Part:BBa_K1909004
NOD1 LRR Domain
BBa_K1909004 is a protein internal domain part designed as an alternative ligand binding domain for osmolarity sensor protein EnvZ, which is part of the well-studied and commonly used two-component system EnvZ/OmpR in Escherichia coli, offering a simple way of signal transduction.
Usage and Biology
It was shown that the periplasmic domain of EnvZ can be exchanged by ligand binding domains of other receptors without compromising its activity (ref. iGEM Tokyo 2012). This alternative ligand binding domain is derived from the LRR domain of the human nucleotide-binding oligomerization domain protein 1 (NOD1). NOD1 is a pattern recognition receptor activating the NF-κB pathway upon recognition of bacterial peptidoglycan, playing an important role in activating the immune response in presence of Chlamydia trachomatis or Chlamydia muridarum. However, NOD1 is part of a vital signal transduction pathway making it not possible to directly use it in bacteria.
Functional Parameters
Since tRNA abundance and consequently the translation speed and co-translational folding in E. coli differs widely from that of the tRNA abundance of human cells we decided to optimize the sequence of this part to harmonize its translation profile for E. coli using the RiboTempo algorithm (Fig 1A, B). Several silent mutations were introduced to harmonize translational speed between E. coli and humans cells. To prevent premature ribosome drop-off, the pair of CTA codons (L77 and L79 of the LRR domain) predicted to strongly stall the translation in E. coli was substituted by TTA codons. Furthermore, the XbaI restriction site was removed by changing L239 from CTA to TTA. To introduce a translational pause, L242 encoding CTG codon was altered to CTA (Fig 1C).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 6
Illegal SapI.rc site found at 358
Functional Parameters
| chassis | E. coli JW 3367-3 |
//cds/membrane/receptor
//cds/receptor
//chassis/prokaryote/ecoli
//proteindomain/binding
//proteindomain/internal
| chassis | E. coli JW 3367-3 |