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

Revision as of 04:15, 29 October 2006

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

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].

Protein Data Bank ID 1ZR4, Jmol image (Li et al. 2005). A Hin protein dimer binds and cleaves DNA at 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 DNA ends are swapped and ligated.

Design Notes

This part is cloned in plasmid pSB1A2.

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

BioBrick 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.	GAATTCGCGGCCGC-TCTAGA-
Hin coding	TGGCTACTATTGGGTATATTCGGGTGTCAACAATTGACCAAAATATCGAT TTACAGCGTAATGCGCTTACCAGTGCAAATTGTGACCGCATTTTTGAGGA CCGTATCAGTGGCAAGATTGCAAACCGCCCCGGCCTGAAACGAGCGTTAA AGTATGTAAATAAAGGCGATACTCTTGTCGTCTGGAAATTAGACAGACTG GGCCGCAGCGTGAAAAACCTGGTGGCGTTAATATCAGAATTACATGAACG TGGAGCTCACTTCCATTCTTTAACCGATAGTATTGATACCAGTAGCGCGA TGGGGCGATTCTTTTTTCATGTAATGTCAGCACTGGCCGAGATGGAGCGA GAATTAATTGTCGAGCGAACCCTTGCCGGACTGGCTGCCGCCAGAGCGCA AGGACGACTGGGAGGGCGCCCTCGGGCGATCAACAGACATGAACAGGAAC AGATTAGTCGGCTATTAGAGAAAGGCCATCCTCGGCAGCAACTAGCTATT ATTTTTGGTATTGGCGTATCTACCTTATACAGATATTTTCCGGCAAGCCG TATAAAAAAACGAATGAATAGGCCTGCTGCAAACGACGAAAACTACGCTT TAGTAGCTTA
BioBrick 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	-ACTAGTTGCGGCCGCCTGCAG

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

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

Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks
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. v309 pp.1210-1215 , 2005

@@ Line 8: / Line 8: @@
 {|
 |-
-[[Image:Jmol_Hin_tetrad_DNA.gif|thumb|250px|left|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].]]
+| [[Image:Jmol_Hin_tetrad_DNA.gif|thumb|250px|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].]]
+| Protein Data Bank ID 1ZR4, Jmol image (Li et al. 2005). A Hin protein dimer binds and cleaves DNA at 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 DNA ends are swapped and ligated.
 |}
@@ Line 46: / Line 47: @@
 ===Data===
-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. Inversion occurs with induction of Hin expression. This may be caused by read-through from the vector backbone or leaky transcription from pLac.
+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. Inversion occurs without IPTG induction of pLac-Hin. This may be caused by read-through from the vector backbone or leaky transcription from pLac.
 {|
 |-
-| [[Image:Jmol_Hin_tetrad_DNA.gif|thumb|250px|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].]]
+| [[Image:PBad_flipping.jpg|thumb|250px|'''Figure 2.''' An NheI digest detects Hin-mediated flipping of a HixC-flanked pBad promoter.]]
-| [[Image:Jmol_Hin_tetrad_DNA.gif|thumb|250px|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].]]
+| [[Image:Tet_flipping.jpg|thumb|250px|'''Figure 3.''' An NheI digest detects Hin-mediated flipping of a HixC-flanked coding region (RBS-Tet).]]
 |}
 ===Source===
-Salmonella typhimurium and the the Hin part without LVA.
+Hin invertase (<partinfo>BBa_J31000</partinfo>) from Salmonella typhimurium and the LVA degredation tag (<partinfo>BBa_M0040</partinfo>).
 ===References===
 *[https://dspace.mit.edu/handle/1721.1/21168|  Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks]
 *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. v309 pp.1210-1215 , 2005