Team:Heidelberg/Project Natural promoters


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=Natural Promoters=

Introduction
Eukaryotic promoters are very complex and difficult to characterize. They usually lie upstream of their target gene, but they can have regulatory elements that are several kilobases away from the promoter. Fig. 1 shows some of the factors involved in the transcriptional regulation of polymerase II [1]. For our assay we want to utilize several natural promoters to sense certain conditions in the cell, such as hypoxia, DNA damage and heat. The selection criteria for these promoters are that they have to be very specific to a transcription factor and they have to be easily induced and inhibited. These natural promoters will give us a chance to start testing and fine-tuning our output system early on which can then be applied to our synthetic promoters.



Results
After cloning the natural promoters in our screening plasmid pSMB_MEASURE, they were tested in different cell lines with appropriate induction substances. The transfected cells were measured by flow cytometry and TECAN. Thereby, the c-Jun promoter and the CYP1A1 promoter were tested by flow cytometry and the LDL receptor promoter, HSP70 promoter and HMG CoA synthase promoter were measured by TECAN. The CYP1A1 measurement could not be analyzed, due to the low cell number. In Fig. 2, the flow cytometry measurement data of the c-Jun promoter is displayed, which was induced by Epidermal Growth Factor for three hours. The results present an induction of more than 100%. Fig. 3 presents the TECAN results of the both SREBP responsive promoters, LDL receptor and HMG CoA synthase promoter.



Discussion
The evaluation of the c-Jun natural promoter looks very promising. The flow cytometry measurement should be repeated at least three times, which could not be accomplished in the given time frame. Besides, this measurement could be also analyzed by fluorescence microscopy and image analysis for validation. The TECAN measurement of the SREBP responsive promoters, LDL receptor and HMG CoA synthase promoter, show a significant induction. This should also be validated by microscopy and flow cytometry. Unfortunately, the appropriate induction conditions for the other natural promoters could not be established in the remaining time.

Methods
The different natural promoter inserts (see table 1) were integrated in the pSMB_MEASURE. For this purpose, the promoters of the ordered plasmids/genomic DNA were amplified by PCR and primers with BBb standard. These PCR products were digested by restriction enzymes and purified. The purified inserts were ligated into the pSMB_MEASURE plasmid and transformed into DH5alpha bacterial cells. After mini-preparation, the plasmids were sequenced, which revealed the right sequences. The natural promoters were transfected in different cell lines and induced with appropriate substances, which is presented in table 1. The analysis was made by flow cytometry or TECAN measurement. More details about the method are found in the Notebook.