Difference between revisions of "Part:BBa J31001:Design"
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| colspan="3" | '''Inversion of HixC-flanked DNA in the presence of HinLVA'''<br>HinLVA has been assembled with a pLac promoter and RBS (see <partinfo>BBa_S03536</partinfo>) 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. The forward orientation of pBad yields a 200 bp fragment, while the reverse orientation yields a larger 300 bp fragment (Figure 3, lanes 2 and 3, respectively). In the presence of Hin, a plasmid originally containing pBad in the forward orientation shows restriction fragments for both orientations (Figure 3, lane 4). Similar results are seen for a HixC-flanked RBS-Tet resistance segment (Figure 4). The occurrence of the two 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. | | colspan="3" | '''Inversion of HixC-flanked DNA in the presence of HinLVA'''<br>HinLVA has been assembled with a pLac promoter and RBS (see <partinfo>BBa_S03536</partinfo>) 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. The forward orientation of pBad yields a 200 bp fragment, while the reverse orientation yields a larger 300 bp fragment (Figure 3, lanes 2 and 3, respectively). In the presence of Hin, a plasmid originally containing pBad in the forward orientation shows restriction fragments for both orientations (Figure 3, lane 4). Similar results are seen for a HixC-flanked RBS-Tet resistance segment (Figure 4). The occurrence of the two 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. | ||
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− | | [[Image:NheI_map.gif|thumb| | + | | [[Image:NheI_map.gif|thumb|400px|'''Figure 2.''' Predicted NheI fragments for different conformations of the different HixC-flanked DNA fragments tested.]] |
− | | [[Image:PBad_flipping.jpg|thumb| | + | | [[Image:PBad_flipping.jpg|thumb|200px|'''Figure 3.''' An NheI digest detects Hin-mediated flipping of a HixC-flanked pBad promoter.]] |
− | | [[Image:Tet_flipping.jpg|thumb| | + | | [[Image:Tet_flipping.jpg|thumb|200px|'''Figure 4.''' An NheI digest detects Hin-mediated flipping of a HixC-flanked coding region (RBS-Tet).]] |
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Revision as of 16:47, 29 October 2006
DNA invertase Hin tagged with LVA
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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). |
Design Notes
The Biobricks on this part are not wildtype but the cut sites are still viable.
Standard BioBrick Cloning Sites (Knight) | 5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG-- 3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC-- |
BBa_J31001 Cloning Sites | 5'--GAATTC GCGGCCGC * TCTAGA * --Hin coding-- * ACTAGT T GCGGCCGCCTGCAG-- 3'--CTTAAG CGCCGGCG * AGATCT * -------------- * TGATCA A CGCCGGCGGACGTC-- Prefix |
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. The forward orientation of pBad yields a 200 bp fragment, while the reverse orientation yields a larger 300 bp fragment (Figure 3, lanes 2 and 3, respectively). In the presence of Hin, a plasmid originally containing pBad in the forward orientation shows restriction fragments for both orientations (Figure 3, lane 4). Similar results are seen for a HixC-flanked RBS-Tet resistance segment (Figure 4). The occurrence of the two 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. | ||
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