Difference between revisions of "Part:BBa K1080004:Design"

(→‎Design Notes)
(→‎Design Notes)
Line 9: Line 9:
  
 
ChlM Clone: DNA sequence from translation start site:
 
ChlM 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 15: Line 15:
 
"ProtParam" at http://au.expasy.org/tools
 
"ProtParam" at http://au.expasy.org/tools
  
−
No Biobrick sites: PstI, XbaI, SpeI or EcoRI
+
No Biobrick restriction sites: PstI, XbaI, SpeI or EcoRI
  
−
<b>ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT GGT GGA
+
<FONT FACE="courier">VGAVVGLGAL LVATDHQKRR SEQMKSFDGD EKEAVKDYFN TAGFERWRKI YGETDEVNKV QLDIRTGHAQ TVDKVLRWVD EEGSVQGITV ADCGCGTGSL<br> AIQLALRGAA VSASDISAAM ASEAEQRYQQ AVAAGQGKAP KVAPKFEALD LESVKGKYDT VTCLDVMIHY PQDKVDAMIT HLAGLSDRRL IISFAPKTLS<br> YSILKRIGEL FPGPSKATRA YLHREEDVEA ALKRAGFKVT KREMTATSFY FSRLLEAIRE 
 +
References and documentation are available. </FONT>
  
−
CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT CAT CCC TTC ACC</b>
+
Please note the modified algorithm for extinction coefficient.
  
 +
--------------------------------------------------------------------------------
 +
Number of amino acids: 260
  
−
<FONT FACE="courier">ATGGCTTCTGAGATTGCTCAAACTGCCGATGTTGGCTCGCTGACCTTCGCGGTTGGCGGA
+
Molecular weight: 28533.5
  
−
GTGGGCGCTGTTGTGGGCCTGGGCGCCCTGCTTGTCGCTACTGACCCTCAGAAGCGCCGC
+
Theoretical pI: 7.12
  
−
TCGGAGCAGATGAAGTCATTCGACGGCGATGAGAAGGAGGCGGTGAAGGACTACTTCAAC
+
Amino acid composition: Ala (A)  32 12.3%
 +
Arg (R)  17   6.5%
 +
Asn (N)  2   0.8%
 +
Asp (D)  17   6.5%
 +
Cys (C)  3   1.2%
 +
Gln (Q)  11   4.2%
 +
Glu (E)  19   7.3%
 +
Gly (G)  20   7.7%
 +
His (H)  5   1.9%
 +
Ile (I)  12   4.6%
 +
Leu (L)  21   8.1%
 +
Lys (K)  19   7.3%
 +
Met (M)  5   1.9%
 +
Phe (F)  9   3.5%
 +
Pro (P)  6   2.3%
 +
Ser (S)  16   6.2%
 +
Thr (T)  15   5.8%
 +
Trp (W)  2   0.8%
 +
Tyr (Y)  8   3.1%
 +
Val (V)  21   8.1%
 +
Pyl (O)  0   0.0%
 +
Sec (U)  0   0.0%
  
−
ACTGCCGGCTTCGAGCGCTGGCGCAAGATCTACGGCGAGACGGACGAGGTGAACAAGGTG
+
(B)  0   0.0%
 +
(Z)  0   0.0%
 +
(X)  0   0.0%
  
−
CAGCTGGACATCCGCACTGGCCACGCGCAGACGGTGGACAAGGTGCTGCGCTGGGTGGAC
 
  
−
GAGGAGGGCAGCGTGCAGGGCATCACCGTGGCGGACTGCGGCTGCGGGACAGGCAGCCTG
+
Total number of negatively charged residues (Asp + Glu): 36
−
 
+
Total number of positively charged residues (Arg + Lys): 36
−
GCCATCCCGCTGGCGCTGCGCGGCGCCGCCGTCAGCGCCTCCGACATCTCCGCCGCCATG
+
−
 
+
−
GCGTCTGAGGCGGAGCAGCGCTACCAGCAGGCGGTGGCGGCCGGGCAGGGCAAGGCGCCC
+
−
 
+
−
AAGGTGGCGCCCAAGTTCGAGGCGCTGGACCTGGAGTCCGTCAAGGGCAAGTACGACACG
+
−
 
+
−
GTCACTTGCCTGGACGTGATGATTCACTACCCCCAGGACAAGGTGGACGCCATGATCACG
+
−
 
+
−
CACCTGGCGGGTCTGTCCGACCGCCGCCTCATCATCTCGTTCGCACCCAAGACGCTGTCG
+
−
 
+
−
TACAGCATCCTCAAGCGCATTGGCGAGCTGTTCCCCGGCCCCAGCAAGGCCACCCGCGCC
+
−
 
+
−
TACCTGCACCGTGAGGAGGACGTGGAGGCTGCCCTCAAGCGCGCCGGCTTCAAGGTCACC
+
−
 
+
−
AAGCGTGAGATGACCGCCACCAGCTTCTACTTCTCGCGCCTGCTGGAGGCCATCCGCGAG
+
−
 
+
−
TAATCTCGCAAACGTTCTCGTGTAAATAGCGTAGCAGTGCAGGACTGGTGTAAGGACTGA
+
−
 
+
−
TGGACTCGGACCGGCGCTGCGGCCGGGTATGAGGGCATGGGCGTGGGCGCTGCTAGGGTG
+
−
 
+
−
CTACGGTGCAGCGCGGTTTTGACGGGCTATAGACATTTATGTGACTGAGTGACGGGCGGA
+
−
 
+
−
TAGCCAAAACGATTGTGTGAACG</FONT>
+
−
 
+
−
<b>Amino acid sequence</b>
+
−
 
+
−
<FONT FACE="courier">MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDHPFTMASE IAQTADVGSL TFAVGGVGAV
+
−
 
+
−
VGLGALLVAT DPQKRRSEQM KSFDGDEKEA VKDYFNTAGF ERWRKIYGET DEVNKVQLDI
+
−
 
+
−
RTGHAQTVDK VLRWVDEEGS VQGITVADCG CGTGSLAIPL ALRGAAVSAS DISAAMASEA
+
−
 
+
−
EQRYQQAVAA GQGKAPKVAP KFEALDLESV KGKYDTVTCL DVMIHYPQDK VDAMITHLAG
+
−
 
+
−
LSDRRLIISF APKTLSYSIL KRIGELFPGP SKATRAYLHR EEDVEAALKR AGFKVTKREM
+
−
 
+
−
TATSFYFSRL LEAIRE</FONT>
+
−
 
+
−
Number of amino acids: 316
+
−
 
+
−
Molecular weight: 34495.0
+
−
 
+
−
Theoretical pI: 6.14
+
−
 
+
−
Amino acid composition:
+
−
 
+
−
Ala (A)  37 11.7%
+
−
Arg (R)  19   6.0%
+
−
Asn (N)  2   0.6%
+
−
Asp (D)  24   7.6%
+
−
Cys (C)  3   0.9%
+
−
Gln (Q)  13   4.1%
+
−
Glu (E)  20   6.3%
+
−
Gly (G)  28   8.9%
+
−
His (H)  11   3.5%
+
−
Ile (I)  13   4.1%
+
−
Leu (L)  23   7.3%
+
−
Lys (K)  20   6.3%
+
−
Met (M)  10   3.2%
+
−
Phe (F)  11   3.5%
+
−
Pro (P)  9   2.8%
+
−
Ser (S)  20   6.3%
+
−
Thr (T)  19   6.0%
+
−
Trp (W)  2   0.6%
+
−
Tyr (Y)  9   2.8%
+
−
Val (V)  23   7.3%
+
−
 
+
−
Asx (B)  0   0.0%
+
−
Glx (Z)  0   0.0%
+
−
Xaa (X)  0   0.0%
+
−
 
+
−
Total number of negatively charged residues (Asp + Glu): 44
+
−
Total number of positively charged residues (Arg + Lys): 39
+
  
 
Atomic composition:
 
Atomic composition:
  
−
Carbon      C       1509
+
Carbon      C       1258
−
Hydrogen    H       2396
+
Hydrogen    H       2019
−
Nitrogen    N       432
+
Nitrogen    N       355
−
Oxygen      O       468
+
Oxygen      O       385
−
Sulfur      S         13
+
Sulfur      S         8
  
−
Formula: C1509H2396N432O468S13
+
Formula: C1258H2019N355O385S8
−
Total number of atoms: 4818
+
Total number of atoms: 4025
  
 
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    24535
+
−
Abs 0.1% (=1 g/l)  0.711, assuming ALL Cys residues appear as half cystines
+
−
 
+
  
 +
Ext. coefficient    23045
 +
Abs 0.1% (=1 g/l)  0.808, assuming all pairs of Cys residues form cystines
  
−
Ext. coefficient    24410
 
−
Abs 0.1% (=1 g/l)  0.708, assuming NO Cys residues appear as half cystines
 
  
 +
Ext. coefficient    22920
 +
Abs 0.1% (=1 g/l)  0.803, assuming all Cys residues are reduced
  
 
Estimated half-life:
 
Estimated half-life:
  
−
The N-terminal of the sequence considered is M (Met).
+
The N-terminal of the sequence considered is V (Val).
  
 
The estimated half-life is:  
 
The estimated half-life is:  
−
                             30 hours (mammalian reticulocytes, in vitro).
+
                             100 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 144: Line 94:
 
Instability index:
 
Instability index:
  
−
The instability index (II) is computed to be 33.76
+
The instability index (II) is computed to be 34.87
 
This classifies the protein as stable.
 
This classifies the protein as stable.
  
  
  
−
Aliphatic index: 77.25
+
Aliphatic index: 85.23
  
−
Grand average of hydropathicity (GRAVY): -0.339
+
Grand average of hydropathicity (GRAVY): -0.267
  
 
===Source===
 
===Source===

Revision as of 02:48, 25 September 2013


ChlM


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 230
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

ChlM 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: PstI, XbaI, SpeI or EcoRI

VGAVVGLGAL LVATDHQKRR SEQMKSFDGD EKEAVKDYFN TAGFERWRKI YGETDEVNKV QLDIRTGHAQ TVDKVLRWVD EEGSVQGITV ADCGCGTGSL
AIQLALRGAA VSASDISAAM ASEAEQRYQQ AVAAGQGKAP KVAPKFEALD LESVKGKYDT VTCLDVMIHY PQDKVDAMIT HLAGLSDRRL IISFAPKTLS
YSILKRIGEL FPGPSKATRA YLHREEDVEA ALKRAGFKVT KREMTATSFY FSRLLEAIRE References and documentation are available.

Please note the modified algorithm for extinction coefficient.

Number of amino acids: 260

Molecular weight: 28533.5

Theoretical pI: 7.12

Amino acid composition: Ala (A) 32 12.3% Arg (R) 17 6.5% Asn (N) 2 0.8% Asp (D) 17 6.5% Cys (C) 3 1.2% Gln (Q) 11 4.2% Glu (E) 19 7.3% Gly (G) 20 7.7% His (H) 5 1.9% Ile (I) 12 4.6% Leu (L) 21 8.1% Lys (K) 19 7.3% Met (M) 5 1.9% Phe (F) 9 3.5% Pro (P) 6 2.3% Ser (S) 16 6.2% Thr (T) 15 5.8% Trp (W) 2 0.8% Tyr (Y) 8 3.1% Val (V) 21 8.1% 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): 36 Total number of positively charged residues (Arg + Lys): 36

Atomic composition:

Carbon C 1258 Hydrogen H 2019 Nitrogen N 355 Oxygen O 385 Sulfur S 8

Formula: C1258H2019N355O385S8 Total number of atoms: 4025

Extinction coefficients:

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

Ext. coefficient 23045 Abs 0.1% (=1 g/l) 0.808, assuming all pairs of Cys residues form cystines


Ext. coefficient 22920 Abs 0.1% (=1 g/l) 0.803, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is V (Val).

The estimated half-life is: 

                            100 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 34.87 This classifies the protein as stable.


Aliphatic index: 85.23

Grand average of hydropathicity (GRAVY): -0.267

Source

Chlamydomonas reinhardtii

References