Team:Munich/Improve

Improved Parts

Improved mTurquoise

Our improved part is the mTurquoise2 (mTq2) expression construct (BBa_K2722004). This part is composed of five subparts, namely a constitutive promoter (BBa_J23106), a self-cleaving ribozyme (BBa_K2722006), a Ribosome Binding Site (BBa_B0034), an mTurquoise2 coding sequence (BBa_K2722003) and a terminator (BBa_K2722007).

Improvements

Improving the mTq2 expression construct BBa_M50070, which is regulated by a pH sensitve promoter BBa_K116001, involved several steps.

Due to our application of the BioBrick the promoter was changed to a constitutive promoter to enable a steady expression of mTq2 during TX-TL measurements.
Furthermore, the mTq2 sequence was codon optimized for E.Coli to ensure higher translation efficiency and therefore an overall higher protein expression rate.
Adding the PmlJ ribozyme to the expression construct served as an additional improvement to the overall protein expression. This is a self-cleaving ribozyme is used to enhance the translation rate by removing the 5'untranslated region (UTR) of the mRNA and thereby eliminating context dependencies. This means that potential inhibitory secondary structures of the UTR are prevented and the translation rates of mTq2 are enhanced and comparable between different experiments due to fluctuations of the forming secondary structures.
The final addition of a strong terminator (380 Terminator) enhanced the overall protein expression rate by avoiding the production of unnecessary long mRNA product s during transcription. Therefore, no rNTPs are wasted during the transcription process, which in turn enables protein expression for a longer time.

Characterisation

Tab.1 and Tab.2 show the data for calibration curves of mTurquoise in buffer (20 mM TRIS, pH 8) and TX-TL, respectively. The fluorescence of mTurquoise2 was measured at 480 nm at an excitation at 440 nm. All the measurements were performed in triplicates.
(Abbreviations: Conc. = protein concentration, SD = standard deviation, SEM = standard error of mean.)

Table 1: Measurement of standard curve of mTurquoise in 20mM TRIS (pH 8)

Table 2: Measurement of standard curve of mTurquoise in TXTL

Both calibration curves are illustrated in Fig. 1. The provided equations can be used to determine the mTurquoise2 concentration in quantitative experiments.

Figure 1: Standard curves of mTurquoise2 in 20 mM TRIS, pH 8 (blue) and in TX-TL (green)

The extinction coefficient ε is calculated with following formula:
$\begin{array}{ccc}& \hfill ϵ=\genfrac{}{}{0.1ex}{}{A}{c\cdot l}& \hfill \text{(1)}\end{array}$ Whereby A is the Absorption, c is the concentration in Mol and l is the pathlength of the probe. We measured the Absorption at 434 nm of six samples using a commercial Nanophotometer (l = 1 mm). Three having a concentration of 22 µM and another three having a concentration of 11 µM mTurquoise2. And calculated the extinction coefficient.

Table 3:Extinction coefficients determined with Nanophotometer measurements

We generated a mean value from our six samples of 30.379 1/Mcm. Which is consistent with the literature value.

References

2. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening.
3. mTurquoise, FPbase (retrieved on 10.10.2019)
4. id="ref_3"> Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.
5. Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells.

<groupparts>iGEM18 Munich</groupparts>

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