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. The most important feature of luminous bacteria which can produce the luciferase is called LuxAB, which can catalyzes the bioluminescence reactions. Almost all luminous bacteria have been classified into the three genera Vibrio, Photobacterium, and Xenorhabdus. In this part, the luciferase what we use is from the Xenorhabdus luminescens.

3D-Structure of luxAB luciferase

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.

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 O₂ 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 FMNH₂, so it is not necessary to add luxG for E.coli host.

In short, this luciferase can be used together with capraldehyde, producing luminescent and working as a reporter.

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.

Characterization

This BioBrick has been characterized. To see more information, you can visit the Experience page: https://parts.igem.org/Part:BBa_K2310100:Experience

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
  

Characterization of BBa_K2310100

Experimental objective

This experiment is designed to explore the influence of IPTG induction dosage, induction time, induction temperature, substrate dosage, microbial concentration (OD600 value), and other conditions to the chemiluminescence of luxAB luciferase.

Induction temperature

We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter+luxAB (BBa_K2310103)in the fluid medium. When the value of OD600 reached about 1.2, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced at 30℃, 33℃ and 37℃, 190rpm shaking table for 3 hours respectively. Took 1ml bacteria solution in an EP tube, added 5ul substrate capraldehyde into EP tubes respectively and then took 100ulinto 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde. We also did the experiment again under the same condition and induced in 30℃ and 37℃ for 3 hours.

Figure 1: Induction temperature –standard luminescence value	Figure 2: Induction temperature –standard luminescence value

(Note: standard luminescence value= luminescence value-background value)

From the diagram, we can draw the conclusion that 30℃ is the best induction temperature among these three induction temperatures.

IPTG induction dosage

We cultured the DH5a bacteria which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul, 8ul, 12ul, 16ul, 20ul IPTG in the tubes respectively. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.

Figure 3: IPTG induction dosage-relative luminescence intensity

(Note1: relative luminescence intensity= luminescence value/background value)

(Note2: The relationship between the lines is incommensurable)

The diagram shows that when OD600 is low, the relative luminescence intensity will rise with the increase of the IPTG induction dosage within a certain range. However, when OD600 is high, the relative luminescence intensity may not rise with the increase of the IPTG induction dosage because the IPTG dosage is nearly saturated. By the way, 1.88 may be the best value of OD600 to test the influence of IPTG induction dosage. We guess that these two factors have an exponential function when OD600 is low; so we try to fit a curve under the first three values of OD600.

Figure 4: Induced at OD600=0.8	Figure 5: Induced at OD600=1.24	Figure 6: Induced at OD600=1.88

Microbial concentration (OD600 value)

We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.

Figure 7: OD600 value-relative luminescence intensity

(Note: relative luminescence intensity= luminescence value/background value)

The diagram shows that the relative luminescence intensity will rise with the increase of OD600 value within a certain range. Besides, it seems that these two factors have an exponential function; as a result we try to fit a curve under different IPTG induction dosages.

Figure 8(a): OD600 value-relative luminescence intensity (1/k IPTG)	Figure 8(b): OD600 value-relative luminescence intensity (2/k IPTG)	Figure 8(c): OD600 value-relative luminescence intensity (3/k IPTG)

Figure 8(a): OD600 value-relative luminescence intensity (4/k IPTG)	Figure 8(b): OD600 value-relative luminescence intensity (5/k IPTG)

When the concentration of IPTG is low, the exponential function between OD600 value and relative luminescence intensity is significant relatively. However, when the concentration of IPTG is high, the function is not significant. This is also because that the IPTG dosage is saturated relatively for the high OD600 value.

Induction time

We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteria solution in the glass test tubes and added 4ul IPTG in the tubes. Induced1, 2, 3 hours in 30℃, 190rpm respectively, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 10ul substrate capraldehyde into EP tubes and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.

Figure 9: Induction time-relative luminescence intensity

(Note1: relative luminescence intensity= luminescence value/background value)

(Note2: The relationship between the lines is incommensurable)

This group of data is a bit odd; we cannot say the relationship between the induction time and relative luminescence intensity, so we tested the data in a different way.

Figure 10: Induction time-relative luminescence intensity value

(Note1: relative luminescence value= (luminescence value-background value)/OD600 value after induction)

(Note2: The relationship between the lines is incommensurable)

The diagram shows that the induction time has little influence on the relative luminescence value. Consequently, we can choose 1 hour as our induction time in order to improve the efficiency.

Substrate dosage

We cultured the E.coli BL21(DE3) which contain the plasmid of T7 promoter + luxAB (BBa_K2310103) in the fluid medium. When the OD600 reached the 0.68, 1.24, 1.88, 2.48, 3.32 and 3.88, we took 4ml bacteriasolution in the glass test tubes and added 4ul IPTG in the tubes. Induced 3 hours in 30℃ , 190rpm, and then measured OD600 again. Took 1ml bacteria solution in an EP tube, added 5ul, 10ul, 15ul, 20ul substrate capraldehyde into EP tubes respectively and then took 100ul into 96-well plates to measure the luminescence. We also took 100ul bacteria solution to measure the value without capraldehyde.

Figure 11: Substrate dosage-relative luminescence intensity

(Note1: relative luminescence intensity= luminescence value/background value)

(Note2: The relationship between the lines is incommensurable)

The diagram shows that when OD600 is low, the influence of substrate dosage on relative luminescence intensity is minimal. However, when OD600 is high, the relative luminescence intensity will rise with the increase of the substrate dosage within some certain range because of the accumulation of the luxAB luciferase.