High input score automatic selector: PoPS->luxI-LVA->K176228
PoPS->luxI-LVA->tetR-LVA + pCon 0.36->luxI-LVA + pCon 0.70->luxR + pLux/Tet->lacZalpha-ccdB(weak RBS) Cell Death = (Cell Density * (pCon 0.36 + Population Average Input PoPS) + Input AHL) / (1 + Input PoPS) Cells with relative high input score in the population will be selected, cells with relative low input score will die. The first luxI-LVA is used to calculate average score in the population.
PoPS->luxI-LVA->tetR-LVA + pCon 0.36->luxI-LVA + pCon 0.70->luxR + pLux/Tet->lacZalpha-ccdB(weak RBS) Input1: PoPS Input2: AHL Output1: AHL Output2: Cell Density Design: Input1 PoPS generates LuxI-LVA and TetR-LVA. pCon 0.36(K176009) also generates LuxI-LVA, LuxI-LVA generates Output1 AHL, the concentration of AHL in the medium depends on both LuxI-LVA level per cell and cell density. pCon 0.70(J23101) generates LuxR. pLux/Tet(K176000) generates lacZalpha-ccdB (weak RBS) to kill the cell, activated by LuxR and (Output1 AHL generated by LuxI-LVA + Input2 AHL), repressed by TetR-LVA and no aTc.
Repressible cell density controller: PoPS->tetR-LVA->K176095
PoPS->tetR-LVA+pCon 0.36->luxI-LVA+pCon 0.70->luxR+pLux/Tet->lacZalpha-ccdB(weak RBS) Cell Death = ((Cell Density * pCon 0.36) + Input AHL) / (1 + Input PoPS) High Input PoPS -> High Steady State Cell Density High Input AHL -> Low Steady State Cell Density
PoPS->tetR-LVA+pCon 0.36->luxI-LVA+pCon 0.70->luxR+pLux/Tet->lacZalpha-ccdB(weak RBS) Input1: PoPS Input2: AHL Output1: AHL Output2: Cell Density Design: Input1 PoPS generates TetR-LVA. pCon 0.36(K176009) generates LuxI-LVA, LuxI-LVA generates Output1 AHL, the concentration of AHL in the medium depends on both LuxI-LVA level per cell and cell density. pCon 0.70(J23101) generates LuxR. pLux/Tet(K176000) generates lacZalpha-ccdB (weak RBS) to kill the cell, activated by LuxR and (Output1 AHL generated by LuxI-LVA + Input2 AHL), repressed by TetR-LVA and no aTc.
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.
V. fischeri <genbank>AF170104</genbank>
Synthesizes 3OC6HSL, which binds to LuxR. The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
An LVA tail (sequence: AANDENYALVA) was added to increase protein degradation. .
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
Cell density controller: pCon 0.36->luxI-LVA+pCon 0.70->luxR+pLux/Tet->lacZalpha-ccdB(weak RBS)
Cell Death = (Cell Density * pCon 0.36) + Input AHL
Input: AHL Output1: AHL Output2: Cell Density Design: pCon 0.36(K176009) generates LuxI-LVA, LuxI-LVA generates Output1 AHL, the concentration of AHL in the medium depends on both LuxI-LVA level per cell and cell density. pCon 0.70(J23101) generates LuxR. pLux/Tet(K176000) generates ccdB to kill the cell, activated by LuxR and (Output1 AHL generated by LuxI-LVA + Input AHL), repressed by TetR and no aTc.
Double terminator consisting of BBa_B0010 and BBa_B0012
weak RBS + lacZalpha-ccdB coding + strong terminator
cell density->PoPS: pCon 0.36->luxI-LVA+pCon 0.70->luxR+pLux/Tet->PoPS
Input: AHL Output1: AHL Output2: PoPS Design: pCon 0.36(K176009) generates LuxI-LVA, LuxI-LVA generates Output1 AHL, the concentration of AHL in the medium depends on both LuxI-LVA level per cell and cell density. pCon 0.70(J23101) generates LuxR. pLux/Tet(K176000) generates Output2 PoPS, activated by LuxR and (Output1 AHL generated by LuxI-LVA + Input AHL), repressed by TetR and no aTc
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.
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.
Medium RBS based on Ron Weiss thesis. Strength considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-1" in figure 4-14 of thesis). 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 Cho
PCR from the plasmid pluxCcdB3(a gift from Prof. Lingchong You). Forward Primer: GTTTCT TCTAG ATG accatgattacgg attcactggccgtcgttttac Reverse Primer: GTTTCTTC CTGCAG CGGCCGC T ACTAGT A TTATTA tattccccagaacatcaggtta Digest with XbaI and SpeI, ligate into pSB1A3.
A fusion protein of lacZalpha(I732006) and ccdB(K145151). It should have the function of ccdB to cause E. coli cells to die, but have less toxicity because of the lacZalpha part. It should also have the function of lacZalpha as a reporter for measurement. This part is similar to J32026 but the multiple cloning sites are eliminated. It is constructed by PCR from the plasmid pluxCcdB3(a gift from Prof. Lingchong You).
The part is similar to BBa_J32026 and the original coding sequence in pluxCcdB3, but the multiple cloning sites are eliminated in order to avoid site-specific mutagenesis. The multiple cloning sites are from lacZalpha in pZErO-2. After eliminate the MCS, the lacZalpha part is identical to the N-terminal of the wild type gene(BBa_I732006 & BBa_I732005), and fused with the ccdB part (BBa_K145151) with a 3aa linker.
TetR repressible AHL->PoPS Receiver (Very High LuxR): pCon 0.70->luxR+pLux/Tet->PoPS
AHL -> PoPS Receiver TetR repressible Similar to F2620, but use pLux/Tet hybrid promoter.
Input: AHL Output: PoPS Design: pCon 0.70(J23101) generate LuxR pLux/Tet(K176000) generate PoPS output, activated by LuxR and AHL, repressed by TetR and no aTc
pLux/Tet Hybrid Promoter: (LuxR+,TetR-)->PoPS
sense TWO INPUTS, activation by AHL and repression by tetR
constitutive promoter family member J23107 actual sequence (pCon 0.36)
The sequence of J23107 is wrong according to sequencing result of the registry and ours. This part K176009 is created for the actual promoter. http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=3628 http://partsregistry.org/cgi/sequencing/one_blast.cgi?id=1184 J23107: tttacggctagctcagCcctaggtattatgctagc K176009: tttacggctagctcagTcctaggtattatgctagc
Produces LuxR protein which can sense 3OC6HSL in the media and activate transcription from R0062, the right hand Lux promoter
In complex with HSL, LuxR binds to the Lux promoter, activating transcription from Pr <bb_part>BBa_R0062</bb_part>, and repressing transcription from Pl <bb_part>BBa_R0063</bb_part>. The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the Lux activator, LuxR complexed to HSL. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
2 silent point mutants were introduced in the coding sequence to remove internal XbaI and PstI sites. Mutation sites were chosen to replace codons commonly used in E. coli with codons used at a similar frequency.
Conversion of the iGEM parts registry to SBOL2.1