Difference between revisions of "Part:BBa K590063"

Revision as of 23:25, 28 September 2011

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 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 into an E. coli BL21 lacI^q 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.cell chain formation was observed in microscopy(below) when this plasmid was transformed in E.coli.

Sequence and Features