Difference between revisions of "Part:BBa K1976001"
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Revision as of 00:14, 27 October 2016
λ-Integrase
Contents
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The λ‑integrase, originally derived from the λ‑phage, catalyzes the recombination of the phage genome with the chromosomal genome of its host in combination with several assisting proteins. Therefore, two attachment sites are necessary: one located on the bacterial genome (attB) and the other located on the λ‑genome (attP), which also contains several binding sites for regulatory proteins. The attachment sites contain homologous recognition sequences, called BOB' region (attB) and COC' region (attP). These regions can be connected by the λ‑integrase and the bacterial integration host factor (IHF) via Holliday junction, forming an intasome, a DNA‑protein‑complex, producing hybrid attachment sites attL and attR. |
Figure 1: Crystal structure of the λ-Integrase in interaction with DNA. Created with VMD (Visual Molecular Dynamics).PDB entry, Biswas, T. et al. 2005 |
Recombination Process
Figure 2: The mechanism of λ-integrase mediated attP/attB-recombination
Figure 3:
Usage and Biology
The λ-Integrase can be used for specific genomic integration of a variable gene of interest (GOI) via attP/attB recombination. For Integration of a certain GOI it has to be cloned into a vector carrying the λ-attP-site. The cloned plasmid together with the presented Part must be transformed into a λ-negative E. Coli-Strain with attB-site. The presented λ-Integrase is specific for the λ-attP-site used in BBa_K1976000.
Characterisation
Figure 4: SDS-Page after expression of λ-integrase gene. The gel band of λ-integrase is highlighted by the red circle.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| uniprot | P03700 |