Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Matthew Jones
Date created: 2011-09-18 11:00:00
Date modified: 2015-05-08 01:12:57
Arabinose inducible Poly-His Reflectin A1 generator
| Types | DnaRegion |
| Roles | Generator engineered_region |
| Sequences | BBa_K638202_sequence (Version 1) |
Description
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.Notes
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.Source
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.| Sequence Annotation | Location | Component / Role(s) |
| AraC BBa_I0500 PC Pbad BBa_B0034 conserved BBa_K128005 BBa_K638001 reflectin A1 stop codon BBa_B0010 stem_loop BBa_B0012 T7 TE polya stop ATG | 1,1079 1,1210 1043,1114 1080,1210 1219,1230 1223,1226 1239,1257 1258,2319 1258,2319 2317,2319 2328,2407 2339,2382 2416,2457 2423,2442 2443,2458 2456,2456 1237,1239 | sequence_feature feature/misc feature/BioBrick engineered_region feature/promoter promoter feature/promoter promoter engineered_region feature/BioBrick conserved_region feature/conserved engineered_region feature/BioBrick engineered_region feature/BioBrick CDS feature/cds feature/stop stop_codon feature/BioBrick engineered_region feature/stem_loop stem_loop feature/BioBrick engineered_region stem_loop feature/stem_loop feature/polya polyA_site stop_codon feature/stop start_codon feature/start |