Part:BBa_K257011

PBad + clyA_CTerm + RFP_NTerm

This part allows us to express the fusion protein clyA-RFP. In a mutant strain which constitutively produce vesicles, the fusion protein inserted in the outer membrane of the bacteria will be incorporated, during vesiculation, into the lumen of the vesicles. The promoter pBad is inductible with L-arabinose.

Safety Note

ClyA is a toxin. For more information see here: BBa_K257002

Experience

Pbad-clyA-RFP on arabinose medium

Pbad-clyA-RFP on LB medium (without arabinose)

Pfec-RFP on medium with citrate (without clyA, fluorescence isn't localize on the membrane exclusively)

On the first image, we observed fluorescence localized to the membrane when growing bacteria transformed with PBad-ClyA-RFP on a medium with arabinose. We can compare it to 2 others images from microcoscopy : with Pbad-clyA-RFP without arabinose, the fluorescent is less intense (pBad isn't repressed so there is little expression of clyA-RFP). The third image demonstrate that without clyA, RFP isn't localize exclusively in the membrane (here, it's an example with BBa_K257023)

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1205
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1302
Illegal BamHI site found at 1144
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 979
Illegal AgeI site found at 2712
Illegal AgeI site found at 2824
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961

References

J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. [http://www.ncbi.nlm.nih.gov/pubmed/18511069 18511069]
N.C.Kesty, M.J.Kuehn. Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles, 2004, J Biol Chem. 279(3):2069-76.[http://www.ncbi.nlm.nih.gov/pubmed/14578354 14578354]

[1] J.Y. Kim, A.M. Doody, D. J. Chen, G.H. Cremona, M.L. Shuler, D.Putnam,and M.P. DeLisa.Engineered. Bacterial Outer Membrane Vesicles with Enhanced Functionality, 2008, J. Mol. Biol. 380, 51–66. [http://www.ncbi.nlm.nih.gov/pubmed/18511069 18511069]

[2] N.C.Kesty, M.J.Kuehn. Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles, 2004, J Biol Chem. 279(3):2069-76.[http://www.ncbi.nlm.nih.gov/pubmed/14578354 14578354]