Difference between revisions of "Part:BBa K2310100"

Latest revision as of 16:07, 31 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. 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.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2310100 short</partinfo>
-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.
+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.
-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.
+[[Image:Structure-BBa_K2310100.gif|thumb|350px| 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 FMNH<sub>2</sub>, 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.
-https://static.igem.org/mediawiki/parts/2/24/Structure-BBa_K2310100.gif
-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:
+As a luciferase, the light-emitting reaction which catalyzed by the LuxAB involves the oxidation of reduced riboflavin phosphate (FMNH<sub>2</sub>) and a long chain fatty aldehyde with the emission of blue-green light (490nm). This reaction is as follows:
 https://static.igem.org/mediawiki/parts/e/e6/M-luxab-BBa_K2310100.jpeg
-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.
+The reduced flavin, FMNH<sub>2</sub>, bound to the enzyme, reacts with O<sub>2</sub> 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.
+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<sub>2</sub>, 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===
+<html><!--- Please copy this table containing parameters for BBa_ at the end of the parametrs section ahead of the references. ---><style type="text/css">table#AutoAnnotator {border:1px solid black; width:100%; border-collapse:collapse;} th#AutoAnnotatorHeader { border:1px solid black; width:100%; background-color: rgb(221, 221, 221);} td.AutoAnnotator1col { width:100%; border:1px solid black; } span.AutoAnnotatorSequence { font-family:'Courier New', Arial; } td.AutoAnnotatorSeqNum	{ text-align:right; width:2%; } td.AutoAnnotatorSeqSeq { width:98% } td.AutoAnnotatorSeqFeat1 { width:3% } td.AutoAnnotatorSeqFeat2a { width:27% } td.AutoAnnotatorSeqFeat2b { width:97% } td.AutoAnnotatorSeqFeat3 { width:70% } table.AutoAnnotatorNoBorder { border:0px; width:100%; border-collapse:collapse; } table.AutoAnnotatorWithBorder { border:1px solid black; width:100%; border-collapse:collapse; } td.AutoAnnotatorOuterAmino { border:0px solid black; width:20% } td.AutoAnnotatorInnerAmino { border:1px solid black; width:50% } td.AutoAnnotatorAminoCountingOuter { border:1px solid black; width:40%;  } td.AutoAnnotatorBiochemParOuter { border:1px solid black; width:60%; } td.AutoAnnotatorAminoCountingInner1 { width: 7.5% } td.AutoAnnotatorAminoCountingInner2 { width:62.5% } td.AutoAnnotatorAminoCountingInner3 { width:30% } td.AutoAnnotatorBiochemParInner1 { width: 5% } td.AutoAnnotatorBiochemParInner2 { width:55% } td.AutoAnnotatorBiochemParInner3 { width:40% } td.AutoAnnotatorCodonUsage1 { width: 3% } td.AutoAnnotatorCodonUsage2 { width:14.2% } td.AutoAnnotatorCodonUsage3 { width:13.8% } td.AutoAnnotatorAlignment1 { width: 3% } td.AutoAnnotatorAlignment2 { width: 10% } td.AutoAnnotatorAlignment3 { width: 87% } td.AutoAnnotatorLocalizationOuter {border:1px solid black; width:40%} td.AutoAnnotatorGOOuter {border:1px solid black; width:60%} td.AutoAnnotatorLocalization1 { width: 7.5% } td.AutoAnnotatorLocalization2 { width: 22.5% } td.AutoAnnotatorLocalization3 { width: 70% } 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font-family:Arial,Helvetica,sans-serif; background:rgb(255, 200, 0); background-color:rgba(255, 200, 0, 1); height:16px; width:16px; top:80px; border-radius:50%; } </style><div id='AutoAnnotator_container_1509219352187'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1509219352187 --><th id="AutoAnnotatorHeader" colspan="2">Protein data table for LuxA automatically created by the <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">BioBrick-AutoAnnotator</a> version 1.0</th></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Nucleotide sequence</strong> in <strong>RFC 10</strong>: (underlined part encodes the protein)<br><span class="AutoAnnotatorSequence">&nbsp;<u>ATGAAATTT&nbsp;...&nbsp;CTATTATAT</u>TAG</span><br>&nbsp;<strong>ORF</strong> from nucleotide position 1 to 1080 (excluding stop-codon)</td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid sequence:</strong> (RFC 25 scars in shown in bold, other sequence features underlined; both given below)<br><span class="AutoAnnotatorSequence"><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqNum">1&nbsp;<br>101&nbsp;<br>201&nbsp;<br>301&nbsp;</td><td class="AutoAnnotatorSeqSeq">MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHHFTEFGLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQMSKGR<br>FRFGICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYMEADNEHIKFHKVKVNPAAYSRGGAPVYVVAESASTTEWAAQFGLPMILSWIINTNE<br>KKAQLELYNEVAQEYGHDIHNIDHCLSYITSVDHDSIKAKEICRKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFVLKGHKDTNRRIDYSYEINP<br>VGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASMKLFQSDVMPFLKEKQRSLLY*</td></tr></table></span></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Sequence features:</strong> (with their position in the amino acid sequence, see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software/FeatureList">list of supported features</a>)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqFeat1"></td><td class="AutoAnnotatorSeqFeat2b">None of the supported features appeared in the sequence</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid composition:</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Ala (A)</td><td class="AutoAnnotatorInnerAmino">24 (6.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">15 (4.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">20 (5.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">23 (6.4%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">8 (2.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">14 (3.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">22 (6.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">26 (7.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">11 (3.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">24 (6.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">29 (8.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">23 (6.4%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">9 (2.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">19 (5.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">11 (3.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">18 (5.0%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">20 (5.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">6 (1.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">17 (4.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">21 (5.8%)</td></tr></table></td></tr></table></td></tr><tr><td class="AutoAnnotatorAminoCountingOuter"><strong>Amino acid counting</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Total number:</td><td class="AutoAnnotatorAminoCountingInner3">360</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">38 (10.6%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">45 (12.5%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">53 (14.7%)</td></tr></table></td><td class="AutoAnnotatorBiochemParOuter"><strong>Biochemical parameters</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Atomic composition:</td><td class="AutoAnnotatorBiochemParInner3">C<sub>1841</sub>H<sub>2818</sub>N<sub>490</sub>O<sub>540</sub>S<sub>17</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">41000.6</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">5.90</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Extinction coefficient at 280 nm [M<sup>-1</sup> cm<sup>-1</sup>]:</td><td class="AutoAnnotatorBiochemParInner3">58330 / 58830 (all Cys red/ox)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges</strong>&nbsp;<input type='button' id='hydrophobicity_charge_button' onclick='show_or_hide_plot_1509219352187()' value='Show'><span id="hydrophobicity_charge_explanation"></span><div id="hydrophobicity_charge_container" style='display:none'><div id="hydrophobicity_charge_placeholder0" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder1" style="width:100%;height:150px"></div></div></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Codon usage</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Organism:</td><td class="AutoAnnotatorCodonUsage3"><i>E. coli</i></td><td class="AutoAnnotatorCodonUsage3"><i>B. subtilis</i></td><td class="AutoAnnotatorCodonUsage3"><i>S. cerevisiae</i></td><td class="AutoAnnotatorCodonUsage3"><i>A. thaliana</i></td><td class="AutoAnnotatorCodonUsage3"><i>P. patens</i></td><td class="AutoAnnotatorCodonUsage3">Mammals</td></tr><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Codon quality (<a href="http://en.wikipedia.org/wiki/Codon_Adaptation_Index">CAI</a>):</td><td class="AutoAnnotatorCodonUsage3">good (0.74)</td><td class="AutoAnnotatorCodonUsage3">good (0.78)</td><td class="AutoAnnotatorCodonUsage3">good (0.69)</td><td class="AutoAnnotatorCodonUsage3">good (0.76)</td><td class="AutoAnnotatorCodonUsage3">good (0.76)</td><td class="AutoAnnotatorCodonUsage3">good (0.64)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><tr><td class="AutoAnnotator1col" colspan="2"> The BioBrick-AutoAnnotator was created by <a href="http://2013.igem.org/Team:TU-Munich">TU-Munich 2013</a> iGEM team. For more information please see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software">documentation</a>.<br>If you have any questions, comments or suggestions, please leave us a <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">comment</a>.</td></tr></table></div><br><!-- IMPORTANT: DON'T REMOVE THIS LINE, OTHERWISE NOT SUPPORTED FOR IE BEFORE 9 --><!--[if lte IE 8]><script language="javascript" type="text/javascript" src="http://2013.igem.org/Team:TU-Munich/excanvas.js"></script><![endif]--><script type='text/javascript' src='http://code.jquery.com/jquery-1.10.0.min.js'></script><script type='text/javascript' src='http://2013.igem.org/Team:TU-Munich/Flot.js?action=raw&ctype=text/js'></script><script>var jqAutoAnnotator = jQuery.noConflict(true);function show_or_hide_plot_1509219352187(){hydrophobicity_datapoints = 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= 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= undefined;trans_datapoints = undefined;sec_helix_datapoints = undefined;sec_strand_datapoints = undefined;acc_exposed_datapoints = undefined;acc_buried_datapoints = undefined;flot_plot_options = []; flot_plot_options[0] = {grid: {borderWidth: {top: 0,right: 0,bottom: 0,left: 0}},legend: {show: false},xaxes: [{show: true,min: 0,max: 200,ticks: [[0.5, '1'], [24.5, '25'], [49.5, '50'], [74.5, '75'], [99.5, '100'], [124.5, '125'], [149.5, '150'], [174.5, '175'], [199.5, '200']],tickLength: -5}],yaxes: [{show: true,ticks: [[0, '0'], [4.5,'hydro-<br>phobic&nbsp;&nbsp;'], [-4.5,'hydro-<br>philic&nbsp;&nbsp;']],min: -4.5,max: +4.5,font: {size: 12,lineHeight: 14,style: 'italic',weight: 'bold',family: 'sans-serif',variant: 'small-caps',color: 'rgba(100,149,237,1)'}},{show: true,ticks: [[0, ''], [1,'positive<br>&nbsp;charge'], [-1,'negative<br>&nbsp;charge']],position: 'right',min: -1,max: 1,font: {size: 12,lineHeight: 14,style: 'italic',weight: 'bold',family: 'sans-serif',variant: 'small-caps',color: 'rgba(255,99,71,1)'}}]};number_of_plots = 2;for ( plot_num = 1 ; plot_num < number_of_plots ; plot_num ++){flot_plot_options[plot_num] = jqAutoAnnotator.extend(true, {} ,flot_plot_options[0]);flot_plot_options[plot_num].xaxes = [{min: plot_num*200,max: (plot_num + 1)*200,ticks: [ [plot_num*200 +   0.5, (plot_num*200 +   1).toString()], [plot_num*200 +  24.5, (plot_num*200 +  25).toString()], [plot_num*200 +  49.5, (plot_num*200 +  50).toString()], [plot_num*200 +  74.5, (plot_num*200 +  75).toString()], [plot_num*200 +  99.5, (plot_num*200 + 100).toString()], [plot_num*200 + 124.5, (plot_num*200 + 125).toString()], [plot_num*200 + 149.5, (plot_num*200 + 150).toString()], [plot_num*200 + 174.5, (plot_num*200 + 175).toString()], [plot_num*200 + 199.5, (plot_num*200 + 200).toString()] ],tickLength: -5}];};try {if( jqAutoAnnotator('#AutoAnnotator_container_1509219352187 #hydrophobicity_charge_button').val() =='Show' ){jqAutoAnnotator('#AutoAnnotator_container_1509219352187 #hydrophobicity_charge_container').css('display','block');jqAutoAnnotator('#AutoAnnotator_container_1509219352187 #hydrophobicity_charge_button').val('Hide');var description_html = '<div id=\'AutoAnnotator_plot_selectors\'>';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'hydrophobicity_checkbox\' checked=\'checked\'>&nbsp;Moving average over 5 amino acids for hydrophobicity (<img src=\'https://static.igem.org/mediawiki/2013/e/e9/TUM13_hydrophobicity_icon.png\' alt=\'blue graph\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'charge_checkbox\' checked=\'checked\'>&nbsp;Moving average over 5 amino acids for charge (<img src=\'https://static.igem.org/mediawiki/2013/3/3e/TUM13_charge_icon.png\' alt=\'red graph\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'dis_checkbox\' checked=\'checked\'>&nbsp;Predicted disulfid bridges (<img src=\'https://static.igem.org/mediawiki/2013/2/28/TUM13_dis_icon.png\' alt=\'yellow circle\' height=\'10\'></img>) with the number of the bridge in the center';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'trans_checkbox\' checked=\'checked\'>&nbsp;Predicted transmembrane helices (<img src=\'https://static.igem.org/mediawiki/2013/7/78/TUM13_trans_icon.png\' alt=\'turquois bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'sec_checkbox\' checked=\'checked\'>&nbsp;Predicted secondary structure: Helices (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_helix_icon.png\' alt=\'violet bars\' height=\'10\'></img>) and beta-strands (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_strand_icon.png\' alt=\'yellow bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'acc_checkbox\' checked=\'checked\'>&nbsp;Predicted solvent accessability: Exposed (<img src=\'https://static.igem.org/mediawiki/2013/1/16/TUM13_exposed_icon.png\' alt=\'blue bars\' height=\'10\'></img>) and buried (<img src=\'https://static.igem.org/mediawiki/2013/0/0b/TUM13_buried_icon.png\' alt=\'green bars\' height=\'10\'></img>) residues';description_html = description_html + '<br></div>';jqAutoAnnotator('#AutoAnnotator_container_1509219352187 #hydrophobicity_charge_explanation').html(description_html);plot_according_to_selectors_1509219352187();jqAutoAnnotator('#AutoAnnotator_container_1509219352187 #AutoAnnotator_plot_selectors').find('input').click(plot_according_to_selectors_1509219352187);}else{jqAutoAnnotator('#AutoAnnotator_container_1509219352187 #hydrophobicity_charge_container').css('display','none');jqAutoAnnotator('#AutoAnnotator_container_1509219352187 #hydrophobicity_charge_button').val('Show');jqAutoAnnotator('#AutoAnnotator_container_1509219352187 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+<html><!--- Please copy this table containing parameters for BBa_ at the end of the parametrs section ahead of the references. ---><style type="text/css">table#AutoAnnotator {border:1px solid black; width:100%; border-collapse:collapse;} th#AutoAnnotatorHeader { border:1px solid black; width:100%; background-color: rgb(221, 221, 221);} td.AutoAnnotator1col { width:100%; border:1px solid black; } span.AutoAnnotatorSequence { font-family:'Courier New', Arial; } td.AutoAnnotatorSeqNum	{ text-align:right; width:2%; } td.AutoAnnotatorSeqSeq { width:98% } td.AutoAnnotatorSeqFeat1 { width:3% } td.AutoAnnotatorSeqFeat2a { width:27% } td.AutoAnnotatorSeqFeat2b { width:97% } td.AutoAnnotatorSeqFeat3 { width:70% } table.AutoAnnotatorNoBorder { border:0px; width:100%; border-collapse:collapse; } table.AutoAnnotatorWithBorder { border:1px solid black; width:100%; border-collapse:collapse; } td.AutoAnnotatorOuterAmino { border:0px solid black; width:20% } td.AutoAnnotatorInnerAmino { border:1px 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td.AutoAnnotatorGO1 { width: 5% } td.AutoAnnotatorGO2 { width: 35% } td.AutoAnnotatorGO3 { width: 60% } td.AutoAnnotatorPredFeat1 { width:3% } td.AutoAnnotatorPredFeat2a { width:27% } td.AutoAnnotatorPredFeat3 { width:70% } div.AutoAnnotator_trans { position:absolute; background:rgb(11,140,143); background-color:rgba(11,140,143, 0.8); height:5px; top:100px; } div.AutoAnnotator_sec_helix { position:absolute; background:rgb(102,0,102); background-color:rgba(102,0,102, 0.8); height:5px; top:110px; } div.AutoAnnotator_sec_strand { position:absolute; background:rgb(245,170,26); background-color:rgba(245,170,26, 1); height:5px; top:110px; } div.AutoAnnotator_acc_buried { position:absolute; background:rgb(89,168,15); background-color:rgba(89,168,15, 0.8); height:5px; top:120px; } div.AutoAnnotator_acc_exposed { position:absolute; background:rgb(0, 0, 255); background-color:rgba(0, 0, 255, 0.8); height:5px; top:120px; } div.AutoAnnotator_dis { position:absolute; text-align:center; font-family:Arial,Helvetica,sans-serif; background:rgb(255, 200, 0); background-color:rgba(255, 200, 0, 1); height:16px; width:16px; top:80px; border-radius:50%; } </style><div id='AutoAnnotator_container_1509219803788'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1509219803788 --><th id="AutoAnnotatorHeader" colspan="2">Protein data table for LuxB automatically created by the <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">BioBrick-AutoAnnotator</a> version 1.0</th></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Nucleotide sequence</strong> in <strong>RFC 10</strong>: (underlined part encodes the protein)<br><span class="AutoAnnotatorSequence">&nbsp;<u>ATGAAATTT&nbsp;...&nbsp;GAATATACC</u>TAA</span><br>&nbsp;<strong>ORF</strong> from nucleotide position 1 to 981 (excluding stop-codon)</td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid sequence:</strong> (RFC 25 scars in shown in bold, other sequence features underlined; both given below)<br><span class="AutoAnnotatorSequence"><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqNum">1&nbsp;<br>101&nbsp;<br>201&nbsp;<br>301&nbsp;</td><td class="AutoAnnotatorSeqSeq">MKFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHFSDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVAIAEEACLLDQLSEGR<br>FILGFSDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYTPGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDV<br>RYEYAERYKAVADKYDVDLSEIDHQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITAAKLAIEKCGAKSVLLSFE<br>PMNDLMSQKNVINIVDDNIKKYHMEYT*</td></tr></table></span></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Sequence features:</strong> (with their position in the amino acid sequence, see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software/FeatureList">list of supported features</a>)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqFeat1"></td><td class="AutoAnnotatorSeqFeat2b">None of the supported features appeared in the sequence</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid composition:</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Ala (A)</td><td class="AutoAnnotatorInnerAmino">20 (6.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">7 (2.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">24 (7.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">20 (6.1%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">6 (1.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">11 (3.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">31 (9.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">14 (4.3%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">11 (3.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">26 (8.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">25 (7.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">22 (6.7%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">8 (2.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">18 (5.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">11 (3.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">17 (5.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">16 (4.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">2 (0.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">17 (5.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">21 (6.4%)</td></tr></table></td></tr></table></td></tr><tr><td class="AutoAnnotatorAminoCountingOuter"><strong>Amino acid counting</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Total number:</td><td class="AutoAnnotatorAminoCountingInner3">327</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">29 (8.9%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">51 (15.6%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">48 (14.7%)</td></tr></table></td><td class="AutoAnnotatorBiochemParOuter"><strong>Biochemical parameters</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Atomic composition:</td><td class="AutoAnnotatorBiochemParInner3">C<sub>1690</sub>H<sub>2579</sub>N<sub>429</sub>O<sub>515</sub>S<sub>14</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">37595.5</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">4.84</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Extinction coefficient at 280 nm [M<sup>-1</sup> cm<sup>-1</sup>]:</td><td class="AutoAnnotatorBiochemParInner3">36330 / 36705 (all Cys red/ox)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges</strong>&nbsp;<input type='button' id='hydrophobicity_charge_button' onclick='show_or_hide_plot_1509219803788()' value='Show'><span id="hydrophobicity_charge_explanation"></span><div id="hydrophobicity_charge_container" style='display:none'><div id="hydrophobicity_charge_placeholder0" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder1" style="width:100%;height:150px"></div></div></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Codon usage</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Organism:</td><td class="AutoAnnotatorCodonUsage3"><i>E. coli</i></td><td class="AutoAnnotatorCodonUsage3"><i>B. subtilis</i></td><td class="AutoAnnotatorCodonUsage3"><i>S. cerevisiae</i></td><td class="AutoAnnotatorCodonUsage3"><i>A. thaliana</i></td><td class="AutoAnnotatorCodonUsage3"><i>P. patens</i></td><td class="AutoAnnotatorCodonUsage3">Mammals</td></tr><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Codon quality (<a href="http://en.wikipedia.org/wiki/Codon_Adaptation_Index">CAI</a>):</td><td class="AutoAnnotatorCodonUsage3">good (0.78)</td><td class="AutoAnnotatorCodonUsage3">good (0.79)</td><td class="AutoAnnotatorCodonUsage3">good (0.77)</td><td class="AutoAnnotatorCodonUsage3">excellent (0.81)</td><td class="AutoAnnotatorCodonUsage3">good (0.75)</td><td class="AutoAnnotatorCodonUsage3">good (0.65)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Alignments</strong> (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)<br>&nbsp;&nbsp;&nbsp;There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.</td></tr><tr><th id='AutoAnnotatorHeader' colspan="2"><strong>Predictions</strong> (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)</th></tr><tr><td class="AutoAnnotator1col" colspan="2">&nbsp;&nbsp;&nbsp;There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.</td><tr><td class="AutoAnnotator1col" colspan="2"> The BioBrick-AutoAnnotator was created by <a href="http://2013.igem.org/Team:TU-Munich">TU-Munich 2013</a> iGEM team. For more information please see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software">documentation</a>.<br>If you have any questions, comments or suggestions, please leave us a <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">comment</a>.</td></tr></table></div><br><!-- IMPORTANT: DON'T REMOVE THIS LINE, OTHERWISE NOT SUPPORTED FOR IE BEFORE 9 --><!--[if lte IE 8]><script language="javascript" type="text/javascript" src="http://2013.igem.org/Team:TU-Munich/excanvas.js"></script><![endif]--><script type='text/javascript' src='http://code.jquery.com/jquery-1.10.0.min.js'></script><script type='text/javascript' src='http://2013.igem.org/Team:TU-Munich/Flot.js?action=raw&ctype=text/js'></script><script>var jqAutoAnnotator = jQuery.noConflict(true);function show_or_hide_plot_1509219803788(){hydrophobicity_datapoints = 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= undefined;trans_datapoints = undefined;sec_helix_datapoints = undefined;sec_strand_datapoints = undefined;acc_exposed_datapoints = undefined;acc_buried_datapoints = undefined;flot_plot_options = []; flot_plot_options[0] = {grid: {borderWidth: {top: 0,right: 0,bottom: 0,left: 0}},legend: {show: false},xaxes: [{show: true,min: 0,max: 200,ticks: [[0.5, '1'], [24.5, '25'], [49.5, '50'], [74.5, '75'], [99.5, '100'], [124.5, '125'], [149.5, '150'], [174.5, '175'], [199.5, '200']],tickLength: -5}],yaxes: [{show: true,ticks: [[0, '0'], [4.5,'hydro-<br>phobic&nbsp;&nbsp;'], [-4.5,'hydro-<br>philic&nbsp;&nbsp;']],min: -4.5,max: +4.5,font: {size: 12,lineHeight: 14,style: 'italic',weight: 'bold',family: 'sans-serif',variant: 'small-caps',color: 'rgba(100,149,237,1)'}},{show: true,ticks: [[0, ''], [1,'positive<br>&nbsp;charge'], [-1,'negative<br>&nbsp;charge']],position: 'right',min: -1,max: 1,font: {size: 12,lineHeight: 14,style: 'italic',weight: 'bold',family: 'sans-serif',variant: 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){jqAutoAnnotator('#AutoAnnotator_container_1509219803788 #hydrophobicity_charge_container').css('display','block');jqAutoAnnotator('#AutoAnnotator_container_1509219803788 #hydrophobicity_charge_button').val('Hide');var description_html = '<div id=\'AutoAnnotator_plot_selectors\'>';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'hydrophobicity_checkbox\' checked=\'checked\'>&nbsp;Moving average over 5 amino acids for hydrophobicity (<img src=\'https://static.igem.org/mediawiki/2013/e/e9/TUM13_hydrophobicity_icon.png\' alt=\'blue graph\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'charge_checkbox\' checked=\'checked\'>&nbsp;Moving average over 5 amino acids for charge (<img src=\'https://static.igem.org/mediawiki/2013/3/3e/TUM13_charge_icon.png\' alt=\'red graph\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'dis_checkbox\' checked=\'checked\'>&nbsp;Predicted disulfid bridges (<img src=\'https://static.igem.org/mediawiki/2013/2/28/TUM13_dis_icon.png\' alt=\'yellow circle\' height=\'10\'></img>) with the number of the bridge in the center';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'trans_checkbox\' checked=\'checked\'>&nbsp;Predicted transmembrane helices (<img src=\'https://static.igem.org/mediawiki/2013/7/78/TUM13_trans_icon.png\' alt=\'turquois bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'sec_checkbox\' checked=\'checked\'>&nbsp;Predicted secondary structure: Helices (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_helix_icon.png\' alt=\'violet bars\' height=\'10\'></img>) and beta-strands (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_strand_icon.png\' alt=\'yellow bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'acc_checkbox\' checked=\'checked\'>&nbsp;Predicted solvent accessability: Exposed (<img src=\'https://static.igem.org/mediawiki/2013/1/16/TUM13_exposed_icon.png\' alt=\'blue bars\' height=\'10\'></img>) and buried (<img src=\'https://static.igem.org/mediawiki/2013/0/0b/TUM13_buried_icon.png\' alt=\'green bars\' height=\'10\'></img>) residues';description_html = description_html + '<br></div>';jqAutoAnnotator('#AutoAnnotator_container_1509219803788 #hydrophobicity_charge_explanation').html(description_html);plot_according_to_selectors_1509219803788();jqAutoAnnotator('#AutoAnnotator_container_1509219803788 #AutoAnnotator_plot_selectors').find('input').click(plot_according_to_selectors_1509219803788);}else{jqAutoAnnotator('#AutoAnnotator_container_1509219803788 #hydrophobicity_charge_container').css('display','none');jqAutoAnnotator('#AutoAnnotator_container_1509219803788 #hydrophobicity_charge_button').val('Show');jqAutoAnnotator('#AutoAnnotator_container_1509219803788 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+===Characterization===
+This BioBrick has been characterized. To see more information, you can visit the  Experience page: https://parts.igem.org/Part:BBa_K2310100:Experience
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K2310100 SequenceAndFeatures</partinfo>
 <!-- Uncomment this to enable Functional Parameter display
@@ Line 32: / Line 43: @@
 <partinfo>BBa_K2310100 parameters</partinfo>
 <!-- -->
+===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 (<partinfo>BBa_K2310103</partinfo>)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.
+<br><table><tr><th>
+[[Image:XMU-luxf1.png|thumb|400px|'''Figure 1''': Induction temperature –standard luminescence value]]</th><th>
+[[Image:XMU-luxf2.png|thumb|390px|'''Figure 2''': Induction temperature –standard luminescence value]]</th><th></table>
+(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.
+<br><table><tr><th>
+[[Image:XMU-luxf3.png|thumb|750px|'''Figure 3''': IPTG induction dosage-relative luminescence intensity]]</th><th></table>
+(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.<br><table><tr><th>
+[[Image:XMU-luxf4.png|thumb|265px|'''Figure 4''': Induced at OD600=0.8]]</th><th>
+[[Image:XMU-luxf5.png|thumb|248px|'''Figure 5''': Induced at OD600=1.24]]</th><th>
+[[Image:XMU-luxf6.png|thumb|255px|'''Figure 6''': Induced at OD600=1.88]]</th></tr></table>
+====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.
+<br><table><tr><th>
+[[Image:XMU-luxf7.png|thumb|750px|'''Figure 7''': OD600 value-relative luminescence intensity]]</th><th></table>
+(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. <br><table><tr><th>
+[[Image:Lux8.png|thumb|265px|'''Figure 8(a)''': OD600 value-relative luminescence intensity (1/k IPTG)]]</th><th>
+[[Image:XMU-luxf8b.png|thumb|265px|'''Figure 8(b)''': OD600 value-relative luminescence intensity (2/k IPTG)]]</th><th>
+[[Image:XMU-luxf8c.png|thumb|265px|'''Figure 8(c)''': OD600 value-relative luminescence intensity (3/k IPTG)]]</th></tr></table><br><table><tr><th>
+[[Image:XMU-luxf8d.png|thumb|265px|'''Figure 8(a)''': OD600 value-relative luminescence intensity (4/k IPTG)]]</th><th>
+[[Image:XMU-luxf8e.png|thumb|265px|'''Figure 8(b)''': OD600 value-relative luminescence intensity (5/k IPTG)]]</th><th></th></tr></table>
+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.
+<br><table><tr><th>
+[[Image:XMU-luxf9.png|thumb|750px|'''Figure 9''': Induction time-relative luminescence intensity]]</th><th></table>
+(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.
+<br><table><tr><th>
+[[Image:XMU-luxf10.png|thumb|750px|'''Figure 10''': Induction time-relative luminescence intensity value]]</th><th></table>
+(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.
+<br><table><tr><th>
+[[Image:XMU-luxf11.png|thumb|750px|'''Figure 11''': Substrate dosage-relative luminescence intensity]]</th><th></table>
+(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.