Team:Tuebingen/Results/Overview
From 2013.igem.org
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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 TU Munich in order to fuse promoter 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 promoters (Pfet3-mOrange-pTUM100, Panb1-mOrange-pTUM100, Psuc2-mOrange-pTUM100, Padh1-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!
![](https://static.igem.org/mediawiki/2013/1/19/TueAssemblyStrategy.png)
Readout
For the characterization of our repressible promoters Pfet3, Panb1 and Psuc2 we transformed pTUM100 constructs with mOrange under the control of the promoter of interest in the laboratory yeast strain w303. We used the constitutive promoter Padh1 (sequence identical with BBa_J63005) as positive control. The promoter activity was analyzed qualitatively using fluorescence microscopy (fig. 2).
![](https://static.igem.org/mediawiki/2013/7/71/TueNurKontrollen.png)
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.
![](https://static.igem.org/mediawiki/2013/d/d2/TueExcitation_spectrum_of_mOrange.png)
Repressible Promotors
![](https://static.igem.org/mediawiki/2013/6/6f/TueWt_Staemme.png)
![](https://static.igem.org/mediawiki/2013/4/40/TueAll_strains.png)
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 promoter activity of Pfet3 had increased after 4 h of incubation in low-iron medium compared to cells which were grown in normal synthetic complete medium (fig. 6). After 8 h of incubation in LIM the promoter activity had increased 3-fold. The dependence of Pfet3 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.
![](https://static.igem.org/mediawiki/2013/d/dc/TueLIM_Induction.png)
Panb1 and Psuc2
First, we transformed the constructs Psuc2-mOrange-pTUM100 and Panb1-mOrange-pTUM100 in w303. The promoter activity was analysed in fluorescence microscopy and using the plate reader. The activity equals 3.8% of Padh1 activity for Psuc2 and 1.9% of Padh1 activity for Panb1. The maximal activity of these promotors also limits the maximal reporter expression and therefore also the sensitivity of the measurement system. Thus, a higher promoter activity was desirable.
Next, we investigated which other factors influence the promoter activity, especially the intrinsic repressor level. Therefore, we transformed the same constructs in mutant yeast strains with deficient rox1 (YPR065W) or mig1 (systematic gene name: YGL035C), see fig. 7 + 8. The promoter activity was enhanced by a factor of 2.6 for Psuc2 and 13.8 for Panb1. 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 Psuc2 together with a GAL-promotoer in further characterization.
![](https://static.igem.org/mediawiki/2013/d/d5/TueDefiziente_staemme.png)
![](https://static.igem.org/mediawiki/2013/8/83/TuePanb_Psuc_deficient_strains.png)
![](https://static.igem.org/mediawiki/2013/e/ee/TuePsuc_mO_GALinduction.png)
![](https://static.igem.org/mediawiki/2013/7/74/TueGALinduction_Psuc_mig.png)
References
SANTANGELO, G. M. 2006. Glucose signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev, 70, 253-82.