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 (&#8710;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, &#8710;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 annotation2521691 1 c range2521691 1 99 99 annotation2522553 1 g range2522553 1 996 996 annotation2501346 1 PctA LBD + HAMP range2501346 1 1 1062 annotation2521679 1 Tar signaling domain range2521679 1 1063 1919 annotation2521700 1 a range2521700 1 402 402 annotation2521701 1 a range2521701 1 405 405 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 annotation1687 1 stop range1687 1 34 34 annotation7020 1 BBa_B0012 range7020 1 1 41 annotation1686 1 T7 TE range1686 1 8 27 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_K1992007 1 BBa_K1992007 PctA-Tar expression system (promoter+RBS+coding+terminator) 2016-10-13T11:00:00Z 2016-10-18T03:25:21Z see K1992001 part Introduction Here is the plasmid which serves as the plasmid of the PctA-Tar chimera. The plasmid is comprised of a promoter, a strong RBS , the chimera and a double terminator. Usage and Biology Essentially, the expression of the chimera is enabled independently by this device, while taking advantage of the bacterium expression resources. For methods and results please go to PctA-Tar (K1992001) false false _2459_ 30463 30463 9 false none false Shilo Ohayon, Tzila Davidov component2501851 1 BBa_B0015 component2501840 1 BBa_J23100 component2501842 1 BBa_B0034 component2501844 1 BBa_K1992001 annotation2501851 1 BBa_B0015 range2501851 1 1990 2118 annotation2501840 1 BBa_J23100 range2501840 1 1 35 annotation2501842 1 BBa_B0034 range2501842 1 44 55 annotation2501844 1 BBa_K1992001 range2501844 1 62 1981 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_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_J23100_sequence 1 ttgacggctagctcagtcctaggtacagtgctagc BBa_B0010_sequence 1 ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc BBa_B0034_sequence 1 aaagaggagaaa BBa_K1992001_sequence 1 atgatcaaaagtctgaagttcagccacaagatcctcctcgcggcctccctcgtggtgttcgccgctttcgcactgttcaccctctacaacgattacctccagcgcaacgcgatccgcgaggacctcgaaagctacctgcgggaaatgggtgacgtgacttccagcaacattcagaactggctcggcggccgtctgctgctggtcgagcagaccgcccagaccctggcccgcgaccacagcccggaaaccgtcagcgccctgctcgaacagccggccctgacctcgaccttcagcttcacctacctcggccagcaggacggcgtcttcaccatgcgtccggacagcccgatgccggccggctacgatccgcgcagccggccctggtacaaggacgccgtggcagcaggcggcctgaccctgaccgaaccctacgtcgacgccgccacccaggaattgatcatcaccgcggcgacaccggtgaaggccgctggcaacaccctcggcgtggtaggcggcgacctcagcctgaagaccctggtgcagatcatcaattcgctggacttcagcggcatgggctacgccttcctggtcagcggcgacggcaagatcctggtgcacccggacaaagagcaggtgatgaagaccctgagcgaggtctacccgcagaacacgccgaagatcgccaccggtttcagcgaggccgaattgcacgggcatacccgcatcctcgcctttacccctatcaagggcctgccttcggtgacctggtacctggcgctgtccatcgacaaggacaaggcctacgccatgctcagcaagttccgcgtctcggccatcgccgccgcgctgatctccatcgtcgccatcctggtcctgctcggcctgctgatccgcctgctgatgcagccgctgcacctgatgggccgtgccatgcaggacatcgcccagggcgagggcgacctgaccaagcgcctcgccgtgaccagccgcgatgagttcggagtgctcggcgatgccttcaaccagttcgtcgagcgtatccatcgctcgatccgggaagtccgcgaaggttcagatgccatctatgccggtacccgtgaaattgcggcgggcaacaccgatctttcctcccgtactgaacagcaggcatccgcgctggaagaaactgccgccagcatggagcagctcaccgcgacagtgaagcaaaacgccgataacgcccgccaggcctcgcaactggcgcaaagtgcctccgacaccgcccagcacggcggcaaagtggtggatggcgtagtgaaaacgatgcatgagatcgccgatagttcgaagaaaattgccgacattatcagcgttatcgacggtattgccttccagactaatatcctcgcgctgaatgccgcggttgaagccgcgcgtgcgggtgaacagggccgtggttttgccgtggtggcgggtgaagtgcgtaatcttgccagtcgcagcgcccaggcggcaaaagagatcaaagccctcattgaagactccgtctcacgcgttgataccggttcggtgctggtcgaaagcgccggggaaacaatgaacaatatcgtcaatgctgtcactcgcgtgactgacattatgggcgagattgcatcggcatcggatgaacagagccgtggcatcgatcaagtcgcattggcggtttcggaaatggatcgcgtcacgcaacagaacgcatcgctggtgcaggaatcagctgccgccgccgctgcgctggaagaacaggcgagtcgtttaacgcaagcagtttccgcgttccgtctggcagccagcccactcaccaataaaccgcaaacaccatcccgtcctgccagtgagcaaccaccggctcagccacgactgcgaattgctgaacaagatccaaactgggaaacattttga BBa_K1992007_sequence 1 ttgacggctagctcagtcctaggtacagtgctagctactagagaaagaggagaaatactagatgatcaaaagtctgaagttcagccacaagatcctcctcgcggcctccctcgtggtgttcgccgctttcgcactgttcaccctctacaacgattacctccagcgcaacgcgatccgcgaggacctcgaaagctacctgcgggaaatgggtgacgtgacttccagcaacattcagaactggctcggcggccgtctgctgctggtcgagcagaccgcccagaccctggcccgcgaccacagcccggaaaccgtcagcgccctgctcgaacagccggccctgacctcgaccttcagcttcacctacctcggccagcaggacggcgtcttcaccatgcgtccggacagcccgatgccggccggctacgatccgcgcagccggccctggtacaaggacgccgtggcagcaggcggcctgaccctgaccgaaccctacgtcgacgccgccacccaggaattgatcatcaccgcggcgacaccggtgaaggccgctggcaacaccctcggcgtggtaggcggcgacctcagcctgaagaccctggtgcagatcatcaattcgctggacttcagcggcatgggctacgccttcctggtcagcggcgacggcaagatcctggtgcacccggacaaagagcaggtgatgaagaccctgagcgaggtctacccgcagaacacgccgaagatcgccaccggtttcagcgaggccgaattgcacgggcatacccgcatcctcgcctttacccctatcaagggcctgccttcggtgacctggtacctggcgctgtccatcgacaaggacaaggcctacgccatgctcagcaagttccgcgtctcggccatcgccgccgcgctgatctccatcgtcgccatcctggtcctgctcggcctgctgatccgcctgctgatgcagccgctgcacctgatgggccgtgccatgcaggacatcgcccagggcgagggcgacctgaccaagcgcctcgccgtgaccagccgcgatgagttcggagtgctcggcgatgccttcaaccagttcgtcgagcgtatccatcgctcgatccgggaagtccgcgaaggttcagatgccatctatgccggtacccgtgaaattgcggcgggcaacaccgatctttcctcccgtactgaacagcaggcatccgcgctggaagaaactgccgccagcatggagcagctcaccgcgacagtgaagcaaaacgccgataacgcccgccaggcctcgcaactggcgcaaagtgcctccgacaccgcccagcacggcggcaaagtggtggatggcgtagtgaaaacgatgcatgagatcgccgatagttcgaagaaaattgccgacattatcagcgttatcgacggtattgccttccagactaatatcctcgcgctgaatgccgcggttgaagccgcgcgtgcgggtgaacagggccgtggttttgccgtggtggcgggtgaagtgcgtaatcttgccagtcgcagcgcccaggcggcaaaagagatcaaagccctcattgaagactccgtctcacgcgttgataccggttcggtgctggtcgaaagcgccggggaaacaatgaacaatatcgtcaatgctgtcactcgcgtgactgacattatgggcgagattgcatcggcatcggatgaacagagccgtggcatcgatcaagtcgcattggcggtttcggaaatggatcgcgtcacgcaacagaacgcatcgctggtgcaggaatcagctgccgccgccgctgcgctggaagaacaggcgagtcgtttaacgcaagcagtttccgcgttccgtctggcagccagcccactcaccaataaaccgcaaacaccatcccgtcctgccagtgagcaaccaccggctcagccacgactgcgaattgctgaacaagatccaaactgggaaacattttgatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata 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