Composite
mamHIEJKL

Part:BBa_K590063

Designed by: Michael Brasino, Alicia Wong, Rashmi Ravichandran   Group: iGEM11_Washington   (2011-09-15)
Revision as of 07:40, 22 September 2011 by Saushun (Talk | contribs)

mamHIEJKL_pLacGFP_pGA3K3

This part is made by [http://2011.igem.org/Team:Washington UW iGEM Team 2011] using Gibson cloning method. It includes mamH, I, E, J, K, L all in pGA3k3_placGFP. This part is contributed to the [http://2011.igem.org/Team:Washington/Magnetosomes/Magnet_Toolkit Magnetosome toolkit], it also serves as an evidence that Gibson Cloning method is capable of combining multiple inserts.

Usage and Biology

We first extracted mamHI, mamE, mamJ, mamKL from the Magnetospirillum magneticum strain AMB-1, then we used Gibson cloning method to fuse the 4 groups of genes together to make a big HIEJKL insert. Followed by another gibson cloning reaction to combine the insert with the plasmid backbone pGA3k3_placGFP.

Below is the gel image of HIEJKL(~9000bp), we extracted this band and "gibsoned" it with pGA3k3_placGFP plasmid backbone.

1kb Ladder (left), mamHIEJKL (right)


After Gibson cloning and transformation, we induced the product with IPTG for the gene expression, cell chain formation was observed in microscopy(below) when this plasmid was transformed in E.coli.

HIEJKL 1b wiki.png


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 12370
    Illegal PstI site found at 5183
    Illegal PstI site found at 8860
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 12370
    Illegal NheI site found at 11025
    Illegal PstI site found at 5183
    Illegal PstI site found at 8860
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 12370
    Illegal BglII site found at 8564
    Illegal BglII site found at 12385
    Illegal BamHI site found at 9642
    Illegal XhoI site found at 89
    Illegal XhoI site found at 6383
    Illegal XhoI site found at 9657
    Illegal XhoI site found at 9818
    Illegal XhoI site found at 10661
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 12370
    Illegal PstI site found at 5183
    Illegal PstI site found at 8860
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 12370
    Illegal PstI site found at 5183
    Illegal PstI site found at 8860
    Illegal NgoMIV site found at 238
    Illegal NgoMIV site found at 1254
    Illegal NgoMIV site found at 2589
    Illegal NgoMIV site found at 3920
    Illegal NgoMIV site found at 6770
    Illegal NgoMIV site found at 8586
    Illegal NgoMIV site found at 8953
    Illegal AgeI site found at 5857
    Illegal AgeI site found at 6272
    Illegal AgeI site found at 11111
    Illegal AgeI site found at 11434
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 651
    Illegal BsaI site found at 1986
    Illegal BsaI site found at 3317
    Illegal BsaI.rc site found at 6963
    Illegal BsaI.rc site found at 12139
    Illegal SapI site found at 825
    Illegal SapI site found at 2160
    Illegal SapI site found at 3491
    Illegal SapI.rc site found at 9232
    Illegal SapI.rc site found at 9553


[edit]
Categories
Parameters
None