Team:TU-Munich/Results/Recombinant

From 2013.igem.org

(Difference between revisions)
(→Eryhtromycin Esterase)
(→Analytical präparation)
Line 98: Line 98:
[...] Characterization
[...] Characterization
<br><br>
<br><br>
-
 
+
[[File:TUM13_Analytprep_Laccase.png|thumb|right|320px| Figure 3:]]
 +
[[File:TUM13_Preparative_Laccase.png|thumb|right|320px| Figure 3:]]

Revision as of 17:42, 25 September 2013


Text

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 <partinfo>BBa_E0040</partinfo> RFC25 c-term. Streptag II
Laccase <partinfo>BBa_E0040</partinfo> RFC25 c-term. Streptag II
Nano Luciferase <partinfo>BBa_E0040</partinfo> RFC25 c-term. Streptag II
XylE


Eryhtromycin Esterase

File:TUM13 Animation EreB.gif
Figure 3:

[...] 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)
 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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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

Figure 3:
Figure 3:

[...] Characterization


Structure of the Nano Luciferase

Figure 3:


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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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

Figure 3:
--->


Analytical präparation

[...] Characterization

Figure 3:
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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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

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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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




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. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
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.



Retrieved from "http://2013.igem.org/Team:TU-Munich/Results/Recombinant"