BBa_K638202 1 BBa_K638202 Arabinose inducible Poly-His Reflectin A1 generator 2011-09-18T11:00:00Z 2015-05-08T01:12:57Z The aim of this design was to produce a his-tagged variant of reflectin A1 so that the protein could be purified by his-affinity column purification. In addition, pBAD was used to allow us to induce expression of the protein with arabinose. The promoter and his-tag sequences were taken from the registry (see subpart list) as were the RBS and the terminator. We used Gibson Assembly to insert the reflectin gene, after we had checked for illeagal restriction sites. We found none, and thanks to the sequence being codon optimised for expression in E.coli had little trouble expressing and purifying the protein. Released HQ 2013 Arabinose inducible Poly-His Reflectin A1 generator produces a poly-histidine (his-tagged) form of reflectin A1. Bacteria expressing this construct will produce Reflectin A1 with a poly-histidine tag to enable purification with a his-affinity column used in conjunction with nickel or cobalt resins. The purified protein has optical properties, best viewed when made into a thin film. First the protein must be dissolved in an ionic solvent such as HFIP (hazardous - use in fume hood) and then can be coated onto a highly clean smooth substrate. Silicon Wafers and glass microscope slides have been shown to work, a razorblade can be used to scrape across a single drop to form a thin film, but more uniform results can be achieved with the use of a flow coater or spin coater device. false false _814_ 0 9698 9 In stock false This part was built from a codon optimised variant of the Reflectin A1 gene provided by Wendy Crookes. We added a his-tag by incorporating the part Part:BBa_K128005 into Gibson Assembly primers. false Matthew Jones annotation2135475 1 PC range2135475 1 1043 1114 annotation2135488 1 stop range2135488 1 2456 2456 annotation2135482 1 stop codon range2135482 1 2317 2319 annotation2135543 1 ATG range2135543 1 1237 1239 annotation2135478 1 conserved range2135478 1 1223 1226 annotation2135473 1 AraC range2135473 1 1 1079 annotation2135486 1 T7 TE range2135486 1 2423 2442 annotation2135474 1 BBa_I0500 range2135474 1 1 1210 annotation2135483 1 BBa_B0010 range2135483 1 2328 2407 annotation2135487 1 polya range2135487 1 2443 2458 annotation2135485 1 BBa_B0012 range2135485 1 2416 2457 annotation2135484 1 stem_loop range2135484 1 2339 2382 annotation2135479 1 BBa_K128005 range2135479 1 1239 1257 annotation2135476 1 Pbad range2135476 1 1080 1210 annotation2135480 1 BBa_K638001 range2135480 1 1258 2319 annotation2135481 1 reflectin A1 range2135481 1 1258 2319 annotation2135477 1 BBa_B0034 range2135477 1 1219 1230 BBa_K638202_sequence 1 ttatgacaacttgacggctacatcattcactttttcttcacaaccggcacggaactcgctcgggctggccccggtgcattttttaaatacccgcgagaaatagagttgatcgtcaaaaccaacattgcgaccgacggtggcgataggcatccgggtggtgctcaaaagcagcttcgcctggctgatacgttggtcctcgcgccagcttaagacgctaatccctaactgctggcggaaaagatgtgacagacgcgacggcgacaagcaaacatgctgtgcgacgctggcgatatcaaaattgctgtctgccaggtgatcgctgatgtactgacaagcctcgcgtacccgattatccatcggtggatggagcgactcgttaatcgcttccatgcgccgcagtaacaattgctcaagcagatttatcgccagcagctccgaatagcgcccttccccttgcccggcgttaatgatttgcccaaacaggtcgctgaaatgcggctggtgcgcttcatccgggcgaaagaaccccgtattggcaaatattgacggccagttaagccattcatgccagtaggcgcgcggacgaaagtaaacccactggtgataccattcgcgagcctccggatgacgaccgtagtgatgaatctctcctggcgggaacagcaaaatatcacccggtcggcaaacaaattctcgtccctgatttttcaccaccccctgaccgcgaatggtgagattgagaatataacctttcattcccagcggtcggtcgataaaaaaatcgagataaccgttggcctcaatcggcgttaaacccgccaccagatgggcattaaacgagtatcccggcagcaggggatcattttgcgcttcagccatacttttcatactcccgccattcagagaagaaaccaattgtccatattgcatcagacattgccgtcactgcgtcttttactggctcttctcgctaaccaaaccggtaaccccgcttattaaaagcattctgtaacaaagcgggaccaaagccatgacaaaaacgcgtaacaaaagtgtctataatcacggcagaaaagtccacattgattatttgcacggcgtcacactttgctatgccatagcatttttatccataagattagcggatcctacctgacgctttttatcgcaactctctactgtttctccatacccgtttttttgggctagctactagagaaagaggagaaatactagatgcatcatcaccatcaccacatgggatccatgaaccgttacctgaatcgtcaacgcctgtataacatgtaccgcaataaatatcgtggcgtaatggaaccgatgtcgcgcatgacgatggatttccagggtcgttatatggattcgcagggccgcatggtagacccgcgctactacgactactacgggcgcatgcatgatcatgatcgctattatggccgtagtatgttcaatcagggtcattcaatggatagccagcgttatgggggctggatggacaatccggagcgctatatggacatgagcggttaccagatggatatgcaggggcgttggatggatgcgcagggtcgcttcaacaatccatttggtcaaatgtggcatggtcgtcagggtcattacccggggtacatgagctcacattccatgtacggtcgcaatatgtataacccatatcatagccattatgcgagccgccattttgattcgccagagcgttggatggatatgtctggttaccagatggacatgcaaggtcgttggatggataattacggtcgctatgtaaatccgttcaatcatcacatgtacggtcgcaacatgtgctatccgtatgggaatcactattataaccgtcacatggaacatccggagcgttatatggacatgtccggctaccaaatggacatgcaaggtcgctggatggatacccatggccgccattgcaacccgtttggccagatgtggcataatcgccatggctactatccgggtcatccacatggtcgcaacatgtttcagccggaacgctggatggatatgtctggttatcagatggatatgcaggggcgctggatggataattatggccgttacgtgaatccattttcacataactatgggcgccacatgaactacccgggtggccattataattaccatcacggtcgttacatgaaccacccagaacgtcacatggatatgagctcctaccagatggacatgcatggccgttggatggataatcagggccgctatattgataacttcgatcgtaattattacgattaccacatgtactaatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata 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