Difference between revisions of "Part:BBa K257011"
(→Experience) |
David.bikard (Talk | contribs) |
||
| (5 intermediate revisions by 2 users not shown) | |||
| Line 4: | Line 4: | ||
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. | 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: <partinfo>BBa_K257002</partinfo> | ||
===Experience=== | ===Experience=== | ||
| + | Pbad-clyA-RFP on arabinose medium | ||
[[Image:TolA clyA Ara 01.jpg]] | [[Image:TolA clyA Ara 01.jpg]] | ||
| − | + | ||
| + | Pbad-clyA-RFP on LB medium (without arabinose) | ||
| + | |||
| + | [[Image:TolA clyA LB 01.jpg]] | ||
| + | |||
| + | Pfec-RFP on medium with citrate (without clyA, fluorescence isn't localize on the membrane exclusively) | ||
| + | |||
| + | |||
| + | [[Image:Citrate30.jpg]] | ||
| + | |||
| + | 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 <partinfo>Bba_K257023</partinfo>) | ||
<!-- --> | <!-- --> | ||
| Line 20: | Line 33: | ||
<partinfo>BBa_K257011 parameters</partinfo> | <partinfo>BBa_K257011 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
| + | |||
| + | ===References=== | ||
| + | <ol class="references"> | ||
| + | <li> 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]</li> | ||
| + | <li> 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]</li> | ||
| + | </ol> | ||
Latest revision as of 07:20, 22 October 2009
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1302
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 2712
Illegal AgeI site found at 2824 - 1000INCOMPATIBLE 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]