Difference between revisions of "Part:BBa K1111012"

Latest revision as of 18:19, 4 October 2013

Ice Nucleation Protein fused to Streptavidin Alive

Introduction

It is a fusion protein between an Ice Nucleation Protein (INP) and Streptavidin Alive made by Gibson Assembly. INP is an outer membrane protein found in the genome of Pseudomonas syringae, that is recognized by the E.coli protein secretion machinery, and thus also exported in that organism.

Figure 1: outline of the expected outcome of the plasmid

Usage and Biology

This part was designed to express streptavidin on the outer membrane of E.coli in order to attach biotinylated nanoparticles to the cells.
Thus, it can be used in any experiment involving biotin-streptavidin affinity (attachment of cargo is one example, but we could also think of purification etc...).

Datasheet

For further Characterization, refer to the datasheet below.

CharacterizationSheet Page1

CharacterizationSheet Page2

File:Team-EPF-Lausanne Sheet.pdf

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 1727
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1649
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 1727
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 1727
Illegal NgoMIV site found at 1036
Illegal AgeI site found at 1691
Illegal AgeI site found at 1742
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 ==Introduction==
-What it is : INP sequence fused with Streptavidin Alive by gibson assembly.
+It is a fusion protein between an Ice Nucleation Protein (INP) and Streptavidin Alive made by Gibson Assembly.
-The INP-streptavidin construct expresses streptavidin at the outer membrane of E.Coli. INP is an outer membrane protein foud in the genome of Pseudomonas syringae. It is under the control of Lac promoter and thus is constitutively expressed in E.Coli.
+INP is an outer membrane protein found in the genome of Pseudomonas syringae, that is recognized by the E.coli protein secretion machinery, and thus also exported in that organism.
-The expression of this plasmid was tested by transforming cells and looking for microscopy at either of a fluorescent antibody again streptavidin or a fluorescent biotin (biotin and streptavidin have a strong affinity, their Kd is 10-15 M).
+<!--The expression of this plasmid was tested by transforming cells and looking for microscopy at either of a fluorescent antibody again streptavidin or a fluorescent biotin (biotin and streptavidin have a strong affinity, their Kd is 10-15 M).-->
+[[File:EPF-Lausanne map.jpg||thumb|400px|center|Figure 1: outline of the expected outcome of the plasmid]]
 ==Usage and Biology==
-For us, the idea was to attach nanoparticles covered with biotin, using the strong affinity between both, but any experience implying biotin can be considered. Nanocapsules have a size comprised in a range from , which is quite big compared to a typical E:Coli cell size, we can therefore imagine attaching large proteins to these bacteria.
+This part was designed to express streptavidin on the outer membrane of E.coli in order to attach biotinylated nanoparticles to the cells.
+<br>Thus, it can be used in any experiment involving biotin-streptavidin affinity (attachment of cargo is one example, but we could also think of purification etc...).
-==Acknowledgements==
+==Datasheet==
-Thanks to the '''Mark Howarth Biochemistry department lab''' in Oxford for providing us the plasmids needed.
+For further Characterization, refer to the datasheet below.
+[[File:Team-EPF-Lausanne_Sheet1.jpg|thumb|270px|left|CharacterizationSheet Page1 ]]
+[[File: Team-EPF-Lausanne_Sheet2.jpg|thumb|270px|left|CharacterizationSheet Page2 ]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+<br><br>[[File: Team-EPF-Lausanne_Sheet.pdf|thumb|270px|left|PDF link ]]
-<span class='h3bb'>Sequence and Features</span>
+==<span class='h3bb'>Sequence and Features</span>==
 <partinfo>BBa_K1111012 SequenceAndFeatures</partinfo>