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control construct - invasin + GFP under J23102
Invasin (INV) plays a key role in the initiation of Yersinia enterocolytica and Yersinia pseudotuberculosis infection. Through interaction with a beta1-integrin receptor present on the surface of eucaryotic cell membranes it triggers a signal-transduction pathway leading to internalisation of the whole bacterium in the endocytosis-dependent manner. The strong affinity of Invasin to it’s receptor results in highly selective binding to the target molecule. Mammalian cells depleted of beta1-integrin cannot be infected.
Green Fluorescent Protein is a noninvasive fluorescent marker for gene expression, protein localisation and intracellulat protein targeting. Originally from Aequorea victoria.
Left: HeLa cells infected with E. coli Top10 harbouring J23102+INV+GFP+TT construct. Thanks to FRET between Hirsch dye and GFP infected cells can be seen as a mildly-glowing areas containing bright spots of GFP inside. More photos at Gallery of Microphotographs.
Authors:Cloned by Joanna Leszczyńska.
Created with the use of part BBa K299810 cloned by Marta Błaszkiewicz.
Work supervised by Michał Lower at all levels.
Construct designThe part consists of J23102 promoter and B0032 RBS ligated to inv gene from Yersinia pestis (horizontal gene transfer form Yersinia pseudotuberculosis). GFP as marker. Double terminator (B0010+B0012) guarantees the stability of polycistronic RNA as a product of transcription. The construct is a control device used in testing of the Invasiveness Operon (BBa_K299813 and BBa_K299815) To find out more about it's background and design click here.
SafetyBacteria transformed with this part are invasive microorganisms. All safety precautions must be taken when manipulating with transformed strain. It involves obligatory use of laboratory gloves. Work under laminar is strongly advised. All waste should be autoclaved to avoid accidental gene transfer to other bacteria and potential rise of pathogenic organism.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21Illegal BglII site found at 813
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 1430
- 1000Illegal BsaI.rc site found at 3245
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