Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Joanna Semrau, Justine Ring, Morgan Litschko, Malak Elbatarny
Date created: 2015-08-29 11:00:00
Date modified: 2015-09-26 08:06:17
NpuDnaE intein for circularization of Type lll AFP+ inker
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K1831000_sequence (Version 1) |
Description
NpuDnaE intein circularization construct using Type III AFP and a computer-optimized linker. To further characterize the circularization construct developed by Team Heidelberg 2014, a moderately active, Type lll antifreeze protein (AFP) from (PDB: 1AME) was used to replace the RFP initially in the part. While the Type lll AFP normally denatures and loses activity at 37C, the AFP expressed from this part retained 78% of its activity even after heating to 90C. This shows that this construct effectively creates circular proteins which have increased stability over a wide range of temperatures.Notes
To ensure that the circular AFP would not be strained, and potentially lose function, several linker sequences ranging from 1 to 6 amino acids were modeled using Growmax. The linkers were developed using a modified version of Team Heidelberg 2014's linker generator software, CRAUT. After the potential linkers were compared in Growmax, it was determined that adding one glycine to the extein scar least disturbed the ice-binding residues of the AFP and this linker was used in part BBa_K1831000This construct was designed to circularize any linear protein, however part BBa_K1362000 from which is was based lacked a T7 promoter. Therefore sub-cloning of the construct would be required by other teams prior to expression of their circular protein. QGEM 2015 was able to add a constitutive T7 promoter, part BBa_I14018, to simplify the use of the NpuDnaE intein for future teams.
Source
This part was derived from BBa_K1362000, a composite part submitted by Team Heidelberg 2014's NpuDnaE intein RFC[105] circularization construct. We modified the part by adding part BBa_I14018, a constitutive T7 promoter with a medium transcription level prior to the RBS.| Sequence Annotation | Location | Component / Role(s) |
| BBa_I712074 BBa_K1362090 NpuDnaE(C) BsaI scar Extein(C) Linker Type III AFP Extein(N) BsaI scar NpuDnaE(N) T7 terminator | 1,46 47,122 123,185 186,188 189,197 198,200 201,401 402,410 411,413 414,714 715,746 | feature/promoter promoter ribosome_entry_site feature/rbs CDS feature/protein feature/scar feature/protein CDS feature/protein CDS feature/protein CDS CDS feature/protein feature/scar feature/protein CDS stem_loop feature/stem_loop |