strength.

The following promoters were sucessfully inserted in front of Spinach2.1 and the constructs 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 stocked to be used for future measurement of promoter activity.

Standard series for DFHBI-1T

A standard series was created to correlate a Spinach-DFHBI-1T complex concentration to a specific flourescence signal. Since we discovered that DFHBI-1T itself caused small amounts of fluorescence, we started by measuring this. To find the background fluorescence associated with unbound DFHBI-1T, a standard curve was made.

We found the slope of the curve to be 0.12 µM^-1, and have thereby obtained a value for the fluorescence which a given concentration of unbound DFHBI-1T is responsible for.

To generate the Spinach2.1-DFHBI-1T standard series, fluorescence was measured with different concentrations of DFHBI-1T, in the presence of excess Spinach2.1 RNA. 5 measurements were made for each concentration. The resulting graph is shown in FIGURE XX.

The slope is here determine to be 1.00 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

We measured the degradation rate of Spinach2.1 in vivo to be able to calculate promoter activity. Three cultures of a strain expressing Spinach2.1 from a strong promoter were grown to exponential phase, and transcription was stopped by adding rifampicin. Production of Spinach2.1 was thus halted, and we could measure the gradual decrease in Spinach2.1 concentration by measuring fluorescence with excess DFHBI-1T.

The three degradation curves are shown in FIGURE XX.

Figure X: …..

The mean of the three calculated degradation rates is 0.74 h^-1 and the standard error of the mean is 0.08 h^-1.

This degradation rate corresponds to an in vivo half-life of 56 minutes.

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.

Figure X: The orange bars show fluorescence/OD for 5 different strains expressing Spinach2.1 from Anderson promoters. The grey bars show the expected fluorescence according to the relative promoter strengths reported in the registry, normalised to J23101.

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