Team:TU-Munich/Results/Recombinant

From 2013.igem.org

(Difference between revisions)
(→Eryhtromycin Esterase)
(→Laccase)
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[...] reaction
[...] reaction
[...] production
[...] production
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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 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% } </style><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1379191798681 --><th id="AutoAnnotatorHeader" colspan="2">Protein data table for BioBrick <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_<!------------------------Enter BioBrick number here------------------------>">BBa_<!------------------------Enter BioBrick number here------------------------></a> automatically created by the <a href="https://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 25</strong>, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)<br><span class="AutoAnnotatorSequence">&nbsp;<u><i>ATGGCCGGC</i>AACCTAGAA&nbsp;...&nbsp;GATATCATC<i>ACCGGT</i></u></span><br>&nbsp;<strong>ORF</strong> from nucleotide position -8 to 1530 (excluding stop-codon)</td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid sequence:</strong> (RFC25 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;</td><td class="AutoAnnotatorSeqSeq">MAGNLEKFVDELPIPEVAKPVKKNPKQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIHANRNEKVKVKWMNKLPLKHFLPVDHTIHEGHHDEPEV</td></tr><tr><td class="AutoAnnotatorSeqNum">101&nbsp;</td><td class="AutoAnnotatorSeqSeq">KTVVHLHGGVTPASSDGYPEAWFSRDFEA<b>TG</b>PFFEREVYEYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKGEYDIPLMIMDRTF</td></tr><tr><td class="AutoAnnotatorSeqNum">201&nbsp;</td><td class="AutoAnnotatorSeqSeq">QEDGALFYPSRPNNTPEDSDIPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRFRILNASNTRTYELHLDNDATILQIGSDGGFLPRPVHHQSFSIAPAE</td></tr><tr><td class="AutoAnnotatorSeqNum">301&nbsp;</td><td class="AutoAnnotatorSeqSeq">RFDVIIDFSAYENKTITLKNKAGCGQEVNPETDANIMQFKVTRPLKGRAPKTLRPIFKPLPPLRPCRADKERTLTLTGTQDKYGRPILLLDNQFWNDPVT</td></tr><tr><td class="AutoAnnotatorSeqNum">401&nbsp;</td><td class="AutoAnnotatorSeqSeq">ENPRLGSVEVWSIVNPTRGTHPIHLHLVQFRVIDRRPFDTEVYQSTGDIVYTGPNEAPPLHEQGYKDTIQAHAGEVIRIIARFVPYSGRYVWHCHILEHE</td></tr><tr><td class="AutoAnnotatorSeqNum">501&nbsp;</td><td class="AutoAnnotatorSeqSeq">DYDMMRPMDIITG*</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="https://2013.igem.org/Team:TU-Munich/Results/Software/FeatureList">list of supported features</a>)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqFeat1"></td><td class="AutoAnnotatorSeqFeat2a">RFC25 scar (shown in bold):&nbsp;</td><td class="AutoAnnotatorSeqFeat3">130 to 131</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">27 (5.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">29 (5.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">23 (4.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">31 (6.0%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">5 (1.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">14 (2.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">37 (7.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">30 (5.8%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">23 (4.5%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">31 (6.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">41 (8.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">27 (5.3%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">10 (1.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">24 (4.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">47 (9.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">18 (3.5%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">33 (6.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">8 (1.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">22 (4.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">33 (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">513</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">56 (10.9%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">68 (13.3%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">77 (15.0%)</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>2671</sub>H<sub>4072</sub>N<sub>718</sub>O<sub>760</sub>S<sub>15</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">58883.0</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">6.03</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">76780 / 77093 (all Cys red/ox)</td></tr></table></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 (0.72)</td><td class="AutoAnnotatorCodonUsage3">good (0.75)</td><td class="AutoAnnotatorCodonUsage3">good (0.68)</td><td class="AutoAnnotatorCodonUsage3">good (0.74)</td><td class="AutoAnnotatorCodonUsage3">good (0.77)</td><td class="AutoAnnotatorCodonUsage3">good (0.67)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"> The BioBrick-AutoAnnotator was created by <a href="https://2013.igem.org/Team:TU-Munich">TU-Munich 2013</a> iGEM team. For more information please see the <a href="https://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="https://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">comment</a>.</td></tr></table><br></html>
===Analytical präparation===
===Analytical präparation===
[...] Characterization
[...] Characterization

Revision as of 20:50, 14 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

[...] 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. 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.

Analytical präparation

[...] Characterization

Nano Luciferase

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

Analytical präparation

[...] Characterization



XylE

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

Analytical präparation

[...] Characterization




DDT-Dehydrochlorinase

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

Analytical präparation

[...] Characterization




SpyCatcher & SpyTag

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

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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