Difference between revisions of "Part:BBa K2310100"

Revision as of 19:45, 28 October 2017

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 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)

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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

Protein data table for BioBrick BBa_ 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)