Difference between revisions of "Part:BBa J31001:Design"

Revision as of 18:03, 29 October 2006

DNA invertase Hin tagged with LVA

Hin Invertase

To the left is a 3-D model of the a Hin/ DNA complex crystal structure (Figure 1, Protein Data Bank ID 1ZR4, Li et al. 2005). A Hin protein dimer binds each HixC sequence flanking the fragment of DNA to be inverted. The two dimers (dimer 1 = leftward green and blue protein structures; dimer 2 = rightward yellow and purple protein structures) come together to form a tetrad complex where cleaved DNA ends are swapped and ligated (Richards and Johnson 2004).
Figure 1. 3-D models of Hin/ DNA complexes based upon crystal structure data (Li et al. 2005, Kamtekar et al. 2006)
3-D structure of a Hin protein dimer bound to cleaved DNA (PDB 2GM4, Kamtekar et al. 2006). View the [http://www.rcsb.org/pdb/explore/explore.do?structureId=1ZR4 interactive 3-D Jmol image].	Figure 1. 3-D structure of a Hin terad complex poised to swap DNA ends for inversion and ligation (PDB 1ZR4, Li et al. 2005). View the [http://www.rcsb.org/pdb/explore/explore.do?structureId=1ZR4 interactive 3-D Jmol image].

Design Notes

The Biobricks on this part are not wildtype but the cut sites are still viable.

Data

Inversion of HixC-flanked DNA in the presence of HinLVA HinLVA has been assembled with a pLac promoter and RBS (see BBa_S03536) to create a HinLVA expression casette. We observe inversion of HixC-flanked segments of DNA in the presence of this casette. Figure 2 shows the sizes of predicted NheI restriction fragments for different confirmations of two HixC-flanked DNA segments. A construct carrying the pBad promoter in the forward orientation (BBa_S03564) yields a 200 bp fragment (Figure 3, lane 2), while the reverse orientation (BBa_S03565) yields a larger 300 bp fragment (Figure 3, lane 3). In the presence of the HinLVA expression casette, the construct carrying pBad in the forward orientation shows restriction fragments for both orientations (Figure 3, lane 4). Similar results are seen for a construct carrying a HixC-flanked RBS-Tet segment (BBa_J3103) (Figure 4). The occurrence of the forward and reverse orientations in roughly equal proportions suggests that inversion has reached a steady state. Inversion occurs without IPTG induction of pLac-Hin. This may be caused by Hin expression via read-through from the vector backbone or leaky transcription from pLac.
Figure 2. Predicted NheI fragments for different conformations of the different HixC-flanked DNA fragments tested (not drawn to scale).	Figure 3. An NheI digest detects Hin-mediated flipping of a HixC-flanked pBad promoter.	Figure 4. An NheI digest detects Hin-mediated flipping of a HixC-flanked coding region (RBS-Tet).

Source

Hin invertase (BBa_J31000) from Salmonella typhimurium and the LVA degredation tag (BBa_M0040).

References

• Li, W., Kamtekar, S., Xiong, Y., Sarkis, G.J., Grindley, N.D., Steitz, T.A. (2005) Structure of a synaptic gamma delta resolvase tetramer covalently linked to two cleaved DNAs. Science. 309: 1210-1215
• Sanders, E.R., Johnson, R.C. (2004) Stepwise Dissection of the Hin-catalyzed Recombination Reaction from Synapsis to Resolution. J. Mol. Biol. 340: 753–766.
• Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks
• Figure 3. The data in Figure 3 was generously provided by Todd Eckdahl (Missouri Western State University iGEM 2006 Team).