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