PctA-Tar GFP tagged expression system (promoter+RBS+coding+terminator)
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)
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.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Double terminator consisting of BBa_B0010 and BBa_B0012
Highly flexible 6 amino acid linker. Translates to gsgsgs. Codon-optimize for E. coli, yeast, mammalian.
* gene synthesis * codon optimized for E. coli
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
gBlocks were not compatible so we had to split them into two fragments
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