Team:Marburg/InterLab
Aim
This year, the iGEM Team Marburg is participating together with many other teams in the InterLab Study. The aim of the InterLab Study is to investigating the reproducibility of BioBrick characterization by collecting data of the same experiment from iGEM Teams all around the globe. All teams are measuring the fluorescence of three different genetic devices constitutively expressing GFP who differ in their promoter strength. These data will allow a comparison of different protocols and lab techniques regarding the variety of the three parts. We transformed the three devices into E. coli cells, diluted to an optical density (OD) of 0.5 at 600 nm using both, a spectrophotometer and a plate reader and measured the fluorescence with a plate reader. The obtained results showed that the promoter J23101 was the strongest one, the promoter J23106 was weaker and the devices with promoter J23117 showed barely fluorescence over the background.
Design
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According to the InterLab Study requirements we measured the three BioBrick parts with promoters of different strengths. The first device consists of the promoter J23101, the GFP coding sequence E0040 and the terminator B0015. The second one is identical to the first device except for the promoter, namely J23106 and the third one uses J23117 as promotor. The promoters are part of a constitutive promoter family that has been introduced to iGEM by the Berkeley Team 2006. The promoters all have the same length and only differ in their sequence. Even small changes have a high impact on the expression level. We used BBa_I20270 with the promoter J23151 as positive control and as negative control BBa_R0040, an empty plasmid with pTetR. The chassis that we used for the characterization was the E.coli strain DH5α. This strain was used as control for the cells’ auto-fluorescence.
Materials and Methods
- We thank the group of Prof. Wolfgang Hillen and Julia Bürger (Friedrich-Alexander-Universität Erlangen-Nürnberg) for sending us the tetR(B) and P5(L9A) promotors.
- We thank the group of Christopher S. Hayes (UC Santa Barbara) for providing us with the e.Coli strain EC93.
- We thank Dr. Knut Drescher (MPI Marburg) for helping isolating the DNA of the CDI-Operon.
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