Difference between revisions of "Part:BBa J31001:Design"
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===Hin Invertase=== | ===Hin Invertase=== | ||
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| [[Image:Jmol_Hin_tetrad_DNA.gif|thumb|250px|'''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].]] | | [[Image:Jmol_Hin_tetrad_DNA.gif|thumb|250px|'''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].]] | ||
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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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| − | | style="width: | + | | style="width:200px" | Standard BioBrick Cloning Sites |
| − | | style="background:lightgrey" | <font face="courier"> | + | | style="background:lightgrey"|<font face="courier">5'---GAATTC GCGGCCGC T TCTAGA ---insert--- T ACTAGT A GCGGCCG CTGCAG---<br>3'---CTTAAG CGCCGGCG A AGATCT ------------ A TGATCA T CGCCGGC GACGTC---<br>EcoRI NotI XbaI SpeI NotI PstI</font> |
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| − | | style="width: | + | | style="width:200px" | Prefix: There is no <font color='red'>T spacer</font color> between the<font color='brown'> NotI site</font color> and the <font color='purple'>XbaI site</font color>. There is no <font color='lime'>G spacer</font color> between the <font color='purple'>XbaI</font> and the <u>coding region</u>.<br> |
| − | + | Suffix: There is no <font color='tan'>T spacer</font color> between the <u>insert</u> and the <font color='green'>SpeI site</font color>. The <font color='gold'>T spacer</font color> between the <font color='green'>SpeI</font color> and the <font color='magenta'>NotI</font color> sites should be an A. The last ''C'' of the <font color='magenta'>NotI</font color> site is not conserved with the initial C from the <font color='cyan'>PstI site</font color>. The BB suffix currently has this sequence for the <font color='magenta'>NotI</font color> and <font color='cyan'>PstI</font color> sites <font color='magenta'>GCGGCCGc</font color><font color='cyan'>CTGCAG</font color> But it should have been: <font color='magenta'>GCGGCCG</font color><font color='purple'>''C''</font color><font color='cyan'>TGCAG</font color> | |
| − | + | | style="background:lightgrey" | <font face="courier"><font color='blue'>GAATTC</font><font color='brown'>GCGGCCGC</font><font color='red'>-</font><font color='purple'>TCTAGA</font><font color='lime'>-</font></font>---Hin coding---<font face="courier"><font color='tan'>-</font><font color='green'>ACTAGT</font><font color='gold'>T</font><font color='magenta'>GCGGCCGC</font>''C''<font color='cyan'>TGCAG</font></font> | |
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| − | | style="background:lightgrey" | <font face="courier"><font color='tan'>-</font><font color='green'>ACTAGT</font><font color='gold'>T</font><font color='magenta'>GCGGCCGC</font>''C''<font color='cyan'>TGCAG</font></font> | + | |
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Revision as of 05:46, 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 (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
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.
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