Team:Goettingen/Parts

Parts

Parts we used in our project

Parts we modified/improved/created

<groupparts>iGEM013 Goettingen</groupparts>

Promoter Characterization

To construct our reporter systems, we needed promoters of different strength. The well-known Anderson promoter collection consists of nineteen constitutive promoters of different strength. Because of their specific strength, we chose four different promoters (Table 1).

Table 1. For our project, we were interested in four different promoters from the Anderson Collection. For each part, we used an intern number, which will be used in the text, as well.

The activity of these promoters was mainly characterized by measurement of the activity of a reporter protein expressed from these promoters. Protein activity is not only determined by the efficiency of transcription. In addition to this, translation efficiency contributes to the protein amounts in a cell. In particular, regarding fluorescent proteins like RFP or GFP, the growth phase might affect their fluorescence (see discussion below). A more direct way to determine the promoter activity is to assess the transcript levels. We were interested in how the mRNA levels of RFP placed downstream of the promoters from the Anderson collection change dependent on the promoter and how the protein activity compares to them.

For this, we transformed E. coli DH5α with the promoter parts present in the plasmid backbone BBa_J61002. As an empty vector control lacking a promoter and RFP, we used E. coli cells transformed with [http://partsregistry.org/Part:BBa_B0034 BBa_B0034(an RBS)] in plasmid Backbone pSB1A2. Subsequently, we characterized three clones of each strain by qRT-PCR and by monitoring the RFP fluorescence in different growth stages. In addition to this, we did fluorescence microscopy of exponentially growing cells (Fig. 1).

For qRT-PCR analysis (Fig. 2), E. coli cells were harvested at an OD_600nm of ca. 0.6 (exponential growth phase) and after 5 – 6 h when the OD_600nm had reached approximately 2 (stationary phase). The cells were incubated over night and a third sample taken the next morning ( > 20 h incubation, OD600nm was ca. 2 – 3). The relative mRNA levels of rfp were normalized to the rpoA levels. Then, the fold change (∆∆C_T) of the promoters compared to promoter 1 (the weakest) was calculated for each phase. The fold enrichments obtained for different growth phases could not be compared since the C_T values determined for rpoA suggested a generally altered transcription during the different growth stages.

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