Difference between revisions of "Part:BBa K1080003:Design"

(→‎Design Notes)
(→‎Design Notes)
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<b>Amino acid sequence</b>
 
<b>Amino acid sequence</b>
  
−
<FONT FACE="courier">MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDHPFTMAMR VTVAAGKLDS VSLFGGDTAS  
+
<FONT FACE="courier">MGRDLYDDDD KDHPFTMAMR VTVAAGKLDS VSLFGGDTAS LMGGSQTVEK KKSGKEAVME
  
−
LMGGSQTVEK KKSGKEAVME VQLSSTAGID YTVLRDHLAN GEFREAEDET RALLIKLAGP  
+
VQLSSTAGID YTVLRDHLAN GEFREAEDET RALLIKLAGP EAVKRNWVYF TEVKNISVTD
  
−
EAVKRNWVYF TEVKNISVTD FQTLDNLWKA SSNNKFGYSV QKEIWVQNQK RWPKFFKQID  
+
FQTLDNLWKA SSNNKFGYSV QKEIWVQNQK RWPKFFKQID WTQGENNNYR KWPMEFIYSM
  
−
WTQGENNNYR KWPMEFIYSM DAPRGHLPLT NALRGTQLFQ AIMEHPAFEK SSTAKTLDQK  
+
DAPRGHLPLT NALRGTQLFQ AIMEHPAFEK SSTAKTLDQK AAEAAGRTQS LF </FONT>
  
−
AAEAAGRTQS LF </FONT>
 
  
 +
________________________________________
  
−
Number of amino acids: 252
 
  
−
Molecular weight: 28476.0
 
  
−
Theoretical pI: 7.18
+
Number of amino acids: 232
  
−
Amino acid composition:
+
Molecular weight: 26272.6
  
−
Ala (A)  22   8.7%
+
Theoretical pI: 6.45
−
Arg (R)  12   4.8%
+
 
−
Asn (N)  11   4.4%
+
Amino acid composition:
−
Asp (D)  16   6.3%
+
 
 +
Ala (A)  21   9.1%
 +
Arg (R)  11   4.7%
 +
Asn (N)  11   4.7%
 +
Asp (D)  16   6.9%
 
Cys (C)  0   0.0%
 
Cys (C)  0   0.0%
−
Gln (Q)  14   5.6%
+
Gln (Q)  12   5.2%
−
Glu (E)  15   6.0%
+
Glu (E)  15   6.5%
−
Gly (G)  19   7.5%
+
Gly (G)  15   6.5%
−
His (H) 10   4.0%
+
His (H)  4   1.7%
−
Ile (I)  7   2.8%
+
Ile (I)  7   3.0%
−
Leu (L)  18   7.1%
+
Leu (L)  18   7.8%
−
Lys (K)  19   7.5%
+
Lys (K)  19   8.2%
−
Met (M) 11   4.4%
+
Met (M)  8   3.4%
−
Phe (F)  12   4.8%
+
Phe (F)  12   5.2%
−
Pro (P)  7   2.8%
+
Pro (P)  7   3.0%
−
Ser (S)  17   6.7%
+
Ser (S)  15   6.5%
−
Thr (T)  17   6.7%
+
Thr (T)  16   6.9%
−
Trp (W)  6   2.4%
+
Trp (W)  6   2.6%
−
Tyr (Y)  6   2.4%
+
Tyr (Y)  6   2.6%
−
Val (V)  13   5.2%
+
Val (V)  13   5.6%
 +
Pyl (O)  0   0.0%
 +
Sec (U)  0   0.0%
 +
 
 +
(B)  0   0.0%
 +
(Z)  0   0.0%
 +
(X)  0   0.0%
  
−
Asx (B)  0   0.0%
 
−
Glx (Z)  0   0.0%
 
−
Xaa (X)  0   0.0%
 
  
 
Total number of negatively charged residues (Asp + Glu): 31
 
Total number of negatively charged residues (Asp + Glu): 31
−
Total number of positively charged residues (Arg + Lys): 31
+
Total number of positively charged residues (Arg + Lys): 30
  
 
Atomic composition:
 
Atomic composition:
  
−
Carbon      C       1255
+
Carbon      C       1167
−
Hydrogen    H       1940
+
Hydrogen    H       1809
−
Nitrogen    N       358
+
Nitrogen    N       321
−
Oxygen      O       380
+
Oxygen      O       355
−
Sulfur      S         11
+
Sulfur      S         8
  
−
Formula: C1255H1940N358O380S11
+
Formula: C1167H1809N321O355S8
−
Total number of atoms: 3944
+
Total number of atoms: 3660
  
 
Extinction coefficients:
 
Extinction coefficients:
  
−
Extinction coefficients are in units of  M-1 cm-1, at 280 nm.
+
Extinction coefficients are in units of  M-1 cm-1, at 280 nm measured in water.
  
 
Ext. coefficient    41940
 
Ext. coefficient    41940
−
Abs 0.1% (=1 g/l)  1.473, assuming ALL Cys residues appear as half cystines
+
Abs 0.1% (=1 g/l)  1.596
−
 
+
  
 
Estimated half-life:
 
Estimated half-life:
Line 92: Line 96:
 
The N-terminal of the sequence considered is M (Met).
 
The N-terminal of the sequence considered is M (Met).
  
−
The estimated half-life is:  
+
The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
−
                            30 hours (mammalian reticulocytes, in vitro).
+
 
                             >20 hours (yeast, in vivo).
 
                             >20 hours (yeast, in vivo).
 
                             >10 hours (Escherichia coli, in vivo).
 
                             >10 hours (Escherichia coli, in vivo).
Line 100: Line 103:
 
Instability index:
 
Instability index:
  
−
The instability index (II) is computed to be 22.16
+
The instability index (II) is computed to be 22.94
 
This classifies the protein as stable.
 
This classifies the protein as stable.
  
  
  
−
Aliphatic index: 62.38
+
Aliphatic index: 67.33
  
−
Grand average of hydropathicity (GRAVY): -0.655
+
Grand average of hydropathicity (GRAVY): -0.594
  
 
===Source===
 
===Source===

Revision as of 02:13, 25 September 2013


GUN4


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 719
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 404
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Incorporated sequence overlap for Gibson assembly and no GC rich region or restriction site in sequence.

GUN4 Clone: DNA sequence from translation start site:

Translated the DNA sequence into a protein sequence using "Translate" at http://au.expasy.org/tools Then used the translated protein sequence to analyse the protein using "ProtParam" at http://au.expasy.org/tools

No Biobrick sites: EcoRI, PstI, SpeI or XbaI


Amino acid sequence

MGRDLYDDDD KDHPFTMAMR VTVAAGKLDS VSLFGGDTAS LMGGSQTVEK KKSGKEAVME

VQLSSTAGID YTVLRDHLAN GEFREAEDET RALLIKLAGP EAVKRNWVYF TEVKNISVTD

FQTLDNLWKA SSNNKFGYSV QKEIWVQNQK RWPKFFKQID WTQGENNNYR KWPMEFIYSM

DAPRGHLPLT NALRGTQLFQ AIMEHPAFEK SSTAKTLDQK AAEAAGRTQS LF


________________________________________


Number of amino acids: 232

Molecular weight: 26272.6

Theoretical pI: 6.45

Amino acid composition:

Ala (A) 21 9.1% Arg (R) 11 4.7% Asn (N) 11 4.7% Asp (D) 16 6.9% Cys (C) 0 0.0% Gln (Q) 12 5.2% Glu (E) 15 6.5% Gly (G) 15 6.5% His (H) 4 1.7% Ile (I) 7 3.0% Leu (L) 18 7.8% Lys (K) 19 8.2% Met (M) 8 3.4% Phe (F) 12 5.2% Pro (P) 7 3.0% Ser (S) 15 6.5% Thr (T) 16 6.9% Trp (W) 6 2.6% Tyr (Y) 6 2.6% Val (V) 13 5.6% 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): 31 Total number of positively charged residues (Arg + Lys): 30

Atomic composition:

Carbon C 1167 Hydrogen H 1809 Nitrogen N 321 Oxygen O 355 Sulfur S 8

Formula: C1167H1809N321O355S8 Total number of atoms: 3660

Extinction coefficients:

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

Ext. coefficient 41940 Abs 0.1% (=1 g/l) 1.596

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 22.94 This classifies the protein as stable.


Aliphatic index: 67.33

Grand average of hydropathicity (GRAVY): -0.594

Source

Chlamydomonas reinhardtii

References