Part:BBa_K303000:Design
Ptet/ara with J23106
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 70
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 30
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The PCR method was used to assemble the part.
Two sets of promoters were designed to respond to tetracycline and lactose, tetracycline and arabinose, and lactose and arabinose, using J23106 and J23110 as bases. According to literature, a promoter with an activation site that is not activated is not entirely inactive. RNA polymerase can still bind, only the interaction is weaker. Promoters J23106 and J23110 were chose because they are of relatively moderate strength. It is predicted that until arabinose is present in the system, the relative strength of these promoters will be fairly weak. When coupled with weak ribosomal binding sites, the rate at which proteins are translated should be negligible, preventing false positives.
Source
The part contains the constitutive promoter, J23106. It has been modified with two new operator sites.
References
Tet:
1. Lutz R and Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res 1997 Mar 15; 25(6) 1203-10. pmid:9092630. PubMed HubMed [Lutz-1997]
Ara:
1. Schlief, R. (2000). Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics. 16(12):559-565.
2. Khlebnikov A, Datsenko KA, Skaug T, Wanner BL, and Keasling JD. (2001). Homogeneous expression of the PBAD promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. Microbiology. 147(12):3241-7.