BBa_K638201
BBa_K638201 Version 1 Component
This part has been discontinued.
Please consider using BBa_K638101 as a replacement.
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_K638201_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
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.Source
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.| Access | Instance | Definition |
| public public public public public | BBa_I0500 BBa_B0034 BBa_K638001 BBa_B0010 BBa_B0012 | pBad/araC BBa_B0034 BBa_K638001 BBa_B0010 BBa_B0012 |