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

(→‎Design Notes)
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
  
−
Incorporated sequence overlap for Gibson assembly and no GC rich region or restriction site in sequence
+
Incorporated sequence overlap for Gibson assembly and no GC rich region or restriction site in sequence.
 +
 
 +
 
 +
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
 +
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
 +
 
 +
Note: Three PstI sites at 368, 758, 920. No EcoRI, XbaI or SpeI sites. Tag needs to be removed to work.
 +
 
 +
 
 +
<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.
 +
 
 +
 
 +
Number of amino acids: 429
 +
 
 +
Molecular weight: 47061.7
 +
 
 +
Theoretical pI: 5.94
 +
 
 +
Amino acid composition:
 +
 
 +
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%
 +
Glx (Z)  0   0.0%
 +
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:
 +
 
 +
Carbon      C       2046
 +
Hydrogen    H       3352
 +
Nitrogen    N       600
 +
Oxygen      O       635
 +
Sulfur      S         17
 +
 
 +
Formula: C2046H3352N600O635S17
 +
Total number of atoms: 6650
 +
 
 +
Extinction coefficients:
 +
 
 +
Extinction coefficients are in units of  M-1 cm-1, at 280 nm.
 +
 
 +
Ext. coefficient    11710
 +
Abs 0.1% (=1 g/l)  0.249, assuming ALL Cys residues appear as half cystines
 +
 
 +
 
 +
 
 +
Ext. coefficient    11460
 +
Abs 0.1% (=1 g/l)  0.244, assuming NO Cys residues appear as half cystines
 +
 
 +
 
 +
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 31.25
 +
This classifies the protein as stable.
 +
 
 +
 
 +
 
 +
Aliphatic index: 89.14
 +
 
 +
Grand average of hydropathicity (GRAVY): -0.396
  
 
===Source===
 
===Source===

Revision as of 04:31, 23 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: Regions in BOLD are the sequence of the leader region in the pET100 plasmid. 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

Note: Three PstI sites at 368, 758, 920. No EcoRI, XbaI or SpeI sites. Tag needs to be removed to work.


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 


 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.


Number of amino acids: 429

Molecular weight: 47061.7

Theoretical pI: 5.94

Amino acid composition:

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% Glx (Z) 0 0.0% 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:

Carbon C 2046 Hydrogen H 3352 Nitrogen N 600 Oxygen O 635 Sulfur S 17

Formula: C2046H3352N600O635S17 Total number of atoms: 6650

Extinction coefficients:

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

Ext. coefficient 11710 Abs 0.1% (=1 g/l) 0.249, assuming ALL Cys residues appear as half cystines


Ext. coefficient 11460 Abs 0.1% (=1 g/l) 0.244, assuming NO Cys residues appear as half cystines


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 31.25 This classifies the protein as stable.


Aliphatic index: 89.14

Grand average of hydropathicity (GRAVY): -0.396

Source

Chlamydomonas reinhardtii

References