Part:BBa_K1976001
λ-Integrase
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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. |
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 |
Contents
Recombination Process
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 2:λ-integrase mediated attP/attB-recombination via Holliday junctions
The attP- and attB-strands are nicked by λ-integrases. The active-site amino-acid of the λ-integrase is its Tyrosine 342. The DNA-strands are cleaved via formation of a high energy 3′-phospho-tyrosine intermediate. After cleavage of the two top-strands a four-way Holliday-junction is formed (Figure 2).Here, two free λ-integrase dimers cut the remaining bottom-strands, also via formation of a 3′-phospho-tyrosine intermediate.
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
References
- 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 |
//plasmid/chromosomalintegration
| uniprot | P03700 |