Difference between revisions of "Part:BBa K540001"

Revision as of 16:52, 21 September 2011

rcn, cobalt-sensitive promoter

Rcn is a promoter that is activated by cobalt. A repressor is fixed to the promoter by default, and cobalt binds to this repressor, which activates the gene under control of this promoter.

Rcn is also activated by nickel, but we have not characterized this aspect.

Characterization

Kinetic Mesurement

Important : this characterization hasn't been done with this biobrick and an iGEM Gfp. It has been done with the promoter before that the silent mutation to remove the PstI site was made. The GFP used here, is a stable GFP.

In order to determine if rcn promoter is inducible by cobalt a kinetics of the fluorescence of the two following strains has been done: NM522+p157 where p157 is a plasmid containing a stable GFP under the control of rcn promoter.
NM522+p115 where p115 is the corresponding empty plasmid.

Each strain is cultivated in two conditions without cobalt and with 25µM of cobalt. 1ml of bacteria is added to 100ml of M63G medium and 1ml of spectinomycin in a 250ml erlen. The four erlens are put in a 37°C bath with a 270rpm agitation.

3ml for the fluorescence test and 1ml for the OD are sampled every hour and every half hour near an OD of 0,2 where the promoter is supposed to be well expressed. The measurements are stopped when the OD reaches 1. Then an other point is taken after the bacterias have grown overnight.

Optical Density : 600nm Fluorescence : excitation 490nm, emission 510nm The experiment has been performed in triplicate and the characterization is done by a kinetic measurement.

The the purpose of this experiment is to determine if the promoter rcn is inducted by cobalt and in what extend in the case there is induction. The results shown below are the mean values obtained from the 3 operators.

It is shown out from these two graphics that the strain NM522+p115 doesn't have any fluorescent activity and does not respond at all to cobalt. Whereas the strain NM522+p157 responds well to cobalt. A fluorescent activity is detected even without cobalt what highlights the leaky nature of the promoter rcn. But this activity is at least two times lower than the one obtained in presence of cobalt.

The standard deviation concerning the values for NM522+p157 + 25µM Cobalt looks big while the one of the other strain is equivalent. The grow state for this strain for each operator is similar as shown bellow so the standard deviation's difference can't be linked to this.

Conclusion

The activation of the rcn promoter by the cobalt increases the transcription by a factor of 2. So this promoter can be used with efficiency in a cobalt sensitive system.

This characterization also points out that the expression is maximum for an OD of 0,2.

Safety

This part is not toxic by itself. However, when using this part, you will probably need to handle cobalt. Cobalt is toxic in all its forms ( ionic or metallic ) by inhalation, ingestion or contact. Wear adapted personal protection equipment ( labcoat, safety glasses, safety gloves ) and dispose of it in appropriate waste containers.

Sequence and Features