Part:BBa_K2310100

LuxAB, emitting luciferase (from X. luminescens)

Luminous bacteria are the most abundant and widely distributed of the light-emitting organisms and are found in marine, freshwater, and terrestrial environments. What their most important feature is they can produce the luciferase called LUXAB, which can catalyzes the bioluminescence reactions. Almost all luminous bacteria have been classified into the three genera Vibrio, Photobacterium, and Xenorhabdus.In our progress, the luciferase what we use is from the Xenorhabdus luminescens. LuxAB is a part of luxCDABEG which is the normal structure of the operon in most bioluminescent bacteria. The LuxCDE gene controls the synthesis/regenerate aldehyde and the FMNH2, which is provided by an FMN reductase such as LuxG. The LuxAB luciferase is a heterodimeric enzyme of almost 80kDa composed of α and β-subunits whose molecular weight is 42kDa and 39kDa. For the two subunits, the α subunit plays a major role which is responsible for the light-emitting reaction and the β-subunit is important for stabling the protein, although there is about 40% identity in the amino acid sequence between the α and β subunits.

The 3D-Structure of luxAB luciferase

Usage and Biology

As a luciferase, the light-emitting reaction which catalyzed by the LuxAB involves the oxidation of reduced riboflavin phosphate (FMNH2) and a long chain fatty aldehyde with the emission of blue-green light (490nm). This reaction is as follows:

The reduced flavin, FMNH2, bound to the enzyme, reacts with 02 to form a 4a-peroxyflavin. This complex interacts with aldehyde to form a highly stable intermediate, which decays slowly, resulting in the emission of light along with the oxidation of the substrates.

There are two ways to use the LuxAB as a reporting system, in heterologous hosts such as Escherichia coli. Either luxAB alone can be used (in which case decanal must be provided as substrate), or luxCDABE can be used, (in which case the organism can synthesize aldehyde itself). Because E.coli is capable for reducing the FMN to FMNH, so it is not necessary to add luxG as E. coli for the host.

Luminescent Wavelength

Substrate: Capraldehyde

Emission: 490nm

Protein data analysis

Protein data table for LuxA automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)  ATGAAATTT ... CTATTATATTAG  ORF from nucleotide position 1 to 1080 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  101  201  301  MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHHFTEFGLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQMSKGR FRFGICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYMEADNEHIKFHKVKVNPAAYSRGGAPVYVVAESASTTEWAAQFGLPMILSWIINTNE KKAQLELYNEVAQEYGHDIHNIDHCLSYITSVDHDSIKAKEICRKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFVLKGHKDTNRRIDYSYEINP VGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASMKLFQSDVMPFLKEKQRSLLY*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) None of the supported features appeared in the sequence
Amino acid composition: Ala (A) 24 (6.7%) Arg (R) 15 (4.2%) Asn (N) 20 (5.6%) Asp (D) 23 (6.4%) Cys (C) 8 (2.2%) Gln (Q) 14 (3.9%) Glu (E) 22 (6.1%) Gly (G) 26 (7.2%) His (H) 11 (3.1%) Ile (I) 24 (6.7%) Leu (L) 29 (8.1%) Lys (K) 23 (6.4%) Met (M) 9 (2.5%) Phe (F) 19 (5.3%) Pro (P) 11 (3.1%) Ser (S) 18 (5.0%) Thr (T) 20 (5.6%) Trp (W) 6 (1.7%) Tyr (Y) 17 (4.7%) Val (V) 21 (5.8%)
Amino acid counting Total number: 360 Positively charged (Arg+Lys): 38 (10.6%) Negatively charged (Asp+Glu): 45 (12.5%) Aromatic (Phe+His+Try+Tyr): 53 (14.7%)	Biochemical parameters Atomic composition: C1841H2818N490O540S17 Molecular mass [Da]: 41000.6 Theoretical pI: 5.90 Extinction coefficient at 280 nm [M-1 cm-1]: 58330 / 58830 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): good (0.74) good (0.78) good (0.69) good (0.76) good (0.76) good (0.64)
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.

Protein data table for LuxB automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)  ATGAAATTT ... GAATATACCTAA  ORF from nucleotide position 1 to 981 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  101  201  301  MKFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHFSDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVAIAEEACLLDQLSEGR FILGFSDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYTPGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDV RYEYAERYKAVADKYDVDLSEIDHQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITAAKLAIEKCGAKSVLLSFE PMNDLMSQKNVINIVDDNIKKYHMEYT*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) None of the supported features appeared in the sequence
Amino acid composition: Ala (A) 20 (6.1%) Arg (R) 7 (2.1%) Asn (N) 24 (7.3%) Asp (D) 20 (6.1%) Cys (C) 6 (1.8%) Gln (Q) 11 (3.4%) Glu (E) 31 (9.5%) Gly (G) 14 (4.3%) His (H) 11 (3.4%) Ile (I) 26 (8.0%) Leu (L) 25 (7.6%) Lys (K) 22 (6.7%) Met (M) 8 (2.4%) Phe (F) 18 (5.5%) Pro (P) 11 (3.4%) Ser (S) 17 (5.2%) Thr (T) 16 (4.9%) Trp (W) 2 (0.6%) Tyr (Y) 17 (5.2%) Val (V) 21 (6.4%)
Amino acid counting Total number: 327 Positively charged (Arg+Lys): 29 (8.9%) Negatively charged (Asp+Glu): 51 (15.6%) Aromatic (Phe+His+Try+Tyr): 48 (14.7%)	Biochemical parameters Atomic composition: C1690H2579N429O515S14 Molecular mass [Da]: 37595.5 Theoretical pI: 4.84 Extinction coefficient at 280 nm [M-1 cm-1]: 36330 / 36705 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): good (0.78) good (0.79) good (0.77) excellent (0.81) good (0.75) good (0.65)
Alignments (obtained from PredictProtein.org)    There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 530
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1049