Difference between revisions of "Part:BBa I12006:Experience"

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

[http://2011.igem.org/Team:Groningen 2011 iGEM team Groningen]

[http://2011.igem.org/Team:Groningen 2011 iGEM team Groningen] characterized the modified lamdba Prm promoter (repressed by 434 cI) (BBa_I12006). The promoter was induced indirectly, with the use of a cI transcription factor generator (BBa_K607001). This generator is working under the pBAD/AraC promoter, which was also characterized by [http://2011.igem.org/Team:Groningen 2011 iGEM team Groningen]. Along with the generator, the cells were carrying a plasmid with BBa_K607038 construct, which is a GFP generator under the modified lamdba Prm promoter (repressed by 434 cI) control.

The cells were induced with different concentrations of arabinose and the fluorescence was measured over time in a fluorimeter.

The measurements were done in DH5alpha strain carrying a pSB1C3 with BBa_K607001 in triplicate.

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  Figure 1. Cells carrying a pSB1C3 plasmid with BBa_K607001 construct and a pSB1A3 plasmid with BBa_K607038 construct, induced at time=0h.

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  Figure 2. Wild type cells, induced at time=0h.

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  Figure 3. Fluorescence from Figure 1. compared to the predicted concentration of the cI transcription factor. The prediction is based on the amount of GFP produced during the pBAD/AraC promoter measurements.

The modified lamdba Prm promoter (repressed by 434 cI) exhibits a dynamic response in a range of arabinose concentrations when induced indirectly. Both the speed of activation and its strength are higher with higher concentrations of the inducing molecule.

Because the promoter was not induced directly, the fluorescence intensity was compared to the predicted concentration of the cI transcription factor. The prediction was based on the measurements of pBAD/AraC promoter. We have assumed that the amount of GFP produced by the arabinose-induced promoter is directly related to that promoter activity and thus to concentration of any protein produced by this promoter when induced with different arabinose concentrations.

The effect of this comparison can be seen in Figure 3. The promoter activity grows exponentially with increasing cI concentrations. We believe that if we could achieve higher concentrations of cI, we would see a bend point and a second part of a sigmoid that would spread among a high concentration range.