Part:BBa_K2540011
Universal Bacterial Expression Resource (UBER)
Universal Bacterial Expression Resource (UBER) is a system containing T7 RNA Polymerase (T7 RNAP) which can be used for the expression of genes under T7 promoter. UBER includes T7 RNAP and TetR repressor which provides negative feedback on the polymerase levels to help reduce toxicity.
Usage and Biology
UBER consists of a TetR gene under control of a T7 promoter and a T7 RNA polymerase gene under the transcriptional control of a broad-host priming promoter as well as a TetR-repressible T7 promoter. The priming promoter starts transcription of the T7 RNAP, which then goes on to transcribe itself, creating a positive feedback loop in which increasing levels of T7 RNAP leads to increased transcription of the T7 RNAP gene. High levels of T7 RNAP may be toxic to cells; to prevent this toxicity, T7 RNAP also transcribes TetR, which binds to the TetR-repressible promoter and blocks transcription of T7 RNAP, creating negative feedback control to keep levels of T7 RNAP consistent. This pool of T7 RNAP can be used to transcribe any additional genes under a T7 promoter. This submitted UBER was adapted from the work of Kushwaha and Salis; the modifications made were: removal of yeast NLSs from the T7 RNAP and TetR genes, replacement of the uncharacterized priming promoter with a characterized broad-host priming promoter, and removal of an internal cut sequence.
The function of UBER containing our modification was compared with the original system from Kushwaha and Salis (Figures 1,2). E. coli DH10B
cells co-transformed with either original or modified UBER and an expression cassette containing mKate2 under T7 promoter were grown for 15 hours at 37 C in LB. Figures 1 and 2 show fluorescence time course for modified (Figure 1) and original (Figure 2) UBER. The results demonstrate that modifications introduced do not interfere with UBER function and result in higher fluorescence/OD compared to the original system.
Figure 1:Fluorescence time course for the modified UBER
Figure 2: Fluorescence time course for UBER from Kushwaha and Salis.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 91
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3
Illegal BsaI.rc site found at 3912
References
Kushwaha, M. & Salis, H. M. A portable expression resource for engineering cross-species genetic circuits and pathways. Nature Communications 6, 7832 (2015).
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