Difference between revisions of "Part:BBa J31001:Design"
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===Design Notes===
 
===Design Notes===
−
This part is cloned in plasmid pSB1A2.
 
  
 
The Biobricks on this part are not wildtype but the cut sites are still viable.
 
The Biobricks on this part are not wildtype but the cut sites are still viable.
  
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{| width="800px" cellspacing="3"
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{| width="800px" cellspacing="5"
 
|- valign="top"
 
|- valign="top"
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| style="width:200px" | '''Standard BioBrick Cloning Sites''' (Knight)
+
| style="width:180px" | '''Standard BioBrick Cloning Sites''' (Knight)
 
| style="background:lightgrey"|<font face="courier">5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG--<br>3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC--</font>
 
| style="background:lightgrey"|<font face="courier">5'--GAATTC GCGGCCGC T TCTAGA G ----insert---- T ACTAGT A GCGGCCG CTGCAG--<br>3'--CTTAAG CGCCGGCG A AGATCT C -------------- A TGATCA T CGCCGGC GACGTC--</font>
 
|- valign="top"
 
|- valign="top"
−
| style="width:200px" | '''BBa_J31001 Cloning Sites'''  
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| style="width:180px" | '''BBa_J31001 Cloning Sites'''  
 
| style="background:lightgrey" |<font face="courier">5'--GAATTC GCGGCCGC <font color='red'>*</font> TCTAGA <font color='blue'>*</font> --Hin coding-- <font color='purple'>*</font> ACTAGT <font color='darkgreen'>T</font> GCGGCCG<font color='magenta'>C</font>CTGCAG--<br>3'--CTTAAG CGCCGGCG <font color='red'>*</font> AGATCT <font color='blue'>*</font> -------------- <font color='purple'>*</font> TGATCA <font color='darkgreen'>A</font> CGCCGGC<font color='magenta'>G</font>GACGTC--</font><br><br>
 
| style="background:lightgrey" |<font face="courier">5'--GAATTC GCGGCCGC <font color='red'>*</font> TCTAGA <font color='blue'>*</font> --Hin coding-- <font color='purple'>*</font> ACTAGT <font color='darkgreen'>T</font> GCGGCCG<font color='magenta'>C</font>CTGCAG--<br>3'--CTTAAG CGCCGGCG <font color='red'>*</font> AGATCT <font color='blue'>*</font> -------------- <font color='purple'>*</font> TGATCA <font color='darkgreen'>A</font> CGCCGGC<font color='magenta'>G</font>GACGTC--</font><br><br>
 
'''Prefix'''<br>There is <font color='red'>no T spacer (*)</font> between the NotI site and the XbaI site.<br>There is <font color='blue'>no G spacer (*)</font> between the XbaI and the Hin coding region.<br>
 
'''Prefix'''<br>There is <font color='red'>no T spacer (*)</font> between the NotI site and the XbaI site.<br>There is <font color='blue'>no G spacer (*)</font> between the XbaI and the Hin coding region.<br>
−
'''Suffix'''<br>There is no <font color='purple'>T spacer (*)</font> between the Hin coding region and the SpeI site.<br>The A spacer between the SpeI and the NotI has changed to a <font color='darkgreen'>T</font>.<br>There is an extra <font color='magenta'>C</font> between the NotI site and the PstI site
+
'''Suffix'''<br>There is <font color='purple'>no T spacer (*)</font> between the Hin coding region and the SpeI site.<br>The A spacer between the SpeI and the NotI has changed to a <font color='darkgreen'>T</font>.<br>There is an extra <font color='magenta'>C</font> between the NotI site and the PstI site
 
|}
 
|}
  
 
===Data===
 
===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 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.
+
{| width="800px"
−
 
+
|-
−
{|
+
| 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 NheI digestInversion 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.
 
|-
 
|-
−
| [[Image:PBad_flipping.jpg|thumb|250px|'''Figure 2.''' An NheI digest detects Hin-mediated flipping of a HixC-flanked pBad promoter.]]
+
| [[Image:NheI_map.jpg|thumb|250px|'''Figure 2.''' Predicted NheI fragments for different conformations of the different HixC-flanked DNA fragments tested.]]
−
| [[Image:Tet_flipping.jpg|thumb|250px|'''Figure 3.''' An NheI digest detects Hin-mediated flipping of a HixC-flanked coding region (RBS-Tet).]]
+
| [[Image:PBad_flipping.jpg|thumb|250px|'''Figure 3.''' An NheI digest detects Hin-mediated flipping of a HixC-flanked pBad promoter.]]
 +
| [[Image:Tet_flipping.jpg|thumb|250px|'''Figure 4.''' An NheI digest detects Hin-mediated flipping of a HixC-flanked coding region (RBS-Tet).]]
 
|}
 
|}
  

Revision as of 06:44, 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

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 (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
There is no T spacer (*) between the NotI site and the XbaI site.
There is no G spacer (*) between the XbaI and the Hin coding region.
Suffix
There is no T spacer (*) between the Hin coding region and the SpeI site.
The A spacer between the SpeI and the NotI has changed to a T.
There is an extra C between the NotI site and the PstI site

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 NheI digestInversion 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.
File:NheI map.jpg
Figure 2. Predicted NheI fragments for different conformations of the different HixC-flanked DNA fragments tested.
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