Team:TU-Munich/Results/Recombinant

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Characterization of recombinant effector proteins

Text. See proteinbiochemical methods for further informatons.

Table 1: Investigated Proteins
Protein	BioBrick	RFC	Affinity tag
Eryhtromycin esterase (EreB)	<partinfo>BBa_K1159000</partinfo>	RFC25	c-term. Streptag II
Laccase	<partinfo>BBa_K1159002</partinfo>	RFC25	c-term. Streptag II
Nano Luciferase	<partinfo>BBa_K1159001</partinfo>	RFC25	c-term. Streptag II
XylE	<partinfo>BBa_E0040</partinfo>
PP1	<partinfo>BBa_K1159004</partinfo>
YFP_TEV_CFP	<partinfo>BBa_K1159112</partinfo>

Eryhtromycin Esterase

[...] description [...] reaction

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0

Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
ATGGCCGGCAGGTTCGAA ... GTTTATGAAACCGGT
ORF from nucleotide position -8 to 1260 (excluding stop-codon)

Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)

1	MAGRFEEWVKDKHIPFKLNHPDDNYDDFKPLRKIIGDTRVVALGENSHFIKEFFLLRHTLLRFFIEDLGFTTFAFEFGFAEGQIINNWIHGQGTDDEIGR
101	FLKHFYYPEELKTTFLWLREYNKAAKEKITFLGIDIPRNGGSYLPNMEIVHDFFRTADKEALHIIDDAFNIAKKIDYFSTSQAALNLHELTDSEKCRLTS
201	QLARVKVRLEAMAPIHIEKYGIDKYETILHYANGMIYLDYNIQAMSGFISGGGMQGDMGAKDKYMADSVLWHLKNPQSEQKVIVVAHNAHIQKTPILYDG
301	FLSCLPMGQRLKNAIGDDYMSLGITSYSGHTAALYPEVDTKYGFRVDNFQLQEPNEGSVEKAISGCGVTNSFVFFRNIPEDLQSIPNMIRFDSIYMKAEL
401	EKAFDGIFQIEKSSVSEVVYETG*

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)	27 (6.4%)
Arg (R)	16 (3.8%)
Asn (N)	20 (4.7%)
Asp (D)	29 (6.9%)

Cys (C)	3 (0.7%)
Gln (Q)	14 (3.3%)
Glu (E)	30 (7.1%)
Gly (G)	31 (7.3%)

His (H)	15 (3.5%)
Ile (I)	36 (8.5%)
Leu (L)	34 (8.0%)
Lys (K)	29 (6.9%)

Met (M)	12 (2.8%)
Phe (F)	31 (7.3%)
Pro (P)	14 (3.3%)
Ser (S)	22 (5.2%)

Thr (T)	19 (4.5%)
Trp (W)	4 (0.9%)
Tyr (Y)	19 (4.5%)
Val (V)	18 (4.3%)

Amino acid counting

	Total number:	423
	Positively charged (Arg+Lys):	45 (10.6%)
	Negatively charged (Asp+Glu):	59 (13.9%)
	Aromatic (Phe+His+Try+Tyr):	69 (16.3%)

Biochemical parameters

	Atomic composition:	C₂₂₀₄H₃₃₄₈N₅₆₈O₆₃₆S₁₅
	Molecular mass [Da]:	48459.2
	Theoretical pI:	5.55
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	50310 / 50498 (all Cys red/ox)

Codon usage

	Organism:	E. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	Codon quality (CAI):	good (0.74)	good (0.77)	good (0.75)	good (0.80)	good (0.76)	good (0.66)

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Analytical präparation

Figure 3:

Degradation of a chromogenic esterase substrate: 4-Nitrophenyl butyrate

Degradation of Erythromycin

Reaction conditions HPLC Kirby Bauer-Assay

[...] Characterization

Laccase

[...] description [...] reaction [...] production

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0

Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
ATGGCCGGCAACCTAGAA ... GATATCATCACCGGT
ORF from nucleotide position -8 to 1530 (excluding stop-codon)

Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)

1	MAGNLEKFVDELPIPEVAKPVKKNPKQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIHANRNEKVKVKWMNKLPLKHFLPVDHTIHEGHHDEPEV
101	KTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFEREVYEYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKGEYDIPLMIMDRTF
201	QEDGALFYPSRPNNTPEDSDIPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRFRILNASNTRTYELHLDNDATILQIGSDGGFLPRPVHHQSFSIAPAE
301	RFDVIIDFSAYENKTITLKNKAGCGQEVNPETDANIMQFKVTRPLKGRAPKTLRPIFKPLPPLRPCRADKERTLTLTGTQDKYGRPILLLDNQFWNDPVT
401	ENPRLGSVEVWSIVNPTRGTHPIHLHLVQFRVIDRRPFDTEVYQSTGDIVYTGPNEAPPLHEQGYKDTIQAHAGEVIRIIARFVPYSGRYVWHCHILEHE
501	DYDMMRPMDIITG*

Sequence features: (with their position in the amino acid sequence, see the list of supported features)

RFC25 scar (shown in bold):

130 to 131

Amino acid composition:

Ala (A)	27 (5.3%)
Arg (R)	29 (5.7%)
Asn (N)	23 (4.5%)
Asp (D)	31 (6.0%)

Cys (C)	5 (1.0%)
Gln (Q)	14 (2.7%)
Glu (E)	37 (7.2%)
Gly (G)	30 (5.8%)

His (H)	23 (4.5%)
Ile (I)	31 (6.0%)
Leu (L)	41 (8.0%)
Lys (K)	27 (5.3%)

Met (M)	10 (1.9%)
Phe (F)	24 (4.7%)
Pro (P)	47 (9.2%)
Ser (S)	18 (3.5%)

Thr (T)	33 (6.4%)
Trp (W)	8 (1.6%)
Tyr (Y)	22 (4.3%)
Val (V)	33 (6.4%)

Amino acid counting

	Total number:	513
	Positively charged (Arg+Lys):	56 (10.9%)
	Negatively charged (Asp+Glu):	68 (13.3%)
	Aromatic (Phe+His+Try+Tyr):	77 (15.0%)

Biochemical parameters

	Atomic composition:	C₂₆₇₁H₄₀₇₂N₇₁₈O₇₆₀S₁₅
	Molecular mass [Da]:	58883.0
	Theoretical pI:	6.03
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	76780 / 77093 (all Cys red/ox)

Codon usage

	Organism:	E. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	Codon quality (CAI):	good (0.72)	good (0.75)	good (0.68)	good (0.74)	good (0.77)	good (0.67)

The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation.
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Laccase a is a secreted enzyme

Figure 3:

--->

Analytical präparation

[...] Characterization

Figure 3:

Structural consideration of EreB

File:TUM13 Animation Laccasex.gif

Figure 3:

Aktivity determination using ABTS

[http://www.rsc.org/ej/OB/2005/b504199f/b504199f-s2.gif|left]

Figure 3:

Nano Luciferase

[...] description [...] reaction [...] production

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0

Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
ATGGCCGGCGTCTTCACA ... ATTCTGGCGACCGGT
ORF from nucleotide position -8 to 516 (excluding stop-codon)

Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)

1	MAGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT
101	LVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILATG*

Sequence features: (with their position in the amino acid sequence, see the list of supported features)

RFC25 scar (shown in bold):

163 to 164

Amino acid composition:

Ala (A)	4 (2.3%)
Arg (R)	7 (4.0%)
Asn (N)	8 (4.6%)
Asp (D)	12 (6.9%)

Cys (C)	1 (0.6%)
Gln (Q)	7 (4.0%)
Glu (E)	8 (4.6%)
Gly (G)	22 (12.6%)

His (H)	4 (2.3%)
Ile (I)	18 (10.3%)
Leu (L)	16 (9.1%)
Lys (K)	7 (4.0%)

Met (M)	3 (1.7%)
Phe (F)	8 (4.6%)
Pro (P)	6 (3.4%)
Ser (S)	6 (3.4%)

Thr (T)	11 (6.3%)
Trp (W)	3 (1.7%)
Tyr (Y)	6 (3.4%)
Val (V)	18 (10.3%)

Amino acid counting

	Total number:	175
	Positively charged (Arg+Lys):	14 (8.0%)
	Negatively charged (Asp+Glu):	20 (11.4%)
	Aromatic (Phe+His+Try+Tyr):	21 (12.0%)

Biochemical parameters

	Atomic composition:	C₈₈₂H₁₃₇₇N₂₂₉O₂₅₄S₄
	Molecular mass [Da]:	19381.3
	Theoretical pI:	5.10
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	25440 / 25503 (all Cys red/ox)

Codon usage

	Organism:	E. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	Codon quality (CAI):	good (0.74)	good (0.76)	acceptable (0.55)	good (0.65)	excellent (0.81)	good (0.77)

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If you have any questions, comments or suggestions, please leave us a comment.

Analytical präparation

[...] Characterization

Figure 3:

Structure of the Nano Luciferase

Figure 3:

--->

XylE

[...] description [...] reaction [...] production

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0

Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
ATGGCCGGCAACAAAGGT ... GTGCTGACCACCGGT
ORF from nucleotide position -8 to 924 (excluding stop-codon)

Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)

1	MAGNKGVMRPGHVQLRVLDMSKALEHYVELLGLIEMDRDDQGRVYLKAWTEVDKFSLVLREADEPGMDFMGFKVVDEDALRQLERDLMAYGCAVEQLPAG
101	ELNSCGRRVRFQAPSGHHFELYADKEYTGKWGLNDVNPEAWPRDLKGMAAVRFDHALMYGDELPATYDLFTKVLGFYLAEQVLDENGTRVAQFLSLSTKA
201	HDVAFIHHPEKGRLHHVSFHLETWEDLLRAADLISMTDTSIDIGPTRHGLTHGKTIYFFDPSGNRNEVFCGGDYNYPDHKPVTWTTDQLGKAIFYHDRIL
301	NERFMTVLTTG*

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)	22 (7.1%)
Arg (R)	19 (6.1%)
Asn (N)	9 (2.9%)
Asp (D)	27 (8.7%)

Cys (C)	3 (1.0%)
Gln (Q)	8 (2.6%)
Glu (E)	21 (6.8%)
Gly (G)	26 (8.4%)

His (H)	15 (4.8%)
Ile (I)	8 (2.6%)
Leu (L)	34 (10.9%)
Lys (K)	14 (4.5%)

Met (M)	11 (3.5%)
Phe (F)	16 (5.1%)
Pro (P)	12 (3.9%)
Ser (S)	10 (3.2%)

Thr (T)	18 (5.8%)
Trp (W)	5 (1.6%)
Tyr (Y)	12 (3.9%)
Val (V)	21 (6.8%)

Amino acid counting

	Total number:	311
	Positively charged (Arg+Lys):	33 (10.6%)
	Negatively charged (Asp+Glu):	48 (15.4%)
	Aromatic (Phe+His+Try+Tyr):	48 (15.4%)

Biochemical parameters

	Atomic composition:	C₁₅₈₅H₂₄₁₈N₄₃₄O₄₆₅S₁₄
	Molecular mass [Da]:	35442.1
	Theoretical pI:	5.41
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	45380 / 45568 (all Cys red/ox)

Codon usage

	Organism:	E. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	Codon quality (CAI):	good (0.73)	good (0.70)	acceptable (0.55)	good (0.63)	excellent (0.82)	good (0.78)

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If you have any questions, comments or suggestions, please leave us a comment.

Analytical präparation

[...] Characterization

DDT-Dehydrochlorinase

[...] description [...] reaction [...] production

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)
ATGGACTTT ... TTCCTGAGCTAGTAG
ORF from nucleotide position 1 to 627 (excluding stop-codon)

Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)

1	MDFYYLPGSAPCRAVQMTAAAVGVELNLKLTDLMKGEHMKPEFLKLNPQHCIPTLVDNGFALWESRAIQIYLAEKYGKDDKLYPKDPQKRAVVNQRLYFD
101	MGTLYQRFADYHYPQIFAKQPANPENEKKMKDAVGFLNTFLEGQEYAAGNDLTIADLSLAATIATYEVAGFDFAPYPNVAAWFARCKANAPGYALNQAGA
201	DEFKAKFLS*

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)	30 (14.4%)
Arg (R)	6 (2.9%)
Asn (N)	11 (5.3%)
Asp (D)	13 (6.2%)

Cys (C)	3 (1.4%)
Gln (Q)	10 (4.8%)
Glu (E)	11 (5.3%)
Gly (G)	12 (5.7%)

His (H)	3 (1.4%)
Ile (I)	6 (2.9%)
Leu (L)	20 (9.6%)
Lys (K)	16 (7.7%)

Met (M)	6 (2.9%)
Phe (F)	13 (6.2%)
Pro (P)	13 (6.2%)
Ser (S)	4 (1.9%)

Thr (T)	8 (3.8%)
Trp (W)	2 (1.0%)
Tyr (Y)	13 (6.2%)
Val (V)	9 (4.3%)

Amino acid counting

	Total number:	209
	Positively charged (Arg+Lys):	22 (10.5%)
	Negatively charged (Asp+Glu):	24 (11.5%)
	Aromatic (Phe+His+Try+Tyr):	31 (14.8%)

Biochemical parameters

	Atomic composition:	C₁₀₇₀H₁₆₁₈N₂₇₂O₃₀₄S₉
	Molecular mass [Da]:	23444.8
	Theoretical pI:	5.94
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	30370 / 30558 (all Cys red/ox)

Codon usage

	Organism:	E. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	Codon quality (CAI):	excellent (0.87)	excellent (0.85)	good (0.62)	good (0.71)	excellent (0.80)	good (0.69)

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Analytical präparation

[...] Characterization

PP1

Figure 3:

SpyCatcher & SpyTag

[...] description [...] reaction [...] production

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0

Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
ATGGCCGGCGTTGATACC ... GCTCATATTACCGGT
ORF from nucleotide position -8 to 345 (excluding stop-codon)

Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)

1	MAGVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQ
101	GQVTVNGKATKGDAHITG*

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)	10 (8.5%)
Arg (R)	2 (1.7%)
Asn (N)	2 (1.7%)
Asp (D)	10 (8.5%)

Cys (C)	0 (0.0%)
Gln (Q)	5 (4.2%)
Glu (E)	9 (7.6%)
Gly (G)	14 (11.9%)

His (H)	2 (1.7%)
Ile (I)	6 (5.1%)
Leu (L)	5 (4.2%)
Lys (K)	8 (6.8%)

Met (M)	3 (2.5%)
Phe (F)	4 (3.4%)
Pro (P)	2 (1.7%)
Ser (S)	10 (8.5%)

Thr (T)	14 (11.9%)
Trp (W)	1 (0.8%)
Tyr (Y)	4 (3.4%)
Val (V)	7 (5.9%)

Amino acid counting

	Total number:	118
	Positively charged (Arg+Lys):	10 (8.5%)
	Negatively charged (Asp+Glu):	19 (16.1%)
	Aromatic (Phe+His+Try+Tyr):	11 (9.3%)

Biochemical parameters

	Atomic composition:	C₅₄₃H₈₄₈N₁₄₄O₁₉₂S₃
	Molecular mass [Da]:	12561.7
	Theoretical pI:	4.49
	Extinction coefficient at 280 nm [M^-1 cm^-1]:	11460 / 11460 (all Cys red/ox)

Codon usage

	Organism:	E. coli	B. subtilis	S. cerevisiae	A. thaliana	P. patens	Mammals
	Codon quality (CAI):	excellent (0.83)	good (0.76)	good (0.78)	good (0.80)	good (0.78)	good (0.67)

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.

Analytical präparation

[...] Characterization

References:

http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984

http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984 Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. Cell, 37(2):629–33.
http://udel.edu/~gshriver/pdf/Pimenteletal1997.pdf Pmentel et al., 1997 Pimentel, D., Wilson, C., McCullum, C., Huang, R., Dwen, P., Flack, J. Tran, Q., Saltman, T., Cliff, T. (1997). Economic and environmental benefits of biodiversity. BioScience, Vol. 47, No. 11., pp. 747-757.

Team:TU-Munich/Results/Recombinant

From 2013.igem.org

Characterization of recombinant effector proteins

Eryhtromycin Esterase

Analytical präparation

Degradation of a chromogenic esterase substrate: 4-Nitrophenyl butyrate

Degradation of Erythromycin

Laccase

Laccase a is a secreted enzyme

Analytical präparation

Structural consideration of EreB

Aktivity determination using ABTS

Nano Luciferase

Analytical präparation

Structure of the Nano Luciferase

XylE

Analytical präparation

DDT-Dehydrochlorinase

Analytical präparation

PP1

SpyCatcher & SpyTag

Analytical präparation

References:

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