Difference between revisions of "Part:BBa K1080011:Design"

(→‎Design Notes)
 
(4 intermediate revisions by the same user not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1080011 short</partinfo>
 
<partinfo>BBa_K1080011 short</partinfo>
Line 12: Line 11:
  
 
ChlI2 Clone: DNA sequence from translation start site:
 
ChlI2 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 18: Line 17:
 
"ProtParam" at http://au.expasy.org/tools
 
"ProtParam" at http://au.expasy.org/tools
  
−
Note: Three PstI sites at 368, 758, 920. No EcoRI, XbaI or SpeI sites. Tag needs to be removed to work.
+
<FONT FACE="courier">
  
−
 
+
MPSTKAAKKP NFPFVKIQGQ EEMKLALLLN VVDPNIGGVL IMGDRGTAKS VAVRALVDML<br> PDIDVVEGDA FNSSPTDPKF MGPDTLQRFR NGEKLPTVRM RTPLVELPLG ATEDRICGTI<br> DIEKALTQGI KAYEPGLLAK ANRGILYVDE VNLLDDGLVD VVLDSSASGL NTVEREGVSI<br> VHPARFIMIG SGNPQEGELR PQLLDRFGMS VNVATLQDTK QRTQLVLDRL AYEADPDAFV<br> DSCKAEQTAL TDKLEAARQR LRSVKISEEL QILISDICSR LDVDGLRGDI VINRAAKALV<br> AFEGRTEVTT NDVERVISGC LNHRLRKDPL DPIDNGTKVA ILFKRMTDPE IMKREEEAKK<br> </FONT>
−
<b>ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT GGT GGA
+
−
 
+
−
CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT CAT CCC TTC ACC
+
−
 
+
−
TTGCCATCCACGAAAGCCGCGAAG</b>
+
−
                                             
+
−
 
+
−
  121 AAGCCGAACTTCCCGTTCGTCAAGATTCAGGGCCAGGAGGAGATGAAGCTTGCACTGCTG
+
−
                                                                 
+
−
 
+
−
  181 CTGAACGTGGTCGACCCCAACATCGGCGGAGTGCTTATTATGGGTGACCGCGGCACTGCC
+
−
                                                                 
+
−
 
+
−
  241 AAGTCGGTCGCGGTCCGCGCCCTGGTGGATATGCTTCCCGACATTGACGTGGTTGAGGGC
+
−
     
+
−
 
+
−
  301 GACGCCTTCAACAGCTCCCCCACCGACCCCAAGTTCATGGGCCCCGACACCCTGCAGCGC
+
−
     
+
−
 
+
−
  361 TTCCGCAACGGCGAGAAGCTGCCCACCGTCCGCATGCGGACCCCCCTGGTGGAGCTGCCT
+
−
     
+
−
 
+
−
  421 CTGGGCGCCACCGAGGACCGCATCTGCGGCACCATCGACATCGAGAAGGCGCTGACGCAG
+
−
     
+
−
 
+
−
  481 GGCATCAAGGCCTACGAGCCCGGCCTGCTGGCCAAGGCCAACCGCGGCATCCTGTATGTG
+
−
     
+
−
 
+
−
  541 GACGAGGTGAACCTGCTGGATGATGGCCTGGTTGATGTCGTGCTGGACTCGTCGGCTAGC
+
−
     
+
−
 
+
−
  601 GGCCTGAACACTGTGGAGCGTGAGGGTGTGTCCATTGTGCACCCTGCCCGCTTCATCATG
+
−
     
+
−
 
+
−
  661 ATTGGCTCAGGCAACCCCCAGGAGGGTGAGCTGCGCCCGCAGCTGCTGGATCGCTTCGGC
+
−
     
+
−
 
+
−
  721 ATGAGCGTCAACGTGGCCACGCTGCAGGACACCAAGCAGCGCACGCAGCTGGTGCTGGAC
+
−
     
+
−
 
+
−
  781 CGGCTTGCGTACGAGGCGGACCCTGACGCATTTGTGGACTCGTGCAAGGCCGAGCAGACG
+
−
     
+
−
 
+
−
  841 GCGCTCACGGACAAGCTGGAGGCGGCCCGCCAGCGCCTGCGGTCCGTCAAGATCAGCGAG
+
−
     
+
−
 
+
−
  901 GAGCTGCAGATCCTGATCTCGGACATTTGCTCGCGCCTGGATGTGGATGGCCTGCGCGGT
+
−
     
+
−
 
+
−
  961 GACATTGTGATCAACCGCGCCGCCAAGGCGCTTGTGGCCTTCGAGGGCCGCACCGAGGTG
+
−
     
+
−
 
+
−
1021 ACCACGAATGACGTGGAGCGCGTCATCTCGGGCTGCCTCAACCACCGCCTGCGCAAGGAC
+
−
     
+
−
 
+
−
1081 CCGCTGGACCCCATTGACAACGGCACCAAGGTGGCCATCCTGTTCAAGCGCATGACCGAC
+
−
     
+
−
 
+
−
1141 CCCGAGATCATGAAGCGCGAGGAGGAGGCCAAGAAGAAGCGCGAGGAGGCGGCCGCCAAG
+
−
     
+
−
 
+
−
1201 GCCAAGGCGGAGGGCAAGGCGGACCGCCCCACGGGCGCCAAGGCTGGCGCCTGGGCTGGC
+
−
     
+
−
 
+
−
1261 TTGCCCCCTCGTCGGTAAAAAAGGCTGGTGTAGGCCTGTCGGGTCGTGTTAAAGGTTGCT
+
−
     
+
−
 
+
−
1321 GCGTGAACGTGTAAGTGTGACAGTGTGCCGGTATGTGTGTGTATACATGTGTTGCGGTGT
+
−
                                             
+
−
 
+
−
1381 GCTTTTGTGGCGGTACATGGTGATGACTGAGCGGGTGGGACAGAGCACGGTTAACTGACG
+
−
                                               
+
−
 
+
−
1441 AGG
+
−
 
+
−
        10        20        30        40        50        60
+
−
MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDHPFTLPST KAAKKPNFPF VKIQGQEEMK
+
−
 
+
−
        70        80        90        100        110        120
+
−
LALLLNVVDP NIGGVLIMGD RGTAKSVAVR ALVDMLPDID VVEGDAFNSS PTDPKFMGPD
+
−
 
+
−
      130        140        150        160        170        180
+
−
TLQRFRNGEK LPTVRMRTPL VELPLGATED RICGTIDIEK ALTQGIKAYE PGLLAKANRG
+
−
 
+
−
      190        200        210        220        230        240
+
−
ILYVDEVNLL DDGLVDVVLD SSASGLNTVE REGVSIVHPA RFIMIGSGNP QEGELRPQLL
+
−
 
+
−
      250        260        270        280        290        300
+
−
DRFGMSVNVA TLQDTKQRTQ LVLDRLAYEA DPDAFVDSCK AEQTALTDKL EAARQRLRSV
+
−
 
+
−
      310        320        330        340        350        360
+
−
KISEELQILI SDICSRLDVD GLRGDIVINR AAKALVAFEG RTEVTTNDVE RVISGCLNHR
+
−
 
+
−
      370        380        390        400        410        420
+
−
LRKDPLDPID NGTKVAILFK RMTDPEIMKR EEEAKKKREE AAAKAKAEGK ADRPTGAKAG
+
−
 
+
−
 
+
−
AWAGLPPRR
+
−
 
+
−
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: 393
  
−
Number of amino acids: 429
+
Molecular weight: 42954.3
  
−
Molecular weight: 47061.7
+
Theoretical pI: 5.78
  
−
Theoretical pI: 5.94
+
Amino acid composition: Ala (A)  38   9.7%
 +
Arg (R)  30   7.6%
 +
Asn (N)  14   3.6%
 +
Asp (D)  32   8.1%
 +
Cys (C)  4   1.0%
 +
Gln (Q)  12   3.1%
 +
Glu (E)  28   7.1%
 +
Gly (G)  29   7.4%
 +
His (H)  2   0.5%
 +
Ile (I)  22   5.6%
 +
Leu (L)  41 10.4%
 +
Lys (K)  27   6.9%
 +
Met (M)  10   2.5%
 +
Phe (F)  10   2.5%
 +
Pro (P)  22   5.6%
 +
Ser (S)  16   4.1%
 +
Thr (T)  21   5.3%
 +
Trp (W)  1   0.3%
 +
Tyr (Y)  3   0.8%
 +
Val (V)  31   7.9%
 +
Pyl (O)  0   0.0%
 +
Sec (U)  0   0.0%
  
−
Amino acid composition:
+
(B)  0   0.0%
 +
(Z)  0   0.0%
 +
(X)  0   0.0%
  
−
Ala (A)  39   9.1%
 
−
Arg (R)  32   7.5%
 
−
Asn (N)  14   3.3%
 
−
Asp (D)  38   8.9%
 
−
Cys (C)  4   0.9%
 
−
Gln (Q)  14   3.3%
 
−
Glu (E)  28   6.5%
 
−
Gly (G)  34   7.9%
 
−
His (H)  9   2.1%
 
−
Ile (I)  22   5.1%
 
−
Leu (L)  43 10.0%
 
−
Lys (K)  28   6.5%
 
−
Met (M)  13   3.0%
 
−
Phe (F)  11   2.6%
 
−
Pro (P)  23   5.4%
 
−
Ser (S)  18   4.2%
 
−
Thr (T)  23   5.4%
 
−
Trp (W)  1   0.2%
 
−
Tyr (Y)  4   0.9%
 
−
Val (V)  31   7.2%
 
  
−
Asx (B)  0   0.0%
+
Total number of negatively charged residues (Asp + Glu): 60
−
Glx (Z)  0   0.0%
+
Total number of positively charged residues (Arg + Lys): 57
−
Xaa (X)  0   0.0%
+
−
 
+
−
Total number of negatively charged residues (Asp + Glu): 66
+
−
Total number of positively charged residues (Arg + Lys): 60
+
  
 
Atomic composition:
 
Atomic composition:
  
−
Carbon      C       2046
+
Carbon      C       1875
−
Hydrogen    H       3352
+
Hydrogen    H       3103
−
Nitrogen    N       600
+
Nitrogen    N       541
−
Oxygen      O       635
+
Oxygen      O       580
−
Sulfur      S         17
+
Sulfur      S         14
  
−
Formula: C2046H3352N600O635S17
+
Formula: C1875H3103N541O580S14
−
Total number of atoms: 6650
+
Total number of atoms: 6113
  
 
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    11710
+
Ext. coefficient    10220
−
Abs 0.1% (=1 g/l)  0.249, assuming ALL Cys residues appear as half cystines
+
Abs 0.1% (=1 g/l)  0.238, assuming all pairs of Cys residues form cystines
  
  
−
 
+
Ext. coefficient     9970
−
Ext. coefficient   11460
+
Abs 0.1% (=1 g/l)  0.232, assuming all Cys residues are reduced
−
Abs 0.1% (=1 g/l)  0.244, assuming NO Cys residues appear as half cystines
+
−
 
+
  
 
Estimated half-life:
 
Estimated half-life:
Line 189: Line 87:
 
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: 30 hours (mammalian reticulocytes, in vitro).
+
The estimated half-life is:  
 +
                            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 196: Line 95:
 
Instability index:
 
Instability index:
  
−
The instability index (II) is computed to be 31.25
+
The instability index (II) is computed to be 32.87
 
This classifies the protein as stable.
 
This classifies the protein as stable.
  
  
  
−
Aliphatic index: 89.14
+
Aliphatic index: 95.06
−
 
+
−
Grand average of hydropathicity (GRAVY): -0.396
+
−
 
+
−
===Source===
+
−
 
+
−
Chlamydomonas reinhardtii
+
  
−
===References===
+
Grand average of hydropathicity (GRAVY): -0.296

Latest revision as of 02:03, 26 September 2013

ChlI2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 523
  • 21
    COMPATIBLE WITH RFC[21]
  • 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.


ChlI2 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

MPSTKAAKKP NFPFVKIQGQ EEMKLALLLN VVDPNIGGVL IMGDRGTAKS VAVRALVDML
PDIDVVEGDA FNSSPTDPKF MGPDTLQRFR NGEKLPTVRM RTPLVELPLG ATEDRICGTI
DIEKALTQGI KAYEPGLLAK ANRGILYVDE VNLLDDGLVD VVLDSSASGL NTVEREGVSI
VHPARFIMIG SGNPQEGELR PQLLDRFGMS VNVATLQDTK QRTQLVLDRL AYEADPDAFV
DSCKAEQTAL TDKLEAARQR LRSVKISEEL QILISDICSR LDVDGLRGDI VINRAAKALV
AFEGRTEVTT NDVERVISGC LNHRLRKDPL DPIDNGTKVA ILFKRMTDPE IMKREEEAKK

Please note the modified algorithm for extinction coefficient.

Number of amino acids: 393

Molecular weight: 42954.3

Theoretical pI: 5.78

Amino acid composition: Ala (A) 38 9.7% Arg (R) 30 7.6% Asn (N) 14 3.6% Asp (D) 32 8.1% Cys (C) 4 1.0% Gln (Q) 12 3.1% Glu (E) 28 7.1% Gly (G) 29 7.4% His (H) 2 0.5% Ile (I) 22 5.6% Leu (L) 41 10.4% Lys (K) 27 6.9% Met (M) 10 2.5% Phe (F) 10 2.5% Pro (P) 22 5.6% Ser (S) 16 4.1% Thr (T) 21 5.3% Trp (W) 1 0.3% Tyr (Y) 3 0.8% Val (V) 31 7.9% 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): 60 Total number of positively charged residues (Arg + Lys): 57

Atomic composition:

Carbon C 1875 Hydrogen H 3103 Nitrogen N 541 Oxygen O 580 Sulfur S 14

Formula: C1875H3103N541O580S14 Total number of atoms: 6113

Extinction coefficients:

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

Ext. coefficient 10220 Abs 0.1% (=1 g/l) 0.238, assuming all pairs of Cys residues form cystines


Ext. coefficient 9970 Abs 0.1% (=1 g/l) 0.232, 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 32.87 This classifies the protein as stable.


Aliphatic index: 95.06

Grand average of hydropathicity (GRAVY): -0.296