Difference between revisions of "Part:BBa K1080001:Design"

(→‎Design Notes)
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
  
−
CR-ChlH sequence with His Tag in pET15b. Sequence from Translation start site.
+
<b> Amino Acid Sequenc:</b>
−
 
+
−
Note 8 PstI sites CTGCAG (785, 1031, 1385, 1652, 2681, 2825, 3458, 3569) No EcoRI or XbaI sites. 1 SpeI (ACTAGT) (4226) site 23 bp after stop codon.
+
−
 
+
−
<b>Amino Acid Sequence</b>
+
−
 
+
−
<FONT FACE="courier">GPAKQQVSLD LRDDGAGMFT STSPEMRRVV PDDVKGRVKV KVVYVVLEAQ YQSAISAAVK NINAKNSKVC FEVVGYLLEE LRDQKNLDML KEDVASANIF<br> IGSLIFIEEL AEKIVEAVSP LREKLDACLI FPSMPAVMKL NKLGTFSMAQ LGQSKSVFSE FIKSARKNND NFEEGLLKLV RTLPKVLKYL PSDKAQDAKN<br> FVNSLQYWLG GNSDNLENLL LNTVSNYVPA LKGVDFSVAE PTAYPDVGIW HPLASGMYED LKEYLNWYDT RKDMVFAKDA PVIGLVLQRS HLVTGDEGHY<br> SGVVAELESR GAKVIPVFAG GLDFSAPVKK FFYDPLGSGR TFVDTVVSLT GFALVGGPAR QDAPKAIEAL KNLNVPYLVS LPLVFQTTEE WLDSELGVHP<br> VQVALQVALP ELDGAMEPIV FAGRDSNTGK SHSLPDRIAS LCARAVNWAN LRKKRNAEKK LAVTVFSFPP DKGNVGTAAY LNVFGSIYRV LKNLQREGYD<br> VGALPPSEED LIQSVLTQKE AKFNSTDLHI AYKMKVDEYQ KLCPYAEALE ENWGKPPGTL NTNGQELLVY GRQYGNVFIG VQPTFGYEGD PMRLLFSKSA<br> SPHHGFAAYY TFLEKIFKAD AVLHFGTHGS LEFMPGKQVG MSGVCYPDSL IGTIPNLYYY AANNPSEATI AKRRSYANTI SYLTPPAENA GLYKGLKELK<br> ELISSYQGMR ESGRAEQICA TIIETAKLCN LDRDVTLPDA DAKDLTMDMR DSVVGQVYRK LMEIESRLLP CGLHVVGCPP TAEEAVATLV NIAELDRPDN<br> NPPIKGMPGI LARAIGRDIE SIYSGNNKGV LADVDQLQRI TEASRTCVRE FVKDRTGLNG RIGTNWITNL LKFTGFYVDP WVRGLQNGEF ASANREELIT<br> LFNYLEFCLT QVVKDNELGA LVEALNGQYV EPGPGGDPIR NPNVLPTGKN IHALDPQSIP TQAALKSARL VVDRLLDRER DNNGGKYPET IALVLWGTDN<br> IKTYGESLAQ VMMMVGVKPV ADALGRVNKL EVIPLEELGR PRVDVVVNCS GVFRDLFVNQ MLLLDRAIKL AAEQDEPDEM  NFVRKHAKQQ AAELGLQSLR<br> DAATRVFSNS SGSYSSNVNL AVENSSWSDE SQLQEMYLKR KSYAFNSDRP GAGGEMQRDV FETAMKTVDV TFQNLDSSEI SLTDVSHYFD SDPTKLVASL<br> RNDGRTPNAY IADTTTANAQ VRTLGETVRL DARTKLLNPK WYEGMLASGY EGVREIQKRM TNTMGWSATS GMVDNWVYDE ANSTFIEDAA MAERLMNTNP<br> NSFRKLVATF LEANGRGYWD AKPEQLERLR QLYMDVEDKI EGVE  </FONT>
+
  
 +
<FONT FACE="courier">MCNVATGPRP PMTTFTGGNK GPAKQQVSLD LRDDGAGMFT STSPEMRRVV PDDVKGRVKV<br> KVVYVVLEAQ YQSAISAAVK NINAKNSKVC FEVVGYLLEE LRDQKNLDML KEDVASANIF<br> IGSLIFIEEL AEKIVEAVSP LREKLDACLI FPSMPAVMKL NKLGTFSMAQ LGQSKSVFSE<br> FIKSARKNND NFEEGLLKLV RTLPKVLKYL PSDKAQDAKN FVNSLQYWLG GNSDNLENLL<br> LNTVSNYVPA LKGVDFSVAE PTAYPDVGIW HPLASGMYED LKEYLNWYDT RKDMVFAKDA<br>  PVIGLVLQRS HLVTGDEGHY SGVVAELESR GAKVIPVFAG GLDFSAPVKK FFYDPLGSGR<br> TFVDTVVSLT GFALVGGPAR QDAPKAIEAL KNLNVPYLVS LPLVFQTTEE WLDSELGVHP<br> VQVALQVALP ELDGAMEPIV FAGRDSNTGK SHSLPDRIAS LCARAVNWAN LRKKRNAEKK<br> LAVTVFSFPP DKGNVGTAAY LNVFGSIYRV LKNLQREGYD VGALPPSEED LIQSVLTQKE<br> AKFNSTDLHI AYKMKVDEYQ KLCPYAEALE ENWGKPPGTL NTNGQELLVY GRQYGNVFIG<br>  VQPTFGYEGD PMRLLFSKSA SPHHGFAAYY TFLEKIFKAD AVLHFGTHGS LEFMPGKQVG<br> MSGVCYPDSL IGTIPNLYYY AANNPSEATI AKRRSYANTI SYLTPPAENA GLYKGLKELK<br> ELISSYQGMR ESGRAEQICA TIIETAKLCN LDRDVTLPDA DAKDLTMDMR DSVVGQVYRK<br> LMEIESRLLP CGLHVVGCPP TAEEAVATLV NIAELDRPDN NPPIKGMPGI LARAIGRDIE<br> SIYSGNNKGV LADVDQLQRI TEASRTCVRE FVKDRTGLNG RIGTNWITNL LKFTGFYVDP<br> WVRGLQNGEF ASANREELIT LFNYLEFCLT QVVKDNELGA LVEALNGQYV EPGPGGDPIR<br> NPNVLPTGKN IHALDPQSIP TQAALKSARL VVDRLLDRER DNNGGKYPET IALVLWGTDN<br> IKTYGESLAQ VMMMVGVKPV ADALGRVNKL EVIPLEELGR PRVDVVVNCS GVFRDLFVNQ<br> AVENSSWSDE SQLQEMYLKR KSYAFNSDRP GAGGEMQRDV FETAMKTVDV TFQNLDSSEI<br> SLTDVSHYFD SDPTKLVASL RNDGRTPNAY IADTTTANAQ VRTLGETVRL DARTKLLNPK<br> WYEGMLASGY EGVREIQKRM TNTMGWSATS GMVDNWVYDE ANSTFIEDAA MAERLMNTNP<br> NSFRKLVATF LEANGRGYWD AKPEQLERLR QLYMDVEDKI EGVE </FONT>
 
References and documentation are available.  
 
References and documentation are available.  
 
Please note the modified algorithm for extinction coefficient.
 
Please note the modified algorithm for extinction coefficient.
  
 
--------------------------------------------------------------------------------
 
--------------------------------------------------------------------------------
−
Number of amino acids: 1344
+
Number of amino acids: 1364
  
−
Molecular weight: 148676.1
+
Molecular weight: 150738.5
  
−
Theoretical pI: 5.33
+
Theoretical pI: 5.39
  
−
Amino acid composition: Ala (A) 113   8.4%
+
Amino acid composition: Ala (A) 114   8.4%
−
Arg (R)  68   5.1%
+
Arg (R)  69   5.1%
−
Asn (N)  77   5.7%
+
Asn (N)  79   5.8%
−
Asp (D)  81   6.0%
+
Asp (D)  81   5.9%
−
Cys (C)  12   0.9%
+
Cys (C)  13   1.0%
 
Gln (Q)  46   3.4%
 
Gln (Q)  46   3.4%
−
Glu (E)  91   6.8%
+
Glu (E)  91   6.7%
−
Gly (G) 97   7.2%
+
Gly (G) 100   7.3%
 
His (H)  14   1.0%
 
His (H)  14   1.0%
−
Ile (I)  51   3.8%
+
Ile (I)  51   3.7%
−
Leu (L) 141 10.5%
+
Leu (L) 141 10.3%
−
Lys (K)  78   5.8%
+
Lys (K)  79   5.8%
−
Met (M)  33   2.5%
+
Met (M)  35   2.6%
−
Phe (F)  49   3.6%
+
Phe (F)  50   3.7%
−
Pro (P)  67   5.0%
+
Pro (P)  70   5.1%
 
Ser (S)  84   6.2%
 
Ser (S)  84   6.2%
−
Thr (T)  65   4.8%
+
Thr (T)  69   5.1%
 
Trp (W)  14   1.0%
 
Trp (W)  14   1.0%
 
Tyr (Y)  49   3.6%
 
Tyr (Y)  49   3.6%
−
Val (V) 114   8.5%
+
Val (V) 115   8.4%
 
Pyl (O)  0   0.0%
 
Pyl (O)  0   0.0%
 
Sec (U)  0   0.0%
 
Sec (U)  0   0.0%
Line 55: Line 50:
  
 
Total number of negatively charged residues (Asp + Glu): 172
 
Total number of negatively charged residues (Asp + Glu): 172
−
Total number of positively charged residues (Arg + Lys): 146
+
Total number of positively charged residues (Arg + Lys): 148
  
 
Atomic composition:
 
Atomic composition:
  
−
Carbon      C       6616
+
Carbon      C       6703
−
Hydrogen    H     10441
+
Hydrogen    H     10581
−
Nitrogen    N       1791
+
Nitrogen    N       1817
−
Oxygen      O       2010
+
Oxygen      O       2036
−
Sulfur      S         45
+
Sulfur      S         48
  
−
Formula: C6616H10441N1791O2010S45
+
Formula: C6703H10581N1817O2036S48
−
Total number of atoms: 20903
+
Total number of atoms: 21185
  
 
Extinction coefficients:
 
Extinction coefficients:
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Ext. coefficient  150760
 
Ext. coefficient  150760
−
Abs 0.1% (=1 g/l)  1.014, assuming all pairs of Cys residues form cystines
+
Abs 0.1% (=1 g/l)  1.000, assuming all pairs of Cys residues form cystines
  
  
 
Ext. coefficient  150010
 
Ext. coefficient  150010
−
Abs 0.1% (=1 g/l)  1.009, assuming all Cys residues are reduced
+
Abs 0.1% (=1 g/l)  0.995, assuming all Cys residues are reduced
  
 
Estimated half-life:
 
Estimated half-life:
  
−
The N-terminal of the sequence considered is G (Gly).
+
The N-terminal of the sequence considered is M (Met).
  
 
The estimated half-life is:  
 
The estimated half-life is:  
Line 91: Line 86:
 
Instability index:
 
Instability index:
  
−
The instability index (II) is computed to be 32.91
+
The instability index (II) is computed to be 32.83
 
This classifies the protein as stable.
 
This classifies the protein as stable.
  
  
  
−
Aliphatic index: 88.72
+
Aliphatic index: 87.71
  
−
Grand average of hydropathicity (GRAVY): -0.257
+
Grand average of hydropathicity (GRAVY): -0.260
  
 
===Source===
 
===Source===

Revision as of 10:12, 25 September 2013


ChlH


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1928
    Illegal BglII site found at 2234
    Illegal BglII site found at 3620
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 486
    Illegal NgoMIV site found at 2152
    Illegal AgeI site found at 1132
    Illegal AgeI site found at 2650
    Illegal AgeI site found at 2704
    Illegal AgeI site found at 2923
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2254
    Illegal BsaI.rc site found at 3049
    Illegal SapI.rc site found at 2979


Design Notes

Amino Acid Sequenc:

MCNVATGPRP PMTTFTGGNK GPAKQQVSLD LRDDGAGMFT STSPEMRRVV PDDVKGRVKV
KVVYVVLEAQ YQSAISAAVK NINAKNSKVC FEVVGYLLEE LRDQKNLDML KEDVASANIF
IGSLIFIEEL AEKIVEAVSP LREKLDACLI FPSMPAVMKL NKLGTFSMAQ LGQSKSVFSE
FIKSARKNND NFEEGLLKLV RTLPKVLKYL PSDKAQDAKN FVNSLQYWLG GNSDNLENLL
LNTVSNYVPA LKGVDFSVAE PTAYPDVGIW HPLASGMYED LKEYLNWYDT RKDMVFAKDA
PVIGLVLQRS HLVTGDEGHY SGVVAELESR GAKVIPVFAG GLDFSAPVKK FFYDPLGSGR
TFVDTVVSLT GFALVGGPAR QDAPKAIEAL KNLNVPYLVS LPLVFQTTEE WLDSELGVHP
VQVALQVALP ELDGAMEPIV FAGRDSNTGK SHSLPDRIAS LCARAVNWAN LRKKRNAEKK
LAVTVFSFPP DKGNVGTAAY LNVFGSIYRV LKNLQREGYD VGALPPSEED LIQSVLTQKE
AKFNSTDLHI AYKMKVDEYQ KLCPYAEALE ENWGKPPGTL NTNGQELLVY GRQYGNVFIG
VQPTFGYEGD PMRLLFSKSA SPHHGFAAYY TFLEKIFKAD AVLHFGTHGS LEFMPGKQVG
MSGVCYPDSL IGTIPNLYYY AANNPSEATI AKRRSYANTI SYLTPPAENA GLYKGLKELK
ELISSYQGMR ESGRAEQICA TIIETAKLCN LDRDVTLPDA DAKDLTMDMR DSVVGQVYRK
LMEIESRLLP CGLHVVGCPP TAEEAVATLV NIAELDRPDN NPPIKGMPGI LARAIGRDIE
SIYSGNNKGV LADVDQLQRI TEASRTCVRE FVKDRTGLNG RIGTNWITNL LKFTGFYVDP
WVRGLQNGEF ASANREELIT LFNYLEFCLT QVVKDNELGA LVEALNGQYV EPGPGGDPIR
NPNVLPTGKN IHALDPQSIP TQAALKSARL VVDRLLDRER DNNGGKYPET IALVLWGTDN
IKTYGESLAQ VMMMVGVKPV ADALGRVNKL EVIPLEELGR PRVDVVVNCS GVFRDLFVNQ
AVENSSWSDE SQLQEMYLKR KSYAFNSDRP GAGGEMQRDV FETAMKTVDV TFQNLDSSEI
SLTDVSHYFD SDPTKLVASL RNDGRTPNAY IADTTTANAQ VRTLGETVRL DARTKLLNPK
WYEGMLASGY EGVREIQKRM TNTMGWSATS GMVDNWVYDE ANSTFIEDAA MAERLMNTNP
NSFRKLVATF LEANGRGYWD AKPEQLERLR QLYMDVEDKI EGVE References and documentation are available. Please note the modified algorithm for extinction coefficient.

Number of amino acids: 1364

Molecular weight: 150738.5

Theoretical pI: 5.39

Amino acid composition: Ala (A) 114 8.4% Arg (R) 69 5.1% Asn (N) 79 5.8% Asp (D) 81 5.9% Cys (C) 13 1.0% Gln (Q) 46 3.4% Glu (E) 91 6.7% Gly (G) 100 7.3% His (H) 14 1.0% Ile (I) 51 3.7% Leu (L) 141 10.3% Lys (K) 79 5.8% Met (M) 35 2.6% Phe (F) 50 3.7% Pro (P) 70 5.1% Ser (S) 84 6.2% Thr (T) 69 5.1% Trp (W) 14 1.0% Tyr (Y) 49 3.6% Val (V) 115 8.4% Pyl (O) 0 0.0% Sec (U) 0 0.0% 

(B)   0	  0.0%
(Z)   0	  0.0%
(X)   0	  0.0%


Total number of negatively charged residues (Asp + Glu): 172 Total number of positively charged residues (Arg + Lys): 148

Atomic composition:

Carbon C 6703 Hydrogen H 10581 Nitrogen N 1817 Oxygen O 2036 Sulfur S 48

Formula: C6703H10581N1817O2036S48 Total number of atoms: 21185

Extinction coefficients:

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 150760 Abs 0.1% (=1 g/l) 1.000, assuming all pairs of Cys residues form cystines


Ext. coefficient 150010 Abs 0.1% (=1 g/l) 0.995, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 

                            30 hours (mammalian reticulocytes, in vitro).
                           >20 hours (yeast, in vivo).
                           >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 32.83 This classifies the protein as stable.


Aliphatic index: 87.71

Grand average of hydropathicity (GRAVY): -0.260

Source

Chlamydomonas reinhardtii

References