BBa_M50053 1 BBa_M50053 FRET-based glucose sensor using a glucose binding protein, mRuby3 and cometGFP 2016-12-11T12:00:00Z 2016-12-11T10:00:33Z source This is a device that uses a stable glucose binding protein (GBP) flanked with two compatible fluorescent proteins to indicate glucose levels with a fluorescent resonance energy transfer (FRET) based actuator. The device input is glucose-binding and the device output is fluorescent spectra of the two flurophore. FRET was achieved using mRuby3 as the acceptor fluorophore and cometGFP as the donor fluorophore. Upon glucose binding, this device will undergo a conformational change such that the cometGFP protein in an excited state would transfer energy to the acceptor mRuby3 to be emitted. Quantifying this excitation/emission pair would reveal the extent to which FRET and thus glucose binding that had occurred, relative to the open, unbounded state. false false _848_ 34390 34390 9 false design notes false Amanda Urke, Tofe Alimi, Katherine Vera component2532583 1 BBa_M50031 component2532584 1 BBa_M36771 component2532585 1 BBa_M50009 component2532587 1 BBa_M50037 component2532588 1 BBa_M50036 component2532589 1 BBa_M36936 component2532586 1 BBa_M50036 annotation2532584 1 BBa_M36771 range2532584 1 49 61 annotation2532587 1 BBa_M50037 range2532587 1 791 2029 annotation2532585 1 BBa_M50009 range2532585 1 62 772 annotation2532583 1 BBa_M50031 range2532583 1 1 48 annotation2532589 1 BBa_M36936 range2532589 1 2048 2320 annotation2532588 1 BBa_M50036 range2532588 1 2030 2047 annotation2532586 1 BBa_M50036 range2532586 1 773 790 BBa_M36771 1 BBa_M36771 Strong RBS from DNA 2.0 Gene Designer 2014-10-22T11:00:00Z 2015-05-08T01:14:06Z DNA 2.0 Gene Designer Strong Ribosome Binding Site from DNA 2.0 Gene Designer. false false _848_ 0 24189 9 Not in stock false None. false Preston Lim BBa_M50036 1 BBa_M50036 6 Amino Acid Fusion Protein Linker 2016-12-11T12:00:00Z 2016-12-11T04:55:01Z Linker sequence was suggested and provided by our TA, Salil Bahte, and then optimized for use in E. coli. This sequence encodes for six amino acids that constitute the fusion protein linker for our Stanford BioE44 DNA construct. It is optimized for use in E. coli, and is designed to maintain linearity of the sequence and prevent protein folding. Our design relied on proper orientation of two GBP domains linked on each end to a flurophore. The linkers were included to ensure that glucose-binding caused the conformational change characterized with FRET instead of inherently folded protein structure, the linkers were included. false false _848_ 34390 34390 9 false Codon optimization software was used since we transformed our plasmid into E. coli. Also, the linker needed to be relatively short since we were already designing a long construct and needed to minimize the synthesis time given class constraints. false Amanda Urke BBa_M50009 1 BBa_M50009 mRuby3 fluorescent protein 2016-11-29T12:00:00Z 2016-12-11T04:21:38Z http://www.nature.com/articles/srep20889 mRuby3 is a brighter and more photostable variant of a red fluorescent protein developed by Bryce T. Bajar et al. According to their February 2016 study, mRuby3 has a maximum excitation wavelength of 558 nm, and a maximum emission wavelength of 592 nm. It can be used in FRET analysis as an acceptor fluorophore when paired with a GFP such as cometGFP. false false _848_ 34390 34390 9 false choosing to put it in our custom part of the plasmid rather than GFP as we had originally intended false Amanda Urke annotation2532561 1 Deletion of stop codon to integrate subsequent parts of construct range2532561 1 711 711 BBa_M50037 1 BBa_M50037 Glucose Binding Protein of Thermus thermophilus 2016-12-11T12:00:00Z 2016-12-11T05:29:45Z This GBP comes from the genomic sequence of Thermus thermophilus. The complete bacterial genome was published by Henne et al. in Nature Biotechnology in 2004 (https://www.ncbi.nlm.nih.gov/pubmed/15064768), and the particular GBP was characterized in a 2006 study by Cuneo et al. in the Journal of Molecular Biology (https://www.ncbi.nlm.nih.gov/pubmed/16904687). This bacterial glucose binding protein (GBP) is derived from the genome sequence of Thermus thermophilus that was published in 2004 by Henne et al. In 2006, Cuneo et al. analyzed the sequence of this particular GBP, determined its X-ray crystal structure and characterized the stability of glucose-binding. The GBP is composed of two domains separated by a hinge, maintaining an open conformation when unbound to glucose and a closed conformation when bound. When flanked with an RFP fluorophore on the N-terminus and a GFP fluorophore on the C-terminus, these conformation changes facilitated FRET-based analysis of glucose binding. In addition, this GBP was selected for its reasonably short sequence length (1242bp) and stable glucose binding ability (Kd value of 0.08(+/- 0.03) mM). According to Cuneo et al., this T. thermophilus GBP shares more structural homology with maltose binding proteins than glucose binding proteins. However, we were able to obtain FRET when glucose was added, suggesting that glucose is a ligand for this protein. false false _848_ 34390 34390 9 false This GBP sequence was optimized for use in E. coli. Also, start and stop codons were removed to facilitate fusion to the other components of our construct. false Amanda Urke annotation2532563 1 Deletion of stop codon to facilitate fusion to linker and fluorophore range2532563 1 1239 1239 annotation2532562 1 Deletion of start codon to facilitate fusion to linker and fluorophore range2532562 1 1 1 BBa_M50031 1 BBa_M50031 pP-T5 promoter 2016-11-01T12:00:00Z 2016-11-01T06:29:33Z Gene Designer 2.0 Generic E. coli T5 promoter obtained from Gene Designer 2.0 false false _848_ 34410 34409 9 false None. false Cale Lester, Raquel Freeman, Tristan Yeung BBa_M36936 1 BBa_M36936 CometGFP 2014-10-22T11:00:00Z 2015-05-08T01:14:07Z DNA 2.0 GFP reporter sequence recommended by DNA 2.0. false false _848_ 0 24151 9 Not in stock false None false Emily Kelly BBa_M50053_sequence 1 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BBa_M50037_sequence 1 cgcaagtggttacttgcaatcggcatggtgctgggtttaagtgccttagcccaagggggaaagcttgaaatttttagttggtgggctggcgacgagggccctgcgctggaagcgcttatccgtttgtataaacagaagtacccaggtgtagaggttattaatgccacggtaacgggcggtgcgggtgtgaacgcacgtgcggtgttgaaaacacgcatgttaggaggcgacccaccagatactttccaagtgcatgcgggaatggagttgattggcacatgggtcgtcgcaaaccgtatggaggacctgtcggctttattccgtcaggaaggatggttgcaggcatttcctaagggattgattgacctgatctcttacaaaggcggaatctggtcagtccccgttaatatccaccgcagcaacgtcatgtggtaccttcctgctaagttgaaagagtggggtgtaaacccacctcgtacatgggacgaatttttggccacttgtcaaacattaaagcaaaagggtttagaagcaccccttgcattgggcgaaaattggacccagcagcatctgtgggaatcagtggccttggcggtattgggtccagatgactggaataatctttggaacgggaagttgaagtttacggaccccaaggcagttcgcgcttgggaggtattcggtcgcgtacttgactgtgcgaataaagatgccgctgggctgagctggcaacaagcggtagatcgtgtagtacaaggaaaagccgctttcaacgtgatgggggactgggccgcagggtatatgaccacgaccctgaagttgaagccgggcaccgacttcgcttgggccccttctccgggtactcaaggagtttttatgatgctgtcggactcctttggacttccaaaaggagctaagaatcgtcaaaacgctatcaattggttgcgtttagtgggttcgaaagagggacaagacacatttaacccattaaagggttccattgcagcgcgtttggatagcgatccatccaagtacaatgcatatgggcagtccgccatgcgtgattggcgttctaatcgtatcgttggctcgttggtccacggtgcggtcgctccagagtcattcatgtctcagttcggcacggtaatggaaatctttcttcagacacgcaacccgcaggccgccgcgaacgctgctcaggccattgcggatcaggtgggactgggccgcttggggcag BBa_M50009_sequence 1 atggtgtctaagggcgaagagctgatcaaggaaaatatgcgtatgaaggtggtcatggaaggttcggtcaacggccaccaattcaaatgcacaggtgaaggagaaggcagaccgtacgagggaactcaaaccatgaggatcaaagtcatcgagggaggacccctgccatttgcctttgacattcttgccacgtcgttcatgtatggcagccgtacttttatcaagtacccggccgacatccctgatttctttaaacagtcctttcctgagggttttacttgggaaagagttacgagatacgaagatggtggagtcgtcaccgtcacgcaggacaccagccttgaggatggcgagctcgtctacaacgtcaaggtcagaggggtaaactttccctccaatggtcccgtgatgcagaagaagaccaagggttgggagcctaatacagagatgatgtatccagcagatggtggtctgagaggatacactgacatcgcactgaaagttgatggtggtggccatctgcactgcaacttcgtgacaacttacaggtcaaaaaagaccgtcgggaacatcaagatgcccggtgtccatgccgttgatcaccgcctggaaaggatcgaggagagtgacaatgaaacctacgtagtgcaacgcgaagtggcagttgccaaatacagcaaccttggtggtggcatggacgagctgtacaag BBa_M36771_sequence 1 aggaggtaaaaaa BBa_M50031_sequence 1 aaatcatgaaaaatttatttgctttgtgagcggataacaattataata BBa_M50036_sequence 1 gggggcaccggcgggtcc BBa_M36936_sequence 1 gcgtgctgcggctgcaccggcgcgggctgcgcggcgaccgcggcgtgcaccgcgggctgcgcgaccgcggcgtgctgctgctgcaccaccggcggcggcggctgctgcacctgcaccgcggcggcgtgcggcggcggcacctgcaccaccggcgcgggcggcggcggcaccaccaccaccaccaccggctgcaccggcgcggcggcgggcggcgcgggcggcgcggcgtgcaccgcgaccgcgacctgctgcggcggcggcaccgcggcgtgc 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