Part:BBa_J13002:Experience

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Part J13002: Characterization by UMN

In 2009, the University of Minnesota's iGEM team chose to characterize this part. We were working on engineering a logical AND gate for the iGEM competition and were interested in existing parts that had the same regulators. We used MIT 2004's exemplar characterization of Part F2620 as a template for our characterization and characterized this part in terms of Transfer Function and Stability.

Device Design

Since this part already contained an RBS, we attached GFP, included in the 2009 iGEM Parts Kit, to the promoter and RBS and examined the fluorescence, which is directly proportional to [http://parts2.mit.edu/wiki/index.php/Abstraction_hierarchy_and_PoPS Polymerase Per Second (PoPS)]. We inserted these two parts into the plasmid backbone pSB3k3 and induced the resulting device at increasing concentrations of aTc: 0, 1, 10, 50, 100 and 200 ng/ml.

Results

Transfer Function

As you can see on the graphs above, there was a general upward trend of GFP production with the concentration of aTc. The error bars in the graphs represent the standard error. Interestingly, we observed a spike in GFP production at 50 ng/ml of aTc. During the sampling, the culture growing in 50 ng/ml aTc inducer media did not grow as fast as the others, with the OD595 as low as 0.08 rather than the preferred 0.2. We continued sampling at each hour point for this concentration bu did not see obvious pellets during the resuspension in PFA or PBS. Since the inducer is an antibiotic, we hypothesized that it could be having a toxic effect on the cell and subsequent GFP production. We did perform this experiment again but did not see any induction.

Stability

We also examined the stability of the device, which is how the production of GFP changes for each inducer concentration over multiple rounds of cell division. As you can see from the graphs above, the device is clearly inducible with a general downward trend in GFP production over time. The graph of aTc concentration of 50 ng/ml again demonstrates the spike in GFP production that we observed at this concentration. This is the optimal inducer concentration for the device.

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