Difference between revisions of "Part:BBa K842013"

 
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When transcribed, RBS-fliC-GFP generates the flagellum protein bonded with a green fluorescent protein.  This structure would then be exported to the growing flagella apparatus and used in the construction of the filament.  When strongly expressed, this structure would directly result in the flagella filament containing GFP, allowing for the flagella to be detectable.  Unfortunately we were unable to promote the sequence enough to test whether this construct actually functions in this manner.
 
When transcribed, RBS-fliC-GFP generates the flagellum protein bonded with a green fluorescent protein.  This structure would then be exported to the growing flagella apparatus and used in the construction of the filament.  When strongly expressed, this structure would directly result in the flagella filament containing GFP, allowing for the flagella to be detectable.  Unfortunately we were unable to promote the sequence enough to test whether this construct actually functions in this manner.
  
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Cloning and Uses
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Cloning and Uses:
 
Both the fliC were cloned via PCR from the E. coli strain DH5&#945; and cloned into the vector pSB1C3.  Due to the fact that fliC has multiple S and P restriction enzyme sites located within its DNA sequence, it was generated with XbaI sites on the forward and reverse primer.  This is so as to allow for the placement of a gene promoter in the E-X sites upstream of the construct in an iGEM biobrick vector.  It is important to note though that the current structure consists of the following so as to avoid the accidental loss of a gene during the cloning procedure.
 
Both the fliC were cloned via PCR from the E. coli strain DH5&#945; and cloned into the vector pSB1C3.  Due to the fact that fliC has multiple S and P restriction enzyme sites located within its DNA sequence, it was generated with XbaI sites on the forward and reverse primer.  This is so as to allow for the placement of a gene promoter in the E-X sites upstream of the construct in an iGEM biobrick vector.  It is important to note though that the current structure consists of the following so as to avoid the accidental loss of a gene during the cloning procedure.
  

Revision as of 21:39, 3 October 2012

fliC-GFP

When transcribed, RBS-fliC-GFP generates the flagellum protein bonded with a green fluorescent protein. This structure would then be exported to the growing flagella apparatus and used in the construction of the filament. When strongly expressed, this structure would directly result in the flagella filament containing GFP, allowing for the flagella to be detectable. Unfortunately we were unable to promote the sequence enough to test whether this construct actually functions in this manner.

Cloning and Uses: Both the fliC were cloned via PCR from the E. coli strain DH5α and cloned into the vector pSB1C3. Due to the fact that fliC has multiple S and P restriction enzyme sites located within its DNA sequence, it was generated with XbaI sites on the forward and reverse primer. This is so as to allow for the placement of a gene promoter in the E-X sites upstream of the construct in an iGEM biobrick vector. It is important to note though that the current structure consists of the following so as to avoid the accidental loss of a gene during the cloning procedure.

Used for reconstitution of flagella apparatus in E. coli B strains.

EcoRI-XbaI-RBS-fliC-XbaI-GFP-SpeI-PstI

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 547
    Illegal PstI site found at 872
    Illegal PstI site found at 919
    Illegal PstI site found at 1486
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 547
    Illegal PstI site found at 872
    Illegal PstI site found at 919
    Illegal PstI site found at 1486
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1224
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 547
    Illegal PstI site found at 872
    Illegal PstI site found at 919
    Illegal PstI site found at 1486
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 547
    Illegal PstI site found at 872
    Illegal PstI site found at 919
    Illegal PstI site found at 1486
    Illegal AgeI site found at 301
    Illegal AgeI site found at 709
  • 1000
    COMPATIBLE WITH RFC[1000]