Difference between revisions of "Part:BBa J3101:Design"

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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_J3101 short</partinfo>
 
<partinfo>BBa_J3101 short</partinfo>
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The base pair change of a T from a C occurred during cloning but is located in the spacer region between the fis binding sites; therefore, we believe that it will still work.
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The C > T point mutation occurred during cloning. It is located in the spacer region between the Fis binding sites; therefore, we believe that the mutation will not impact RE function. There is an insertion immediately  before the BioBrick suffix and several bases after the distal fis binding site that should affect RE function. RE is cloned in plasmid pSB1A2.
  
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The insertion is right before the biobrick ends and several bases after the distal fis binding site; therefore, we believe it will function correctly.
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The BioBrick prefix and suffix on this part are not wildtype but the restriction sites are still viable.
  
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We chose this RE to use because after cloning it retained functional full biobricks and the mutations do not seem harmful to function. The other RE that were cloned retained the proper RE sequence but lost critical cut sites in the biobricks.
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{| width="800px" cellspacing="5"
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|- valign="top"
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| style="width:180px" | '''Standard BioBrick Cloning Sites''' (Knight)
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| 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>
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|- valign="top"
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| style="width:180px" | '''BBa_J31001 Cloning Sites'''
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| style="background:lightgrey" |<font face="courier">5'--GAATTC GCGGCCGC T TCTAGA <font color='blue'>*</font> ------RE------ <font color='purple'>G</font> ACTAGT <font color='darkgreen'>T</font> GCGGCCG<font color='magenta'>C</font>CTGCAG--<br>3'--CTTAAG CGCCGGCG A AGATCT <font color='blue'>*</font> -------------- <font color='purple'>C</font> TGATCA <font color='darkgreen'>A</font> CGCCGGC<font color='magenta'>G</font>GACGTC--</font><br><br>
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'''Prefix'''<br>There is <font color='blue'>no G spacer (*)</font> between the XbaI and the RE.<br>
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'''Suffix'''<br>The T spacer between the RE and the SpeI site has changed to a <font color='purple'>G</font>.<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
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|}
  
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RE is cloned in plasmid pSB1A2.
 
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=BioBricks=
 
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The BioBricks designed for this part are not wild type, but the cut sites are still viable. The following is the sequence with its BioBricks:
 
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<font color='navy'>GAATTC</font color><font color='tan'>GCGGCCGC</font color>T<font color='orange'>TCTAGA</font color><font color='red'>-</font color><u>TTCGGGTGTCAACAATTGACCAAAATATTGATTTACAGCGTAATGCGCTTTCTAGTGCAAATTGTGACCGCATTTT</u><font color='blue'>G</font color><font color='gold'>ACTAGT</font color><font color='sky blue'>T</font color><font color='pink'>GCGGCCGC</font color><u>''C''</u><font color='blue'>TGCAG</font color>
 
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There is no <font color='red'>G</font color> spacer between the <font color='orange'>XbaI</font color> and the <u>insert</u>.
 
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The spacer between the <u>insert</u> and the <font color='gold'>SpeI</font color> site is a <font color='blue'>G</font color> instead of a T.
 
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The spacer between the <font color='gold'>SpeI</font color> site and the <font color='pink'>NotI</font color> site is a <font color='sky blue'>T</font color> instead of an A.
 
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The last <u>''C''</u> of the <font color='pink'>NotI</font color> site is not conserved with the initial C from the <font color='blue'>PstI</font color> site.
 
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The BB suffix currently has this sequence for Not I and PstI:
 
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<font color='pink'>GCGGCCG'''c'''</font color><font color='blue'>CTGCAG</font color>
 
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But it should have been:
 
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<font color='pink'>GCGGCCG</font color><font color='purple'>C</font color><font color='blue'>TGCAG</font color>
 
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We compared our BioBricks with those from Tom Knight's paper,<u> Idempotent Vector Design for Standard Assembly of Biobricks</u>. As seen below
 
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[[Image:BioBricks_from_paper.png]]
 
  
 
===Source===
 
===Source===
  
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The Recombination Enhancer sequence from ''Salmonella typhimurium''.
  
 
===References===
 
===References===
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<biblio>
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Haykinson and Johnson. 1993. DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly
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#Johnson pmid=2548848
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[http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=8508775]
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#Haykinson pmid=8508775
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#PerkinsBalding pmid=9244261
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</biblio>
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[[Media:Enhancer_Sequence_Paper.pdf| PERKINS-BALDING D, DIAS D, and GLASGOW A:Location, Degree, and Direction of DNA Bending Associated
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with the Hin Recombinational Enhancer Sequence and
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Fis-Enhancer Complex ]]
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[https://dspace.mit.edu/handle/1721.1/21168|  Knight, Tom. Idempotent Vector Design for Standard Assembly of Biobricks]
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Latest revision as of 09:34, 30 October 2008

Recombinational Enhancer (RE) for Hin/Hix inverting


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]


Design Notes

The C > T point mutation occurred during cloning. It is located in the spacer region between the Fis binding sites; therefore, we believe that the mutation will not impact RE function. There is an insertion immediately before the BioBrick suffix and several bases after the distal fis binding site that should affect RE function. RE is cloned in plasmid pSB1A2.

The BioBrick prefix and suffix on this part are not wildtype but the restriction 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 T TCTAGA * ------RE------ G ACTAGT T GCGGCCGCCTGCAG--
3'--CTTAAG CGCCGGCG A AGATCT * -------------- C TGATCA A CGCCGGCGGACGTC--

Prefix
There is no G spacer (*) between the XbaI and the RE.
Suffix
The T spacer between the RE and the SpeI site has changed to a G.
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


Source

The Recombination Enhancer sequence from Salmonella typhimurium.

References

<biblio>

  1. Johnson pmid=2548848
  2. Haykinson pmid=8508775
  3. PerkinsBalding pmid=9244261

</biblio>