Coding
Hin LVA

Part:BBa_J31001:Design

Designed by: Erin Zwack, Sabriya Rosemond   Group: iGEM06_Davidson   (2006-07-12)
Revision as of 05:46, 29 October 2006 by Kahaynes (Talk | contribs)

DNA invertase Hin tagged with LVA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Hin Invertase

Figure 1. 3-D structure of a Hin protein complex bound to DNA. View the [http://www.rcsb.org/pdb/explore/explore.do?structureId=1ZR4 interactive 3-D Jmol image].
 To the left is a 3-D model of the a Hin/ DNA complex crystal structure (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).

Design Notes

This part is cloned in plasmid pSB1A2.

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

Standard BioBrick Cloning Sites 5'---GAATTC GCGGCCGC T TCTAGA ---insert--- T ACTAGT A GCGGCCG CTGCAG---
3'---CTTAAG CGCCGGCG A AGATCT ------------ A TGATCA T CGCCGGC GACGTC---
EcoRI NotI XbaI SpeI NotI PstI
Prefix: There is no T spacer between the NotI site and the XbaI site. There is no G spacer between the XbaI and the coding region.

Suffix: There is no T spacer between the insert and the SpeI site. The T spacer between the SpeI and the NotI sites should be an A. The last C of the NotI site is not conserved with the initial C from the PstI site. The BB suffix currently has this sequence for the NotI and PstI sites GCGGCCGcCTGCAG But it should have been: GCGGCCGCTGCAG

GAATTCGCGGCCGC-TCTAGA----Hin coding----ACTAGTTGCGGCCGCCTGCAG

We compared our BioBricks with those from Tom Knight's paper, Idempotent Vector Design for Standard Assembly of Biobricks. As seen below

BioBricks from paper.png

Data

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. Inversion occurs without IPTG induction of pLac-Hin. This may be caused by read-through from the vector backbone or leaky transcription from pLac.

Figure 2. An NheI digest detects Hin-mediated flipping of a HixC-flanked pBad promoter.
Figure 3. 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