Composite
mamHIEJKL

Part:BBa_K590063

Designed by: Michael Brasino, Alicia Wong, Rashmi Ravichandran   Group: iGEM11_Washington   (2011-09-15)
Revision as of 23:34, 28 September 2011 by Robere (Talk | contribs) (→‎Usage and Biology)

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 vector. 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 to make large constructs (10 kb in this case).

Usage and Biology

We first extracted mamHI, mamE, mamJ, mamKL from the Magnetospirillum magneticum strain AMB-1, then we used Gibson assembly method to fuse the 4 groups of genes together to make a large HIEJKL insert. We then performed a two-fragment Gibson assembly reaction to combine the insert with a plasmid backbone pGA3k3 with an inducible promoter (R0011).

Below is a gel image of singly-cut mamHIEJKL(~9000bp) on pGA3K3.

1kb Ladder (left), mamHIEJKL (right)

After Gibson cloning and transformation into an E. coli BL21 lacIq strain, we induced gene expression with IPTG. Though these cells do not have any superfolder GFP-tagged mam genes (see sfGFP-mamK and sfGFP-mamI for reference), the cells appeared to be forming chains (see image below). We plan to continue characterizing this subsystem of the mamAB operon.

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


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