pT7+pTet+RBS+tetX-GFP(fusion protein)+RBS+T7RNAP+DT
pT7:PCR amplified from BBa_K525998;T7RNAP:PCR amplified from genome of BL21. As for fusion protein tetX-GFP, tetX is the same as (xxx), the protein linker is provided by our advisor Li Hua, and the GFP is from BBa_E0040. We combine this three parts using overlap extension polymerase chain reaction (OE-PCR). pTet from BBa_R0040, DT from BBa_B0015,RBS from BBa_B0034
A positive feedback device to over-express the fusion protein of tetX and GFP.
PT7 is placed upstream of pTet, regarding that pT7 is a strong promoter while pTet is relatively weak. Linker for fusion protein is carefully selected to avoid repeated sequence that may cause problems in assembly.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Double terminator consisting of BBa_B0010 and BBa_B0012
Bacteriophage T7
Gene 1 coding region from bacteriophage T7 encoding a RNAP polymerase (RNAP). This single-protein RNAP recognizes a 21 bp consensus promoter sequence and is orthogonal to E. coli RNAP.
A fusion protein combining tetX(BBa_K2150101) with GFP(BBa_E0040)
The source of tetX is the same as (xxx), the protein linker is provided by our advisor Li Hua, and the GFP is from BBa_E0040. We combine this three parts using overlap extension polymerase chain reaction (OE-PCR).
This protein(noted as tetX-GFP) combines a tetracycline-degrading enzyme (tetX monooxygenase, ) with a green fluorescent protein (GFPmt3, BBa_E0040). TetX-GFP has activities of both the tetX and the GFP. As a degradation enzyme, it can degrade tetracycline(tc) and its analogs as we describe (here), and also give the tetracycline resistance to E.coli. As a green fluorescent protein, it inherits the normal function of BBa_E0040. However, the expression quantity of this protein may be smaller than BBa_E0040 under the same promoter or it may take longer time for tetX-GFP to mature, for we notice that when it is expressed under the same promoter as BBa_E0040, the fluorescent intensity(FI) of tetX-GFP is weaker than BBa_E0040 at the same growth stage of E.coli, and it takes longer for E.coli expressing tetX-GFP to reach the same FI as E.coli expressing BBa_E0040. We use tetX-GFP as a monitor of the quantity of tetX expressed in E.coli. TetX-GFP can also act as a reporter of whether our system is working (meaning whether our machine is degrading tetracycline). When tetX-GFP is expressed under a tetracycline-regulated promoter such as BBa_R0040, it will become a visible sensor of tetracycline in the presence of tetR, with no need for additional tetracycline-resistance genes.
The protein linker is 3*GGGGS which provides flexibility to the fusion protein. When choosing the codon for this linker, we need to avoid repeated sequence which may cause problems when assemble these parts.
This part is the T7 promoter without RBS. It has been reported that T7 promoter doesn't work with E.coli RNA polymerase, but only with T7 RNA polymerase(T7RNAP). However, we observed leakage of it in the absence of T7RNAP.
We deleted the RBS in BBa_K525998, so that RBS with different efficiency can be used.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1