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

(→‎Design Notes)
(→‎Design Notes)
Line 11: Line 11:
  
 
ChlI1 Clone: DNA sequence from translation start site:
 
ChlI1 Clone: DNA sequence from translation start site:
−
Regions in BOLD are the sequence of the leader region in the pET100 plasmid.
 
 
Translated the DNA sequence into a protein sequence
 
Translated the DNA sequence into a protein sequence
 
using "Translate" at http://au.expasy.org/tools
 
using "Translate" at http://au.expasy.org/tools
Line 19: Line 18:
 
No Biobrick restriction sites (EcoRI, XbaI, SpeI or PstI). Note This won’t work with the highlighted tag in the magnesium chelatase reaction.
 
No Biobrick restriction sites (EcoRI, XbaI, SpeI or PstI). Note This won’t work with the highlighted tag in the magnesium chelatase reaction.
  
−
<b>ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT GGT GGA
+
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.
  
−
CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT CAT CCC TTC ACC</b>
+
--------------------------------------------------------------------------------
 +
Number of amino acids: 362
  
 +
Molecular weight: 39952.7
  
−
<FONT FACE="courier">GCTGCCGCTACTGAGGTCAAGGCTGCTGAGGGCCGCACTGAGAAGGAGCTGGGCCAGGCC
+
Theoretical pI: 5.11
  
−
CGCCCCATCTTCCCCTTCACCGCCATCGTGGGCCAGGATGAGATGAAGCTGGCGCTGATT
+
Amino acid composition: Ala (A)  31   8.6%
−
 
+
Arg (R)  28   7.7%
−
CTGAACGTGATCGACCCCAAGATCGGTGGTGTCATGATCATGGGCGACCGTGGCACTGGC
+
Asn (N)  12   3.3%
−
 
+
Asp (D)  29   8.0%
−
AAGTCCACCACCATTCGTGCCCTGGCGGATCTGCTGCCCGAGATGCAGGTGGTTGCCAAC
+
−
 
+
−
GACCCCTTTAACTCGGACCCCACCGACCCCGAGCTGATGAGCGAGGAGGTGCGCAACCGC
+
−
 
+
−
GTCAAGGCCGGCGAGCAGCTGCCCGTGTCTTCCAAGAAGATTCCCATGGTGGACCTGCCC
+
−
 
+
−
CTGGGCGCCACTGAGGACCGCGTGTGCGGCACCATCGACATCGAGAAGGCGCTGACCGAG
+
−
 
+
−
GGTGTCAAGGCGTTCGAGCCCGGCCTGCTGGCCAAGGCCAACCGCGGCATCCTGTACGTG
+
−
 
+
−
GATGAGGTCAACCTGCTGGACGACCACCTGGTCGATGTGCTGCTGGACTCGGCCGCCTCC
+
−
 
+
−
GGCTGGAACACCGTGGAGCGCGAGGGTATCTCCATCAGCCACCCCGCCCGCTTCATCCTG
+
−
 
+
−
GTCGGCTCGGGCAACCCCGAGGAGGGTGAGCTGCGCCCCCAGCTGCTGGATCGCTTCGGC
+
−
 
+
−
ATGCACGCCCAGATCGGCACCGTCAAGGACCCCCGCCTGCGTGTGCAGATCGTGTCGCAG
+
−
 
+
−
CGCTCGACCTTCGACGAGAACCCCGCCGCCTTCCGCAAGGACTACGAGGCCGGCCAGATG
+
−
 
+
−
GCGCTGACCCAGCGCATCGTGGACGCGCGCAAGCTGCTGAAGCAGGGCGAGGTCAACTAC
+
−
 
+
−
GACTTCCGCGTCAAGATCAGCCAGATCTGCTCGGACCTGAACGTGGACGGCATCCGCGGC
+
−
 
+
−
GACATCGTGACCAACCGCGCCGCCAAGGCCCTGGCCGCCTTCGAGGGCCGCACCGAGGTG
+
−
 
+
−
ACCCCCGAGGACATCTACCGTGTCATTCCCCTGTGCCTGCGCCACCGCCTCCGGAAAGAC
+
−
 
+
−
CCCCTGGCTGAGATCGACGACGGTGACCGCGTGCGTGAGATCTTCAAGCAGGTGTTCGGC
+
−
 
+
−
ATGGAGTAAGCGGCA<br>
+
−
</FONT>
+
−
 
+
−
<b>Amino Acid sequence: </b>
+
−
 
+
−
<FONT FACE="courier">MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFTAAATEVKAAEGRTEKELGQARPIF
+
−
PFTAIVGQDEMKLALILNVIDPKIGGVMIMGDRGTGKSTTIRALADLLPEMQVVANDPFN
+
−
SDPTDPELMSEEVRNRVKAGEQLPVSSKKIPMVDLPLGATEDRVCGTIDIEKALTEGVKA
+
−
FEPGLLAKANRGILYVDEVNLLDDHLVDVLLDSAASGWNTVEREGISISHPARFILVGSG
+
−
NPEEGELRPQLLDRFGMHAQIGTVKDPRLRVQIVSQRSTFDENPAAFRKDYEAGQMALTQ
+
−
RIVDARKLLKQGEVNYDFRVKISQICSDLNVDGIRGDIVTNRAAKALAAFEGRTEVTPED<br>
+
−
IYRVIPLCLRHRLRKDPLAEIDDGDRVREIFKQVFGME<br>
+
−
</FONT>
+
−
 
+
−
Number of amino acids: 398
+
−
 
+
−
Molecular weight: 44018.0
+
−
 
+
−
Theoretical pI: 5.35
+
−
 
+
−
Amino acid composition:
+
−
 
+
−
Ala (A)  33   8.3%
+
−
Arg (R)  30   7.5%
+
−
Asn (N)  12   3.0%
+
−
Asp (D)  35   8.8%
+
 
Cys (C)  3   0.8%
 
Cys (C)  3   0.8%
−
Gln (Q)  15   3.8%
+
Gln (Q)  13   3.6%
−
Glu (E)  29   7.3%
+
Glu (E)  29   8.0%
−
Gly (G)  33   8.3%
+
Gly (G)  28   7.7%
−
His (H) 11   2.8%
+
His (H)  4   1.1%
−
Ile (I)  25   6.3%
+
Ile (I)  25   6.9%
−
Leu (L)  35   8.8%
+
Leu (L)  34   9.4%
−
Lys (K)  20   5.0%
+
Lys (K)  19   5.2%
−
Met (M)  13   3.3%
+
Met (M)  10   2.8%
−
Phe (F)  13   3.3%
+
Phe (F)  12   3.3%
−
Pro (P)  20   5.0%
+
Pro (P)  19   5.2%
−
Ser (S)  16   4.0%
+
Ser (S)  14   3.9%
−
Thr (T)  19   4.8%
+
Thr (T)  17   4.7%
 
Trp (W)  1   0.3%
 
Trp (W)  1   0.3%
−
Tyr (Y)  5   1.3%
+
Tyr (Y)  4   1.1%
−
Val (V)  30   7.5%
+
Val (V)  30   8.3%
 +
Pyl (O)  0   0.0%
 +
Sec (U)  0   0.0%
  
−
Asx (B)  0   0.0%
+
(B)  0   0.0%
−
Glx (Z)  0   0.0%
+
(Z)  0   0.0%
−
Xaa (X)  0   0.0%
+
(X)  0   0.0%
  
−
Total number of negatively charged residues (Asp + Glu): 64
+
 
−
Total number of positively charged residues (Arg + Lys): 50
+
Total number of negatively charged residues (Asp + Glu): 58
 +
Total number of positively charged residues (Arg + Lys): 47
  
 
Atomic composition:
 
Atomic composition:
  
−
Carbon      C       1920
+
Carbon      C       1752
−
Hydrogen    H       3100
+
Hydrogen    H       2857
−
Nitrogen    N       558
+
Nitrogen    N       499
−
Oxygen      O       594
+
Oxygen      O       539
−
Sulfur      S         16
+
Sulfur      S         13
  
−
Formula: C1920H3100N558O594S16
+
Formula: C1752H2857N499O539S13
−
Total number of atoms: 6188
+
Total number of atoms: 5660
  
 
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    13075
+
−
Abs 0.1% (=1 g/l)  0.297, assuming ALL Cys residues appear as half cystines
+
−
 
+
  
 +
Ext. coefficient    11585
 +
Abs 0.1% (=1 g/l)  0.290, assuming all pairs of Cys residues form cystines
  
−
Ext. coefficient    12950
 
−
Abs 0.1% (=1 g/l)  0.294, assuming NO Cys residues appear as half cystines
 
  
 +
Ext. coefficient    11460
 +
Abs 0.1% (=1 g/l)  0.287, assuming all Cys residues are reduced
  
 
Estimated half-life:
 
Estimated half-life:
Line 146: Line 94:
 
Instability index:
 
Instability index:
  
−
The instability index (II) is computed to be 28.58
+
The instability index (II) is computed to be 29.73
 
This classifies the protein as stable.
 
This classifies the protein as stable.
  
  
  
−
Aliphatic index: 88.94
+
Aliphatic index: 96.16
  
−
Grand average of hydropathicity (GRAVY): -0.367
+
Grand average of hydropathicity (GRAVY): -0.250
  
 
===Source===
 
===Source===

Revision as of 01:15, 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.

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