Part:BBa_K1650007:Experience

The composite part contains a lac-inducible CDI operon which gives your strain the capability to inhibit growth of bacterial cells that express BamA in their outer membrane (most gram negative bacteria). Additionally the part contains mRFP1 (BBa_E1010) driven by another lac-inducible promoter as induction control. Moreover the composite part contains LacI. IPTG-induced cells harboring BBa_K1650007 express CdiB which forms a beta-barrel structure. The CdiA protein is transported through CdiB and is displayed on the cell surface. When getting in contact with BamA of a target cell, the C-terminal domain is delivered into the target cell acting as pore forming protein thus destroying the proton motoric force of the target cell. BBa_K1650007 could be used to improve transformation efficiency. Induced CDI+ cells would kill false positive cells.

Effect of CDI expression on the ratio of CDI+ cells to target cells. The x-axis shows the time in coculture of CDI+ and target cells and the y-axis shows the corresponding normalized cell count. Over time the ratio for induced CDI+ cells increases and decreases for uninduced CDI+ cells. The used method was flow cytometry.

Due to the extraordonary size of CdiA the metabolic burden even limited to the leaky epression significantly reduces the growth compared to a control strain if the CDI-operon is used on a high copy backbone such as pSB1C3. Therefore we submitted BBa_K1650007 in the low copy backbone plasmid pSB4C5 and recommend to use this part in this backbone or in backbones with similar amounts of copys per cell. Additionally we recommend to use E.coli strains that express additional LacI to reduce leaky expression as good as possbile.

Applications of BBa_K1650007

BBa_K1650007 could be used to improve transformation efficiency. Induced CDI+ cells would kill false positive cells or to avoid contamination by just inhibiting the growth of possible contaminants.

Up to now we used the part BBa_K1650007 in experiments having the goal to inhibit the growth of certain populations of target cells. This effect was veriefied using flow cytometry measurements as well as fluorescence microscopy.

User Reviews

We are looking forward to see in which way you are going to use our part. UNIQc937d93e13bdeb2a-partinfo-00000000-QINU UNIQc937d93e13bdeb2a-partinfo-00000001-QINU