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Cloning

This year, we succeeded in cloning all missing parts required for our measuring system. While mPR Xl, mPR Dr, mig1, Padh1 and rox1 were available in pUC-IDT vector or pGEM T-easy vector from last year's iGEM-Team Tuebingen, Pfet3, Panb1, Psuc2 were cloned directly from yeast genome. Furthermore we used the fluorescent reporter protein mOrange (BBa_E2050) and the transcriptional terminator Tadh (BBa_K801012) from the parts registry. A galactose inducible promotor (BBa_J63006) from the registry was used to create some plasmids which we want to use for the characterization of some of our parts. The cloning of luciferase succeeded only shortly before wiki freeze. As a result, the assemblies requiring luciferase were not performed anymore.

Assembly

Our general assembly strategy is illustrated in fig. 1. The pRS vectors do not fit the RFC10 criteria and can therefore not be used the for 3A-Assembly. Instead we used the BioBrick RFC10 vector pTUM100 (BBa_K801000) which was kindly provided by the iGEM Team of the TU Munich to fuse promotor and coding sequence. pTUM100 is a high copy shuttle plasmid which is based on the commercially available pYES2 vector and contains the cyc1 transcription terminator after the BioBrick suffix. Thus we could use some of the pTUM constructs directly for characterization of our repressible promotors (Pfet-mOrange-pTUM100, Panb-mOrange-pTUM100, Psuc-mOrange-pTUM100, Padh-mOrange-pTUM100). The parts required for the next steps of characterization were equipped with a the transcriptional terminator Tadh by ligation into a pSB1C3 plasmid containing Tadh. The resulting assemblies were again BioBricks. These inserts were then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix. In total we have created 29 new plasmids!

Fig. 1: General assembly scheme: First, promoter and coding sequence were assembled in pTUM100 using the 3A-Assembly protocol. Constructs with the reporter mOrange were used for characterization directly. Subsequently, the promotor and the fused coding sequence are ligated into a pSB1C3 plasmid with the transcriptional terminator Tadh1. The resulting assemblies are again BioBricks. These inserts are then ligated into the pRS shuttle vectors losing parts of their prefix and/or suffix.

Readout

For the characterization of our repressible promotors Pfet3, Panb1 and Psuc2 we transformed pTUM100 constructs with mOrange under the control of the promotor of interest in the laboratory yeast strain w303. We used the constitutive promotor Padh1 (sequence identical with BBa_J63005) as positive control. The promoter activity was analysed qualitatively using fluorescence microscopy (fig. 2).

Fig. 2: Detection of mOrange expression in the fluorescence microscope. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).

Next, we established a protocol for quantitative read-out of mOrange fluorescence using the plate reader. We recorded an excitation spectrum of non-transformed yeast and yeast expressing mOrange measuring the emission at 581nm (+/- 25nm). Fig. 3 compares the excitation spectra with the ideal excitation spectrum of mOrange. While the fluorescence decreases with higher excitation wavelength for non-transformed yeast, the spectrum of yeast expressing mOrange has a similar shape than the ideal excitation spectrum.

Fig. 3: Excitation spectra of non-transformed w303 yeast (neg. control) and yeast expressing mOrange. Both spectra were recorded in the plate reader (Ex: 400nm-550nm +/-9nm, Em: 581nm +/-20nm). The excitation spectrum of mOrange is indicated by a dashed line (source: http://www.tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls).

Repressible Promotors

Fig. 4: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).

Fig. 5: Basal expression level of mOrange under the control of the promotors Pfet3, Panb1 and Psuc2. Non-transformed yeast was used as negative control. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.

Pfet3

Before we could observe the regulation of Pfet3 through the co-expressed membrane-bound progestin receptors, we needed to investigated the native function of Pfet3 which is the response to intracellular iron concentration. Therefore we grew yeast containing the Pfet-mOrange-pTUM100 construct in a low-ion environment (Low-iron medium (LIM) contained 1 mM EDTA). The promotor activity of Pfet3 is increased after 4 h in low-iron medium compared to cells which were grown in normal synthetic complete medium (fig. 6). After 8 h the activity was 3-fold increased. The dependence of Pfet activity on iron allows modifying the expression level of the repressors mig1 and rox1. This is desirable as sensitivity analysis has shown that the maximal repressor concentration is crucial for the functionality of the measurement system. Nevertheless, this finding brings along new challenges as we need to maintain Pfet3 activity constant to avoid an undesired influence on the measurement.

Fig. 6: Alterations of mOrange expression in low-iron environment of w303 containing Pfet3-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.

Panb1 and Psuc2

First we transformed the constructs Psuc-mOrange-pTUM100 and Panb-mOrange-pTUM100 in w303. The promotor activity was analysed in fluorescence microscopy and using the plate reader. The activity equals 3.8% of Padh activity for Psuc and 1.9% of Padh activity for Panb. The maximal activity of these promotors also limits the maximal reporter expression and therefore also the sensitivity of the measurement system. Thus, a higher promotor activity was desirable.

Next we investigated which other factors influence the promotor activity, especially the intrinsic repressor level. Therefore we transformed the same constructs in mutant yeast strains with deficient Rox(YPR065W) or Mig(systematic gene name: YGL035C), see fig.7+8. The promotor activity was enhanced by a factor of 2.6 for Psuc and 13.8 for Panb. We therefore intend to implement our system in these deficient strains allowing higher reporter expression levels. Furthermore the function of mig1 in glucose repression is well described (Santangelo, 2006). We observed an increase in promotor activity upon changing the medium from SC-Ura(2% glucose) to SC-Ura(2% galactose) for wt as well as the Mig deficient strain (fig.9+10). This effect has to be considered if we intend to use Psuc together with GAL-Promotor in further characterization.

Fig. 7: Increased promoter activity of Psuc and Panb in repressor deficient strains. Fluorescence was detected using a RFP filter set (ET Bandpass 470/40, ET Bandpass 572/35).

Fig. 8: Promoter activity of Psuc and Panb in repressor deficient strains. The promoter activity was enhanced by a factor of 2.6 for Psuc and 13.8 for Panb. Fluorescence was measured using a plate reader (Ex: 548nm +/-9nm, Em: 581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as triplicates.

Fig. 9: Alterations of mOrange expression upon galactose induction of w303 containing Psuc-mOrange-pTUM100. Fluorescence was measured using a plate reader (Ex:548nm +/-9nm, Em:581nm +/-20nm) and normalized to OD600 of the cell suspension. All measurements were performed as duplicates.

References

SANTANGELO, G. M. 2006. Glucose signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev, 70, 253-82.