pCon+RBS+tetR+DT+pTet+RBS+tetX-GFP(fusion protein)+DT
pCon from BBa_J23119, RBS from BBa_B0034, tetR from BBa_C0040, DT from BBa_B0015,PTet from BBa_R0040 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).
In this part, a fusion protein(referred to as tetX-GFP) of tetX(a tetracycline-degrading enzyme) and GFP combined with a 3*GGGGS linker is used to report the expression level of tetX. The first promoter(BBa_J23119) is constitutively on and can express tetR, a protein that can repress the downstream promoter pTet in the absence of tetracycline(Tc). As a result, no fusion protein tetX-GFP is expressed and no fluorescence can be detected. However, in the presence of Tc, tetR is repressed and repression over pTet is removed. This leads to the expression of tetX-GFP, degrading tetracycline and emitting fluorescence at the same time. This part can report activity of tetX in the presence of Tc by emission of green fluorescence.
Linker for fusion protein is carefully designed to avoid repeated sequence which 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
tetracycline repressor from transposon Tn10 (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)
Coding region for the TetR protein without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. TetR binds to the pTet regulator (BBa_R0040). aTc (anhydrotetracycline) binds to TetR and inhibits its operation.
References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). Lutz R, 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 References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). Lutz R, 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 BBa_C0040 TetR Protein is based on the TetR sequence from Elowitz's repressilator. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman.
Overlap extension of synthetic oligonucleotides
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.
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