Part:BBa_K5317011
MREdada-EGFP
Usage and Biology
The MRE-sites containing promoter enables the metal-dependent expression of a downstream positioned reporter gene via the metal ion-dependent transcription factor MTF-1 for cell-based metal detection.
In order to integrate the findings of Searle and colleagues (1985) and Wang and colleagues (2004) regarding the metal inducibility of a promoter with two MREa sites and a high affinity between MREd and MTF-1, we designed a synthetic promoter with two MREa and two MREd sites that alternate. The aim is to enhance the sensitivity and efficiency of the metal-dependent promoter.
Cloning
Theoretical Part Design
Placing the MRE containing promoter upstream of the reporter gene EGFP allows the visualization of primarily metal-dependent activation of MTF-1.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Cloning
To test the MREdada promoter functionality the reporter gene EGFP (K3338006) was cloned downstream of the promoter by inserting the MREdada promoter into the AseI- and NheI-digested EGFP-C2 backbone (K3338020) using NEB Hifi Assembly.
| Primer name | Sequence |
|---|---|
| MREdada_fw | CCGCCATGCATTAGTTATGCACACTGGCGCT |
| MREdada_rev | TGGCGACCGGTAGCGGACGCTTAGAGGACAGC |
The vector map of the assembled construct is shown in figure 1. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |