Difference between revisions of "Part:BBa K1080000:Design"

(→‎Design Notes)
(→‎Design Notes)
Line 20: Line 20:
 
<b>Amino Acid Sequence</b>
 
<b>Amino Acid Sequence</b>
  
−
<FONT FACE="courier">RPIFPFTAIV GQDEMKLALI LNVIDPKIGG VMIMGDRGTG KSTTIRALAD LLPEMQVVAN DPFNSDPTDP ELMSEEVRNR VKAGEQLPVS SKKIPMVDLP<br> LGATEDRVCG TIDIEKALTE GVKAFEPGLL AKANRGILYV DEVNLLDDHL VDVLLDSAAS GWNTVEREGI SISHPARFIL VGSGNPEEGE LRPQLLDRFG<br> MHAQIGTVKD PRLRVQIVSQ RSTFDENPAA FRKDYEAGQM ALTQRIVDAR KLLKQGEVNY DFRVKISQIC SDLNVDGIRG DIVTNRAAKA LAAFEGRTEV<br>  TPEDIYRVIP LCLRHRLRKD PLAEIDDGDR VREIFKQVFG ME </FONT>  
+
<FONT FACE="courier"> MAATEVKAAE GRTEKELGQA RPIFPFTAIV GQDEMKLALI LNVIDPKIGG VMIMGDRGTG KSTTIRALAD LLPEMQVVAN DPFNSDPTDP ELMSEEVRNR VKAGEQLPVS SKKIPMVDLP LGATEDRVCG TIDIEKALTE GVKAFEPGLL AKANRGILYV DEVNLLDDHL VDVLLDSAAS GWNTVEREGI SISHPARFIL VGSGNPEEGE LRPQLLDRFG MHAQIGTVKD PRLRVQIVSQ RSTFDENPAA FRKDYEAGQM ALTQRIVDAR KLLKQGEVNY DFRVKISQIC SDLNVDGIRG DIVTNRAAKA LAAFEGRTEV TPEDIYRVIP LCLRHRLRKD PLAEIDDGDR VREIFKQVFG ME </FONT>
  
 
References and documentation are available.  
 
References and documentation are available.  
Line 26: Line 26:
  
 
--------------------------------------------------------------------------------
 
--------------------------------------------------------------------------------
−
Number of amino acids: 342
+
Number of amino acids: 362
  
−
Molecular weight: 37880.4
+
Molecular weight: 39952.7
  
−
Theoretical pI: 5.14
+
Theoretical pI: 5.11
  
−
Amino acid composition: Ala (A)  26   7.6%
+
Amino acid composition: Ala (A)  31   8.6%
−
Arg (R)  27   7.9%
+
Arg (R)  28   7.7%
−
Asn (N)  12   3.5%
+
Asn (N)  12   3.3%
−
Asp (D)  29   8.5%
+
Asp (D)  29   8.0%
−
Cys (C)  3   0.9%
+
Cys (C)  3   0.8%
−
Gln (Q)  12   3.5%
+
Gln (Q)  13   3.6%
−
Glu (E)  25   7.3%
+
Glu (E)  29   8.0%
−
Gly (G)  26   7.6%
+
Gly (G)  28   7.7%
−
His (H)  4   1.2%
+
His (H)  4   1.1%
−
Ile (I)  25   7.3%
+
Ile (I)  25   6.9%
−
Leu (L)  33   9.6%
+
Leu (L)  34   9.4%
−
Lys (K)  17   5.0%
+
Lys (K)  19   5.2%
−
Met (M)   9   2.6%
+
Met (M) 10   2.8%
−
Phe (F)  12   3.5%
+
Phe (F)  12   3.3%
−
Pro (P)  19   5.6%
+
Pro (P)  19   5.2%
−
Ser (S)  14   4.1%
+
Ser (S)  14   3.9%
−
Thr (T)  15   4.4%
+
Thr (T)  17   4.7%
 
Trp (W)  1   0.3%
 
Trp (W)  1   0.3%
−
Tyr (Y)  4   1.2%
+
Tyr (Y)  4   1.1%
−
Val (V)  29   8.5%
+
Val (V)  30   8.3%
 
Pyl (O)  0   0.0%
 
Pyl (O)  0   0.0%
 
Sec (U)  0   0.0%
 
Sec (U)  0   0.0%
Line 60: Line 60:
  
  
−
Total number of negatively charged residues (Asp + Glu): 54
+
Total number of negatively charged residues (Asp + Glu): 58
−
Total number of positively charged residues (Arg + Lys): 44
+
Total number of positively charged residues (Arg + Lys): 47
  
 
Atomic composition:
 
Atomic composition:
  
−
Carbon      C       1666
+
Carbon      C       1752
−
Hydrogen    H       2711
+
Hydrogen    H       2857
−
Nitrogen    N       473
+
Nitrogen    N       499
−
Oxygen      O       508
+
Oxygen      O       539
−
Sulfur      S         12
+
Sulfur      S         13
  
−
Formula: C1666H2711N473O508S12
+
Formula: C1752H2857N499O539S13
−
Total number of atoms: 5370
+
Total number of atoms: 5660
  
 
Extinction coefficients:
 
Extinction coefficients:
Line 79: Line 79:
  
 
Ext. coefficient    11585
 
Ext. coefficient    11585
−
Abs 0.1% (=1 g/l)  0.306, assuming all pairs of Cys residues form cystines
+
Abs 0.1% (=1 g/l)  0.290, assuming all pairs of Cys residues form cystines
  
  
 
Ext. coefficient    11460
 
Ext. coefficient    11460
−
Abs 0.1% (=1 g/l)  0.303, assuming all Cys residues are reduced
+
Abs 0.1% (=1 g/l)  0.287, assuming all Cys residues are reduced
  
 
Estimated half-life:
 
Estimated half-life:
  
−
The N-terminal of the sequence considered is R (Arg).
+
The N-terminal of the sequence considered is M (Met).
  
 
The estimated half-life is:  
 
The estimated half-life is:  
−
                            1 hours (mammalian reticulocytes, in vitro).
+
                            30 hours (mammalian reticulocytes, in vitro).
−
                             2 min (yeast, in vivo).
+
                             >20 hours (yeast, in vivo).
−
                             2 min (Escherichia coli, in vivo).
+
                             >10 hours (Escherichia coli, in vivo).
  
  
 
Instability index:
 
Instability index:
  
−
The instability index (II) is computed to be 29.48
+
The instability index (II) is computed to be 29.73
 
This classifies the protein as stable.
 
This classifies the protein as stable.
  
  
  
−
Aliphatic index: 98.33
+
Aliphatic index: 96.16
  
−
Grand average of hydropathicity (GRAVY): -0.227
+
Grand average of hydropathicity (GRAVY): -0.250
  
 
===Source===
 
===Source===

Revision as of 09:10, 25 September 2013


ChlI1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 231
    Illegal BglII site found at 887
    Illegal BglII site found at 1082
    Illegal BamHI site found at 279
  • 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

ChlI1 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 restriction sites (EcoRI, XbaI, SpeI or PstI). Note This won’t work with the highlighted tag in the magnesium chelatase reaction.

Amino Acid Sequence

MAATEVKAAE GRTEKELGQA RPIFPFTAIV GQDEMKLALI LNVIDPKIGG VMIMGDRGTG KSTTIRALAD LLPEMQVVAN DPFNSDPTDP ELMSEEVRNR VKAGEQLPVS SKKIPMVDLP LGATEDRVCG TIDIEKALTE GVKAFEPGLL AKANRGILYV DEVNLLDDHL VDVLLDSAAS GWNTVEREGI SISHPARFIL VGSGNPEEGE LRPQLLDRFG MHAQIGTVKD PRLRVQIVSQ RSTFDENPAA FRKDYEAGQM ALTQRIVDAR KLLKQGEVNY DFRVKISQIC SDLNVDGIRG DIVTNRAAKA LAAFEGRTEV TPEDIYRVIP LCLRHRLRKD PLAEIDDGDR VREIFKQVFG ME

References and documentation are available. Please note the modified algorithm for extinction coefficient.

Number of amino acids: 362

Molecular weight: 39952.7

Theoretical pI: 5.11

Amino acid composition: Ala (A) 31 8.6% Arg (R) 28 7.7% Asn (N) 12 3.3% Asp (D) 29 8.0% Cys (C) 3 0.8% Gln (Q) 13 3.6% Glu (E) 29 8.0% Gly (G) 28 7.7% His (H) 4 1.1% Ile (I) 25 6.9% Leu (L) 34 9.4% Lys (K) 19 5.2% Met (M) 10 2.8% Phe (F) 12 3.3% Pro (P) 19 5.2% Ser (S) 14 3.9% Thr (T) 17 4.7% Trp (W) 1 0.3% Tyr (Y) 4 1.1% Val (V) 30 8.3% 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): 58 Total number of positively charged residues (Arg + Lys): 47

Atomic composition:

Carbon C 1752 Hydrogen H 2857 Nitrogen N 499 Oxygen O 539 Sulfur S 13

Formula: C1752H2857N499O539S13 Total number of atoms: 5660

Extinction coefficients:

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

Ext. coefficient 11585 Abs 0.1% (=1 g/l) 0.290, assuming all pairs of Cys residues form cystines


Ext. coefficient 11460 Abs 0.1% (=1 g/l) 0.287, 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 29.73 This classifies the protein as stable.


Aliphatic index: 96.16

Grand average of hydropathicity (GRAVY): -0.250

Source

Chlamydomonas reinhardtii

References