Team:TU-Munich/Results/Recombinant

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Revision as of 19:20, 28 September 2013

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 preparation

Figure 3:

Degradation of a chromogenic esterase substrate: 4-Nitrophenyl butyrate

Figure 3:

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)

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Laccase a is a secreted enzyme

Figure 3:

--->

Analytical präparation

[...] Characterization

Figure 3:

Structural consideration of Laccase from B. pumilus

File:TUM13 Animation Laccasex.gif

Figure 3:

Activity determination using ABTS

File:TUM13 ABTS-oxidation.png

Figure 3:

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. The absorption at 280 nm in the SEC chromatogramm (blue) identifies three main protein peaks, with a first peak corresponding to aggregated protein, a shoulder which also corresponds to higher molecular protein and a single peak which was proposed to be the monomeric laccase. The relative activity obtained for the different elution fractions was plotted in the same diagramm and shows a clear peak which matches the laccase peak in the SEC. Beside this major peak a second smaller peak of active fraction was visible which appeared in earlier elution fractions and might correspond to dimerized laccase. As the laccase is a secreted enzyme which also bears disluphide bonds it was produced in the cytoplasm and subsequently it was oxidized to form the proper disulphide bond. As this process might be only partial there is a possiblity for the formation of disulphid dimers. Never the less the fractions 14 to 17 were pooled for further experiments as they showed the highest enzymatic activity. The protein concentration of the pooled fraction was determined to 0.48 mg/ml after SEC.

Oxidation of relevant xenobiotics

Diclofenac

Figure 3:

Estradiol

File:TUM13 Laccase Estradiol.png

Figure 3:

Nano Luciferase

The Nano Luciferase (NanoLuc) which was introduced in 2013 by Promega is a new member of the luciferase reporter gene/protein familiy and shows some advantages compared to the other family members. The NanoLuc is very small (19 kDa) compared to the firefly luciferase (61 kDa) and the Renilla luciferase (36 kDa). On the other hand it is also said that the specific activity of the NanoLuc is about 150-fold stronger compared to conventional luciferases and the background caused by autoluminescense of the substrate shel be smaller.

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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Production in E. coli and purification

Figure 3:Analytical size exclusion chromatography on a Superdex 200 10/30 column showing a single elution peak for the NanoLuc.

Figure 3:Preperative size exclusion chromatography on a Superdex 75 10/30 column showing a single elution peak for the NanoLuc.

Therefore the NanoLuc was synthesized as a BioBrick in RFC[25] and was produced in E. coli using the pBad expression system with a C-terminal Strep-tag. After the production (2 l of LB-media for analytical and 12 l for preparative preparations) the cells were disrupted using sonification and the lysate was dialysed against 5 l of 1x SA-buffer. Afterwards the lysate was applied to a Streptavidin-Affinity (SA) column and was subsequently washed using SA-Buffer until a baseline was reached and the protein was then eluted using 5 mM of biotin (Attention: These are special columns which are not availible commercially. If you are using commercial colum material you have to use d-Desthiobiotin because usual biotin will elute your protein but you will not be able to regenerate the column after your chromatography). After the SA-chromatography the protein was concentrated using centrifugal concentration units (MWCO: 10 kDa). The concentrated protein was then applied on a Superdex S200/75 size exclusion chromatography.

Structure of the Nano Luciferase

Figure 3:

--->

Activity determination of Luminescense

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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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)

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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.

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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 1379191901701 --><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>GTCTTCACA&nbsp;...&nbsp;ATTCTGGCG<i>ACCGGT</i></u></span><br>&nbsp;<strong>ORF</strong> from nucleotide position -8 to 516 (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">MAGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT</td></tr><tr><td class="AutoAnnotatorSeqNum">101&nbsp;</td><td class="AutoAnnotatorSeqSeq">LVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV<b>TG</b>WRLCERILATG*</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">163 to 164</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">4 (2.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">7 (4.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">8 (4.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">12 (6.9%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">1 (0.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">7 (4.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">8 (4.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">22 (12.6%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">4 (2.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">18 (10.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">16 (9.1%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">7 (4.0%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">3 (1.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">8 (4.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">6 (3.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">6 (3.4%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">11 (6.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">3 (1.7%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">6 (3.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">18 (10.3%)</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">175</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">14 (8.0%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">20 (11.4%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">21 (12.0%)</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>882</sub>H<sub>1377</sub>N<sub>229</sub>O<sub>254</sub>S<sub>4</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">19381.3</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">5.10</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">25440 / 25503 (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 (0.74)</td><td class="AutoAnnotatorCodonUsage3">good (0.76)</td><td class="AutoAnnotatorCodonUsage3">acceptable (0.55)</td><td class="AutoAnnotatorCodonUsage3">good (0.65)</td><td class="AutoAnnotatorCodonUsage3">excellent (0.81)</td><td class="AutoAnnotatorCodonUsage3">good (0.77)</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===
+===Production in ''E. coli'' and purification===
 [[File:TUM13_Analytprep_NanoLuc.png|thumb|right|320px| Figure 3:Analytical size exclusion chromatography on a Superdex 200 10/30 column showing a single elution peak for the NanoLuc.]]
 [[File:TUM13_Preparative_NanoLuc.png|thumb|right|320px| Figure 3:Preperative size exclusion chromatography on a Superdex 75 10/30 column showing a single elution peak for the NanoLuc.]]

Team:TU-Munich/Results/Recombinant

From 2013.igem.org

Revision as of 19:20, 28 September 2013

Characterization of recombinant effector proteins

Eryhtromycin Esterase

Analytical preparation

Degradation of a chromogenic esterase substrate: 4-Nitrophenyl butyrate

Degradation of Erythromycin

Laccase

Laccase a is a secreted enzyme

Analytical präparation

Structural consideration of Laccase from B. pumilus

Activity determination using ABTS

Oxidation of relevant xenobiotics

Diclofenac

Estradiol

Nano Luciferase

Production in E. coli and purification

Structure of the Nano Luciferase

Activity determination of Luminescense

XylE

Analytical präparation

DDT-Dehydrochlorinase

Analytical präparation

PP1

SpyCatcher & SpyTag

Analytical präparation

References:

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