Lead responsive promoter with mRFP
lead responsive promoter with mRFP for detection
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC
IISER Pune 2019 - Characterisation
To check for the activity of the promoter in this construct, fluorescence measurements of cells transformed with the biobrick were taken in different conditions.
DH5 alpha cells transformed with the biobrick were cultured in LB and M9 media and their fluorescence measurements were taken either 12 or 16 hours from the time of inoculation. These data was subsequently compared with the fluorescence data of a negative control (DH5 alpha cells producing no fluorescent proteins) and a positive control (DH5 alpha cells consecutively expressing RFP, transformed with BBa_J04450).
Cells grown in LB were pelleted, washed once with PBS and resuspended in 1x PBS. 100 microlitres of this suspension and 100 microlitres of M9 cultures (directly taken) were loaded onto a 96-well plate.
The fluorescence measurement was taken at emission wavelength-611 nm and excitation wavelength-586 nm.
The following results were obtained:
No significant fluorescence was obtained from the construct despite the lack of the pbrR (repressor) protein.
To check if lead had some effect on the activity of the promoter, DH5 alpha cells containing the construct with grown in LB for 12 hours and then induced with varying concentrations of lead for 4 hours. These cells were then pelleted, washed with PBS and resuspended in 1x PBS.
The following data was obtained:
The fluorescence data didn't seem to be affected by the addition of lead and the promoter still remained in a inactive state.
Conclusion: This part does not constitutively express RFP. Neither does it express RFP in the presence of lead.