BBa_B0032
1
BBa_B0032
RBS.3 (medium) -- derivative of BBa_0030
2003-01-31T12:00:00Z
2015-08-31T04:07:20Z
Released HQ 2013
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
false
true
_41_44_48_46_1_
0
24
7
In stock
false
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). <P>
Contact info for this part: <a href="mailto:(bchow@media.mit.edu)">Brian Chow</a>
true
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
annotation7027
1
BBa_B0032
range7027
1
1
13
annotation1710
1
RBS
range1710
1
7
10
annotation1709
1
RBS-3\Weak
range1709
1
1
13
BBa_K360023
1
BBa_K360023
trpL promoter
2010-10-20T11:00:00Z
2015-05-08T01:12:12Z
LovTAP was assembled by Strickland et al.
Reference
Strickland, D., Moffat, K., & Sosnick, T. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences, 105(31), 10709. National Acad Sciences. Retrieved from http://www.pnas.org/content/105/31/10709.full.
Released HQ 2013
LovTAP
This photoreceptor was assembled by Strickland et al., and consists of a LOV (light-oxygen-voltage) domain of Avena sativa phototropin1 (AsLOV2) that senses blue light, fused to the trpR- DNA binding domain of the transcription factor trp repressor. The resulting protein is called LovTAP: LOV- and tryptophan-activated protein.
LOV domains bind a flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) cofactor, which are used in a wide variety of metabolic pathways as cofactors in redox reactions and are available in most organisms. The cofactor has a broad absorption spectrum, with a maximum at 450 nm. Besides, the core of the LOV domain is often flanked by amino- or carboxy-terminal helices, termed A???α and Jα, respectively.
In the LovTAP construction, AsLOV2 domain via its carboxyl-terminal Jα ???helix was ligated to an amino-terminal truncation of TrpR. The resulting protein has a domain-domain overlap with a shared helix. Thus, photoexcitation would change the conformation of the protein, in turn changing the stability of the shared-helix-domain contacts.
Under the presence of light, absorption of a photon leads to the formation of a covalent adduct between the flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in the AsLOV2 domain, which results in conformational rearrangements in LovTAP. This change impacts the affinity of the shared helix for the two domains: disrupting the contacts between the shared helix and the LOV domain and enabling the association of the shared helix with the TrpR domain, which establishes DNA-binding affinity and LovTAP can then bind DNA as an homodimer, repressing the transcription of the genes downstream of its binding sites.
In the dark, when the shared helix contacts the LOV domain, the TrpR domain's DNA-binding affinity decreases and LovTAP is in an inactive conformation.
false
true
_485_
0
6618
9
In stock
false
We decided to synthesize a new LovTap part, that in comparison with the Part:BBa_K191006[1] that is already at the registry, has the following differences:
1. The 2 '''PstI restriction sites''' were '''removed''' from the coding region of LovTap.
2. We included a punctual mutation to change the '''ILE427 by a PHE427''', as was proposed by the model results of the team iGEM09_EPF-Lausanne [2]. With this mutation LovTAP should react faster and the conformational change should be more stable (the protein stays in the active form for longer, under light induction).
The reason of the conformational change is the following:
Cys450 side chain is involved in light state in bond formation with the cofactor. Cys450 can assume two conformational states, called here ON and OFF, and corresponding respectively, to the Sg being near or far from FMN[2].
The isoleucine 427 is quite big. But not enough to push the cystein's side chain significantly toward the cofactor. So we choose to replace this ILE427 by an PHE427, an amino acid which is much bigger and have more or less the same propreties than the ILE[2].
5. The stop codon tga was changed for two taa.
1. Registry entry: Part:BBa_K191003
2. Wiki Team: EPF-Laussane. Simulations and results of predicted lovTAP mutations.
3. Wiki Team: EPF-Laussane. LovTAP characterization results.
4. Registry entry: Part:BBa_B0030
5. Registry entry: Part:BBa_B0032
6. Registry entry: Part:BBa_R0010
false
Claudia Ivonne Hernandez Armenta, Jorge Zepeda
annotation2091494
1
trpR binding site -11.5
range2091494
1
21
40
annotation2091495
1
trpR binding site -19.5
range2091495
1
13
30
annotation2091493
1
trpR binding site -3.5
range2091493
1
29
45
annotation2091496
1
-35
range2091496
1
3
11
annotation2091497
1
-10
range2091497
1
28
35
BBa_E0040
1
GFP
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
2004-09-29T11:00:00Z
2016-01-26T02:09:38Z
Released HQ 2013
GFP (mut3b) [note that this part does not have a barcode]
false
true
_11_1_
4206
61
7
In stock
false
true
jcbraff
annotation1934520
1
GFP protein
range1934520
1
1
720
BBa_B0010
1
BBa_B0010
T1 from E. coli rrnB
2003-11-19T12:00:00Z
2015-08-31T04:07:20Z
Transcriptional terminator consisting of a 64 bp stem-loop.
false
false
_1_
0
24
7
In stock
false
true
Randy Rettberg
annotation7018
1
BBa_B0010
range7018
1
1
80
annotation4184
1
stem_loop
range4184
1
12
55
BBa_E0240
1
GFP report
GFP generator
2004-10-17T11:00:00Z
2015-08-31T04:07:26Z
Released HQ 2013
B0032.E0040.B0015
false
true
_11_1_
0
61
7
In stock
false
true
Jennifer Braff
component1249216
1
BBa_E0040
component1249221
1
BBa_B0010
component1249213
1
BBa_B0032
component1249231
1
BBa_B0012
annotation1249231
1
BBa_B0012
range1249231
1
836
876
annotation1249216
1
BBa_E0040
range1249216
1
20
739
annotation1249221
1
BBa_B0010
range1249221
1
748
827
annotation1249213
1
BBa_B0032
range1249213
1
1
13
BBa_B0012
1
BBa_B0012
TE from coliphageT7
2003-01-31T12:00:00Z
2015-08-31T04:07:20Z
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Released HQ 2013
Transcription terminator for the <i>E.coli</i> RNA polymerase.
false
false
_1_
0
24
7
In stock
false
<P> <P>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).<P> Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
false
Reshma Shetty
annotation1690
1
polya
range1690
1
28
41
annotation7020
1
BBa_B0012
range7020
1
1
41
annotation1687
1
stop
range1687
1
34
34
annotation1686
1
T7 TE
range1686
1
8
27
BBa_K360124
1
BBa_K360124
trpL promoter + GFP
2010-10-20T11:00:00Z
2015-05-08T01:12:12Z
The seven bases spacer is the one from Escherichia coli's LacZ gene.
Strong RBS (BBa_B0034) with a spacer of seven bases between the RBS and the start codon which makes more efficient the transcription.
false
false
_485_
0
6838
9
Not in stock
false
We haven't characterized t yet, so compared with other spacers we don't know the exact yield.
false
Claudia Ivonne Hernandez Armenta, Jorge Arturo Zepeda Mart??nez
component2091687
1
BBa_K360023
component2091698
1
BBa_E0240
annotation2091698
1
BBa_E0240
range2091698
1
58
933
annotation2091687
1
BBa_K360023
range2091687
1
1
49
BBa_K360124_sequence
1
gctgttgacaattaatcatcgaactagttaactagtacgcaagttcacgtactagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
BBa_B0010_sequence
1
ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc
BBa_K360023_sequence
1
gctgttgacaattaatcatcgaactagttaactagtacgcaagttcacg
BBa_B0032_sequence
1
tcacacaggaaag
BBa_E0240_sequence
1
tcacacaggaaagtactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
BBa_E0040_sequence
1
atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataa
BBa_B0012_sequence
1
tcacactggctcaccttcgggtgggcctttctgcgtttata
igem2sbol
1
iGEM to SBOL conversion
Conversion of the iGEM parts registry to SBOL2.1
Chris J. Myers
James Alastair McLaughlin
2017-03-06T15:00:00.000Z