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_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
annotation1686
1
T7 TE
range1686
1
8
27
annotation1687
1
stop
range1687
1
34
34
BBa_K1992010
1
BBa_K1992010
PctA-Tar GFP tagged expression system (promoter+RBS+coding+terminator)
2016-10-13T11:00:00Z
2016-10-18T03:40:03Z
none
Introduction
A GFP gene was fused to the PctA chimera. The plasmid is comprised of a promoter, a strong RBS, the chimera, a GFP gene and a terminator. In addition, a proper linker ought to be introduced in order to ensure the right correct fold of the protein.
Usage and Biology
This device was used as a proof of concept in order to verify the migration of the new chemoreceptor to the poles of the bacterial membrane.
Design Considerations
A flexible linker was introduced to fuse the GFP gene to the chimera. The linker is essential for the protein to fold in the right manner and for the correct tertiary structure to be obtained.
For methods and results please go to PctA-Tar (K1992001)
false
false
_2459_
30463
30463
9
false
none
false
Shilo Ohayon
component2501982
1
BBa_E0040
component2501977
1
BBa_B0034
component2501989
1
BBa_B0015
component2501979
1
BBa_K1992001
component2501980
1
BBa_J18921
component2501975
1
BBa_J23100
annotation2501975
1
BBa_J23100
range2501975
1
1
35
annotation2501977
1
BBa_B0034
range2501977
1
44
55
annotation2501989
1
BBa_B0015
range2501989
1
2742
2870
annotation2501979
1
BBa_K1992001
range2501979
1
62
1981
annotation2501980
1
BBa_J18921
range2501980
1
1990
2007
annotation2501982
1
BBa_E0040
range2501982
1
2014
2733
BBa_J18921
1
3xGS
6aa [GS]x linker
2010-01-26T12:00:00Z
2015-08-31T04:08:36Z
gene synthesis
Highly flexible 6 amino acid linker. Translates to gsgsgs. Codon-optimize for E. coli, yeast, mammalian.
false
false
_165_
0
2175
165
It's complicated
false
* gene synthesis
* codon optimized for E. coli
false
Raik Gruenberg
BBa_K1992001
1
BBa_K1992001
PctA-Tar hybrid receptor
2016-10-13T11:00:00Z
2016-10-18T03:07:56Z
The sequence for the desired segment of PctA was taken from the Pseudomonas genome database http://www.pseudomonas.com/feature/show?id=111456. Tar???s cytoplasmic region, was obtained from the iGEM parts catalog
PctA-Tar
Introduction
PctA is a chemoreceptor found in the bacterium Pseudomonas Aeruginosa which mediates chemotaxis toward amino acids and away from organic compounds. It is sensitive to all amino acids accept for Aspartate (1).
A PctA-Tar chimera was formed by the replacement of the Tar LBD with the one of PctA. A procedure which previously was conducted in the literature was recovered (1), in order to proof the concept of the S. Tar platform.
Usage and biology
Integration of the new chemoreceptor with the chemotaxis system of E. coli is enabled thank our chimera design. Hence, a chemotactic attractant response to new amino acids and a repellent response to organic compounds, which weren't detected by E. coli before, are now mediated.
Design considerations
When designing a gBlock for the PctA-Tar Chimera an incompatible part is obtained, this according to the IDT gBlock sequence online analyzer (2). As a result, a split part has to be designed. Therefore it is recommended to use the part itself, since its synthesis is problematic.
Methods
The sequence for the desired segment of PctA was taken from the Pseudomonas genome database (3). The second part of the chimera, Tar???s cytoplasmatic region, was obtained from the iGEM parts catalog. Amino acids sequence of the LBD and HAMP of PctA was determined to be from 1 to 134 and this of the signaling region of Tar was determined to be 296-553 (Figure 1a).
Figure 1: (a) Amino acids sequence of PctA-Tar chimera (1) (b) PctA-Tar chimera crystalogaphy
The plasmid carrying the chimera was transformed into receptorless bacteria- UU1250 (Parkinson J S, University of Utah) and the presents of the DNA sequence was verified by a colony PCR and sequencing.
In order to test the motility of our strain, chemotaxis assays were performed. First, swarming assay was conducted using a poor BA medium. Note that with a TB medium the strain failed to show decisive results, provably due to high concentration of amino acid which resulted in no movement.
Following the swarming assay, a GFP gene was fused to the chimera to verify the migration of the chemoreceptor to the poles of the bacterial membrane.
In addition, a protocol for a chemotaxis assay on chip for repellent response was constructed by us and we are the first group to show this kind of assay. A receptorless strain containing a PctA-Tar chimera plasmid (pSB1A2 was served as vector) and a blue chromoprotein plasmid (pSB1C3 was served as a vector with J23100+K592009 biobricks). This clone resulted in blue bacteria expressing the PctA-Tar chimera. The strain was confined into a commercial ibidi microchannel while Tetrachloroethylene (PCE) served as a repellent. PCE in the concentration of 9??10-5M (0.015 g/lit) was inserted through the well and the bacterial movement was monitored throughout the entire experiment.
Results
The strain containing the PctA-Tar chimera showed a significant movement on the swarming assay with a BA medium. On figure 1 the swarming assay of our strain is shown compared to our negative control (UU1250) and our positive control (∆Z). Hence, the PctA-Tar chimera is expressed and bacterial movement is mediated by it.
Figur 2: Swarming assay for attractant response of the PctA-Tar chimera. From left to right: PctA chimera, UU1250, ∆Z.
As for the GFP fusion, the clone exhibited fluorescence on the polar parts of the bacterial membrane (Figure 3), indicating for a proper migration of the chimera to the poles of the membrane, as expected.
Figure 3: GFP fused to the PctA-Chimera is located in the polar part of the bacterial membrane. From left to right: PctA-Tar strain, Cloned Tar strain.
Moreover, a repellent response was shown while using our new chemotaxis assay for repellent response. The bacterial movement away from the PCE was distinguished to the naked eye and significant.
conclution
Using our strain containing the PctA-Tar chimera we showed a modified response of the Tar chemoreceptor to substances which are natural attractants and repellents of PctA as it found originally in the bacterium Pseudomonas
Aeruginosa. Therefore, this work serves as a decisive proof of concept for our S. Tar platform and it has valuable scientific meanings for our project.
1)Reyes-Darias, J.A., Yang, Y., Sourjik, V., and Krell, T. (2015). Correlation between signal input and output in PctA
and PctB amino acid chemoreceptor of Pseudomonas aeruginosa. Mol. Microbiol. 96, 513???525.
2) https://eu.idtdna.com/site/order/gblockentry
3) http://www.pseudomonas.com/feature/show?id=111456
false
false
_2459_
30463
30463
9
Not in stock
true
gBlocks were not compatible so we had to split them into two fragments
false
Shilo Ohayon,Asif Gil
annotation2522553
1
g
range2522553
1
996
996
annotation2521679
1
Tar signaling domain
range2521679
1
1063
1919
annotation2521691
1
c
range2521691
1
99
99
annotation2521700
1
a
range2521700
1
402
402
annotation2521701
1
a
range2521701
1
405
405
annotation2501346
1
PctA LBD + HAMP
range2501346
1
1
1062
BBa_B0015
1
BBa_B0015
double terminator (B0010-B0012)
2003-07-16T11:00:00Z
2015-08-31T04:07:20Z
Released HQ 2013
Double terminator consisting of BBa_B0010 and BBa_B0012
false
true
_1_
0
24
7
In stock
false
true
Reshma Shetty
component1916610
1
BBa_B0010
component1916612
1
BBa_B0012
annotation1916612
1
BBa_B0012
range1916612
1
89
129
annotation1916610
1
BBa_B0010
range1916610
1
1
80
BBa_B0034
1
BBa_B0034
RBS (Elowitz 1999) -- defines RBS efficiency
2003-01-31T12:00:00Z
2015-08-31T04:07:20Z
Released HQ 2013
RBS based on Elowitz repressilator.
false
true
_1_
0
24
7
In stock
false
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. <p>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: <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.
annotation23325
1
conserved
range23325
1
5
8
BBa_J23100
1
BBa_J23100
constitutive promoter family member
2006-08-03T11:00:00Z
2015-08-31T04:08:40Z
Isolated from library of promoters
Released HQ 2013
Replace later
false
true
_52_
0
483
95
In stock
true
N/A
true
John Anderson
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
annotation4184
1
stem_loop
range4184
1
12
55
annotation7018
1
BBa_B0010
range7018
1
1
80
BBa_J23100_sequence
1
ttgacggctagctcagtcctaggtacagtgctagc
BBa_B0010_sequence
1
ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc
BBa_K1992010_sequence
1
ttgacggctagctcagtcctaggtacagtgctagctactagagaaagaggagaaatactagatgatcaaaagtctgaagttcagccacaagatcctcctcgcggcctccctcgtggtgttcgccgctttcgcactgttcaccctctacaacgattacctccagcgcaacgcgatccgcgaggacctcgaaagctacctgcgggaaatgggtgacgtgacttccagcaacattcagaactggctcggcggccgtctgctgctggtcgagcagaccgcccagaccctggcccgcgaccacagcccggaaaccgtcagcgccctgctcgaacagccggccctgacctcgaccttcagcttcacctacctcggccagcaggacggcgtcttcaccatgcgtccggacagcccgatgccggccggctacgatccgcgcagccggccctggtacaaggacgccgtggcagcaggcggcctgaccctgaccgaaccctacgtcgacgccgccacccaggaattgatcatcaccgcggcgacaccggtgaaggccgctggcaacaccctcggcgtggtaggcggcgacctcagcctgaagaccctggtgcagatcatcaattcgctggacttcagcggcatgggctacgccttcctggtcagcggcgacggcaagatcctggtgcacccggacaaagagcaggtgatgaagaccctgagcgaggtctacccgcagaacacgccgaagatcgccaccggtttcagcgaggccgaattgcacgggcatacccgcatcctcgcctttacccctatcaagggcctgccttcggtgacctggtacctggcgctgtccatcgacaaggacaaggcctacgccatgctcagcaagttccgcgtctcggccatcgccgccgcgctgatctccatcgtcgccatcctggtcctgctcggcctgctgatccgcctgctgatgcagccgctgcacctgatgggccgtgccatgcaggacatcgcccagggcgagggcgacctgaccaagcgcctcgccgtgaccagccgcgatgagttcggagtgctcggcgatgccttcaaccagttcgtcgagcgtatccatcgctcgatccgggaagtccgcgaaggttcagatgccatctatgccggtacccgtgaaattgcggcgggcaacaccgatctttcctcccgtactgaacagcaggcatccgcgctggaagaaactgccgccagcatggagcagctcaccgcgacagtgaagcaaaacgccgataacgcccgccaggcctcgcaactggcgcaaagtgcctccgacaccgcccagcacggcggcaaagtggtggatggcgtagtgaaaacgatgcatgagatcgccgatagttcgaagaaaattgccgacattatcagcgttatcgacggtattgccttccagactaatatcctcgcgctgaatgccgcggttgaagccgcgcgtgcgggtgaacagggccgtggttttgccgtggtggcgggtgaagtgcgtaatcttgccagtcgcagcgcccaggcggcaaaagagatcaaagccctcattgaagactccgtctcacgcgttgataccggttcggtgctggtcgaaagcgccggggaaacaatgaacaatatcgtcaatgctgtcactcgcgtgactgacattatgggcgagattgcatcggcatcggatgaacagagccgtggcatcgatcaagtcgcattggcggtttcggaaatggatcgcgtcacgcaacagaacgcatcgctggtgcaggaatcagctgccgccgccgctgcgctggaagaacaggcgagtcgtttaacgcaagcagtttccgcgttccgtctggcagccagcccactcaccaataaaccgcaaacaccatcccgtcctgccagtgagcaaccaccggctcagccacgactgcgaattgctgaacaagatccaaactgggaaacattttgatactagagggtagcggcagcggtagctactagatgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
BBa_B0034_sequence
1
aaagaggagaaa
BBa_K1992001_sequence
1
atgatcaaaagtctgaagttcagccacaagatcctcctcgcggcctccctcgtggtgttcgccgctttcgcactgttcaccctctacaacgattacctccagcgcaacgcgatccgcgaggacctcgaaagctacctgcgggaaatgggtgacgtgacttccagcaacattcagaactggctcggcggccgtctgctgctggtcgagcagaccgcccagaccctggcccgcgaccacagcccggaaaccgtcagcgccctgctcgaacagccggccctgacctcgaccttcagcttcacctacctcggccagcaggacggcgtcttcaccatgcgtccggacagcccgatgccggccggctacgatccgcgcagccggccctggtacaaggacgccgtggcagcaggcggcctgaccctgaccgaaccctacgtcgacgccgccacccaggaattgatcatcaccgcggcgacaccggtgaaggccgctggcaacaccctcggcgtggtaggcggcgacctcagcctgaagaccctggtgcagatcatcaattcgctggacttcagcggcatgggctacgccttcctggtcagcggcgacggcaagatcctggtgcacccggacaaagagcaggtgatgaagaccctgagcgaggtctacccgcagaacacgccgaagatcgccaccggtttcagcgaggccgaattgcacgggcatacccgcatcctcgcctttacccctatcaagggcctgccttcggtgacctggtacctggcgctgtccatcgacaaggacaaggcctacgccatgctcagcaagttccgcgtctcggccatcgccgccgcgctgatctccatcgtcgccatcctggtcctgctcggcctgctgatccgcctgctgatgcagccgctgcacctgatgggccgtgccatgcaggacatcgcccagggcgagggcgacctgaccaagcgcctcgccgtgaccagccgcgatgagttcggagtgctcggcgatgccttcaaccagttcgtcgagcgtatccatcgctcgatccgggaagtccgcgaaggttcagatgccatctatgccggtacccgtgaaattgcggcgggcaacaccgatctttcctcccgtactgaacagcaggcatccgcgctggaagaaactgccgccagcatggagcagctcaccgcgacagtgaagcaaaacgccgataacgcccgccaggcctcgcaactggcgcaaagtgcctccgacaccgcccagcacggcggcaaagtggtggatggcgtagtgaaaacgatgcatgagatcgccgatagttcgaagaaaattgccgacattatcagcgttatcgacggtattgccttccagactaatatcctcgcgctgaatgccgcggttgaagccgcgcgtgcgggtgaacagggccgtggttttgccgtggtggcgggtgaagtgcgtaatcttgccagtcgcagcgcccaggcggcaaaagagatcaaagccctcattgaagactccgtctcacgcgttgataccggttcggtgctggtcgaaagcgccggggaaacaatgaacaatatcgtcaatgctgtcactcgcgtgactgacattatgggcgagattgcatcggcatcggatgaacagagccgtggcatcgatcaagtcgcattggcggtttcggaaatggatcgcgtcacgcaacagaacgcatcgctggtgcaggaatcagctgccgccgccgctgcgctggaagaacaggcgagtcgtttaacgcaagcagtttccgcgttccgtctggcagccagcccactcaccaataaaccgcaaacaccatcccgtcctgccagtgagcaaccaccggctcagccacgactgcgaattgctgaacaagatccaaactgggaaacattttga
BBa_J18921_sequence
1
ggtagcggcagcggtagc
BBa_E0040_sequence
1
atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataataa
BBa_B0012_sequence
1
tcacactggctcaccttcgggtgggcctttctgcgtttata
BBa_B0015_sequence
1
ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
igem2sbol
1
iGEM to SBOL conversion
Conversion of the iGEM parts registry to SBOL2.1
James Alastair McLaughlin
Chris J. Myers
2017-03-06T15:00:00.000Z