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	Size [kDa]	Disulphid bridges	comment
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)
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

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

Laccase a is a secreted enzyme

--->

Analytical präparation

[...] Characterization

Structural consideration of Laccase from B. pumilus

Aktivity determination using ABTS

The enzymatic activity of the purified laccae was determined by the ABTS-assay. In a first pre experiment the appropriate dilution factor was determined to 100-fold. The elution fractions obtained from size exclusion chromatography were diluted 1:100 in PBS and in an ELISA plate 100 µl of the enzyme and 100 µl of ABTS substrate were mixed and a kinetic measurement at 405 nm was performed.

Oxidation of relevant xenobiotics

Diclofenac

Estradiol

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)
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

Structure of the Nano Luciferase

--->

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)
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

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)
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

PP1

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

1. 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.
2. 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.

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