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

(→‎Design Notes)
(→‎Design Notes)
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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
 +
 +
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
 +
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.
 +
 +
'''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'''
 +
 +
GCTGCCGCTACTGAG
 +
 +
GTCAAGGCTGCTGAGGGCCGCACTGAGAAGGAGCTGGGCCAGGCCCGCCCCATCTTCCCC
 +
 +
TTCACCGCCATCGTGGGCCAGGATGAGATGAAGCTGGCGCTGATTCTGAACGTGATCGAC
 +
 +
CCCAAGATCGGTGGTGTCATGATCATGGGCGACCGTGGCACTGGCAAGTCCACCACCATT
 +
 +
CGTGCCCTGGCGGATCTGCTGCCCGAGATGCAGGTGGTTGCCAACGACCCCTTTAACTCG
 +
 +
GACCCCACCGACCCCGAGCTGATGAGCGAGGAGGTGCGCAACCGCGTCAAGGCCGGCGAG
 +
 +
CAGCTGCCCGTGTCTTCCAAGAAGATTCCCATGGTGGACCTGCCCCTGGGCGCCACTGAG
 +
 +
GACCGCGTGTGCGGCACCATCGACATCGAGAAGGCGCTGACCGAGGGTGTCAAGGCGTTC
 +
 +
GAGCCCGGCCTGCTGGCCAAGGCCAACCGCGGCATCCTGTACGTGGATGAGGTCAACCTG
 +
 +
CTGGACGACCACCTGGTCGATGTGCTGCTGGACTCGGCCGCCTCCGGCTGGAACACCGTG
 +
 +
GAGCGCGAGGGTATCTCCATCAGCCACCCCGCCCGCTTCATCCTGGTCGGCTCGGGCAAC
 +
 +
CCCGAGGAGGGTGAGCTGCGCCCCCAGCTGCTGGATCGCTTCGGCATGCACGCCCAGATC
 +
 +
GGCACCGTCAAGGACCCCCGCCTGCGTGTGCAGATCGTGTCGCAGCGCTCGACCTTCGAC
 +
 +
GAGAACCCCGCCGCCTTCCGCAAGGACTACGAGGCCGGCCAGATGGCGCTGACCCAGCGC
 +
 +
ATCGTGGACGCGCGCAAGCTGCTGAAGCAGGGCGAGGTCAACTACGACTTCCGCGTCAAG
 +
 +
ATCAGCCAGATCTGCTCGGACCTGAACGTGGACGGCATCCGCGGCGACATCGTGACCAAC
 +
 +
CGCGCCGCCAAGGCCCTGGCCGCCTTCGAGGGCCGCACCGAGGTGACCCCCGAGGACATC
 +
 +
TACCGTGTCATTCCCCTGTGCCTGCGCCACCGCCTCCGGAAAGACCCCCTGGCTGAGATC
 +
 +
GACGACGGTGACCGCGTGCGTGAGATCTTCAAGCAGGTGTTCGGCATGGAGTAAGCGGCA
 +
 +
'''Amino Acid sequence:'''
 +
 +
MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFTAAATEVKAAEGRTEKELGQARPIF
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PFTAIVGQDEMKLALILNVIDPKIGGVMIMGDRGTGKSTTIRALADLLPEMQVVANDPFN
 +
SDPTDPELMSEEVRNRVKAGEQLPVSSKKIPMVDLPLGATEDRVCGTIDIEKALTEGVKA
 +
FEPGLLAKANRGILYVDEVNLLDDHLVDVLLDSAASGWNTVEREGISISHPARFILVGSG
 +
NPEEGELRPQLLDRFGMHAQIGTVKDPRLRVQIVSQRSTFDENPAAFRKDYEAGQMALTQ
 +
RIVDARKLLKQGEVNYDFRVKISQICSDLNVDGIRGDIVTNRAAKALAAFEGRTEVTPED
 +
IYRVIPLCLRHRLRKDPLAEIDDGDRVREIFKQVFGME
 +
 +
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%
 +
Gln (Q)  15   3.8%
 +
Glu (E)  29   7.3%
 +
Gly (G)  33   8.3%
 +
His (H)  11   2.8%
 +
Ile (I)  25   6.3%
 +
Leu (L)  35   8.8%
 +
Lys (K)  20   5.0%
 +
Met (M)  13   3.3%
 +
Phe (F)  13   3.3%
 +
Pro (P)  20   5.0%
 +
Ser (S)  16   4.0%
 +
Thr (T)  19   4.8%
 +
Trp (W)  1   0.3%
 +
Tyr (Y)  5   1.3%
 +
Val (V)  30   7.5%
 +
 +
Asx (B)  0   0.0%
 +
Glx (Z)  0   0.0%
 +
Xaa (X)  0   0.0%
 +
 +
Total number of negatively charged residues (Asp + Glu): 64
 +
Total number of positively charged residues (Arg + Lys): 50
 +
 +
Atomic composition:
 +
 +
Carbon      C       1920
 +
Hydrogen    H       3100
 +
Nitrogen    N       558
 +
Oxygen      O       594
 +
Sulfur      S         16
 +
 +
Formula: C1920H3100N558O594S16
 +
Total number of atoms: 6188
 +
 +
Extinction coefficients:
 +
 +
Extinction coefficients are in units of  M-1 cm-1, at 280 nm.
 +
 +
Ext. coefficient    13075
 +
Abs 0.1% (=1 g/l)  0.297, assuming ALL Cys residues appear as half cystines
 +
 +
 +
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Ext. coefficient    12950
 +
Abs 0.1% (=1 g/l)  0.294, 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 28.58
 +
This classifies the protein as stable.
 +
 +
 +
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Aliphatic index: 88.94
 +
 +
Grand average of hydropathicity (GRAVY): -0.367
  
 
===Source===
 
===Source===

Revision as of 01:47, 24 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: 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

No Biobrick restriction sites (EcoRI, XbaI, SpeI or PstI). Note This won’t work with the highlighted tag in the magnesium chelatase reaction.

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

GCTGCCGCTACTGAG

GTCAAGGCTGCTGAGGGCCGCACTGAGAAGGAGCTGGGCCAGGCCCGCCCCATCTTCCCC

TTCACCGCCATCGTGGGCCAGGATGAGATGAAGCTGGCGCTGATTCTGAACGTGATCGAC

CCCAAGATCGGTGGTGTCATGATCATGGGCGACCGTGGCACTGGCAAGTCCACCACCATT

CGTGCCCTGGCGGATCTGCTGCCCGAGATGCAGGTGGTTGCCAACGACCCCTTTAACTCG

GACCCCACCGACCCCGAGCTGATGAGCGAGGAGGTGCGCAACCGCGTCAAGGCCGGCGAG

CAGCTGCCCGTGTCTTCCAAGAAGATTCCCATGGTGGACCTGCCCCTGGGCGCCACTGAG

GACCGCGTGTGCGGCACCATCGACATCGAGAAGGCGCTGACCGAGGGTGTCAAGGCGTTC

GAGCCCGGCCTGCTGGCCAAGGCCAACCGCGGCATCCTGTACGTGGATGAGGTCAACCTG

CTGGACGACCACCTGGTCGATGTGCTGCTGGACTCGGCCGCCTCCGGCTGGAACACCGTG

GAGCGCGAGGGTATCTCCATCAGCCACCCCGCCCGCTTCATCCTGGTCGGCTCGGGCAAC

CCCGAGGAGGGTGAGCTGCGCCCCCAGCTGCTGGATCGCTTCGGCATGCACGCCCAGATC

GGCACCGTCAAGGACCCCCGCCTGCGTGTGCAGATCGTGTCGCAGCGCTCGACCTTCGAC

GAGAACCCCGCCGCCTTCCGCAAGGACTACGAGGCCGGCCAGATGGCGCTGACCCAGCGC

ATCGTGGACGCGCGCAAGCTGCTGAAGCAGGGCGAGGTCAACTACGACTTCCGCGTCAAG

ATCAGCCAGATCTGCTCGGACCTGAACGTGGACGGCATCCGCGGCGACATCGTGACCAAC

CGCGCCGCCAAGGCCCTGGCCGCCTTCGAGGGCCGCACCGAGGTGACCCCCGAGGACATC

TACCGTGTCATTCCCCTGTGCCTGCGCCACCGCCTCCGGAAAGACCCCCTGGCTGAGATC

GACGACGGTGACCGCGTGCGTGAGATCTTCAAGCAGGTGTTCGGCATGGAGTAAGCGGCA

Amino Acid sequence:

MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFTAAATEVKAAEGRTEKELGQARPIF PFTAIVGQDEMKLALILNVIDPKIGGVMIMGDRGTGKSTTIRALADLLPEMQVVANDPFN SDPTDPELMSEEVRNRVKAGEQLPVSSKKIPMVDLPLGATEDRVCGTIDIEKALTEGVKA FEPGLLAKANRGILYVDEVNLLDDHLVDVLLDSAASGWNTVEREGISISHPARFILVGSG NPEEGELRPQLLDRFGMHAQIGTVKDPRLRVQIVSQRSTFDENPAAFRKDYEAGQMALTQ RIVDARKLLKQGEVNYDFRVKISQICSDLNVDGIRGDIVTNRAAKALAAFEGRTEVTPED IYRVIPLCLRHRLRKDPLAEIDDGDRVREIFKQVFGME

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% Gln (Q) 15 3.8% Glu (E) 29 7.3% Gly (G) 33 8.3% His (H) 11 2.8% Ile (I) 25 6.3% Leu (L) 35 8.8% Lys (K) 20 5.0% Met (M) 13 3.3% Phe (F) 13 3.3% Pro (P) 20 5.0% Ser (S) 16 4.0% Thr (T) 19 4.8% Trp (W) 1 0.3% Tyr (Y) 5 1.3% Val (V) 30 7.5%

Asx (B) 0 0.0% Glx (Z) 0 0.0% Xaa (X) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 64 Total number of positively charged residues (Arg + Lys): 50

Atomic composition:

Carbon C 1920 Hydrogen H 3100 Nitrogen N 558 Oxygen O 594 Sulfur S 16

Formula: C1920H3100N558O594S16 Total number of atoms: 6188

Extinction coefficients:

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

Ext. coefficient 13075 Abs 0.1% (=1 g/l) 0.297, assuming ALL Cys residues appear as half cystines


Ext. coefficient 12950 Abs 0.1% (=1 g/l) 0.294, 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 28.58 This classifies the protein as stable.


Aliphatic index: 88.94

Grand average of hydropathicity (GRAVY): -0.367

Source

Chlamydomonas reinhardtii

References