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 2 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 2: Fluorescence values normalised by RNA concentration for Spinach2 and Spinach2.1. Error bars denote standard error of the mean, obtained from 9 successive measurements on the samples. Fluorescence units are in arbitrary units.

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 (see our Parts page) 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_J23117
• BBa_J23118
• BBa_J23119

As the list indicates 9 constructs out of the 15 were successfully constructed. 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 as shown in Figure 3.

Figure 3: The graph shows the correlation between DFHBI-1T concentration and fluorescence, in the absence of any Spinach RNA.

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 4.

Figure 4: The graph shows the correlation between DFHBI-1T concentration and fluorescence in the presence of excess Spinach2.1. The background fluorescence for unbound DFHBI-1T is subtracted. The relation can be used to calculate the concentration of Spinach2.1-DFHBI-1T complex.

The slope of the curve in Figure 4 was determined to be 1.00 µM^-1 and the intercept was found to be 0.78.
We have thereby generated a standard curve that can correlate the observed fluorescence signal to the concentration of the DFHBI-1T Spinach2.1 complex.

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 the strong promoter J23119 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 resulting degradation curves are shown in Figure 5.

Figure 5: Degradation curves for three replicates of a strain expressing Spinach2.1 from promoter J23119. Samples were taken each hour and measured with excess DFHBI-1T.

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. An average value of these was used and illustrated in Figure 6 below.

Figure 6: 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 measured fluorescence/OD values are shown in Figure 6 together with the expected value, based on the relative promoter strengths reported in the registry. The expected values have been normalised to the measured fluorescence/OD value of J23101.
The strength of the promoter BBa_J23119 is not yet characterised other than as a very strong consensus promoter. According to our data J23119 should have a relative activity of 238 percent, compared to J23100.
All in all the measured fluorescence/OD values are in accordance with the relative characterisations done previously. This implies that measuring on an RNA level gives results comparable to using reporter proteins, at least for promoters of moderate strength.
It could be theorised that using Spinach as a reporter allows characterisation of stronger promoters than using protein reporters, as Spinach does not need to be translated, and therefore stresses the cell considerably less.

See our modeling page, for the derivations of the following calculations. In the following we conduct these PoPS calculation for the J23101 promotor. With the use of our developed calculatorPoPS values can easily be calculated from fluorescence signal.

To determine the autofluorescence, fluorescence signal and biomass was measured for a strain containing a Spinach2.1 gene with no promoter. The cell autofluorescence per OD was determined to 0.5.

From our generated DFHBI standard curve we know that the slope in the DFHBI vs. fluorescence graph is 1.00 µM^-1 with an intercept of 0.78. For the J23101 promoter we measured a fluorescence signal of 7.6 at OD600 6.3. By using the autofluorescence per OD the background on this measurement is subtracted.

Fluorescence_real = 7.6 - 6.3*0.5 = 4.4

The remaining fluorescence is the Spinach-specific fluorescence, which can be used to calculate the concentration of Spinach-DFHBI complex.

[SpinachDFHBI] = (Fluorescence - 0.78) /1

[SpinachDFHBI] = (4.4-0.78)/1 = 3.7 µM

The total Spinach concentration is found by dividing with the fraction of Spinach that is correctly folded. The folding fraction of Spinach has by Strack et al. been demonstrated to be around 60%. However this is at 37 C, and since we observed that RNA folding is temperature dependent this folding fraction could be higher. In the following we apply a folding fraction at 60%.

[Spinach] = [SpinachDFHBI] / 0.6

[Spinach] = 3.7µM /0.6=6.1 µM

To determine the cell concentration the CFU is approximated from the OD:

CFU ≈ OD*10<12

The number of Spinach molecules per cell is calculated by use of Avogados Constant and the cell density in CFU/L:

Spinach/cell = [Spinach]*CFU/10^6

Spinach/cell = (6.1 µM*6.3*10^12) /10^6 = 584357 spinach molecules per cell

The production rate of Spinach is hereafter found by multiplying with growth rate plus degradation rate. We have found the growth rate to be 0.0002 s^-1 and the degradation rate to be 0.0002 s^-1.

The production rate is calculated:

Production rate = 584357*(0.002+0.002) = 248.68

Production rate = 248.68 spinach/cell/s

If the copy number on the plasmid is known PoPS can be calculated:
In this case we set the copy number at 200 s^-1.

PoPS = 248.68 /200

PoPS =1.24

Below is a list of the calculated PoPS for the 5 examined promoters. The same procedure is used.