Difference between revisions of "Part:BBa J37027:Design"

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===Design Notes===
 
===Design Notes===
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'''This part contains multiple reading frames so cannot be correctly represented in this version of the registary.'''
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'''This part contains coding regions on both strands so cannot be correctly represented in this version of the registry.''' The supplied sequence is correct however. Further information can be found [https://static.igem.org/mediawiki/parts/0/00/Cre_updated.pdf here]
  
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</big>
 
  
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The sequence is correct however the annotations are not. Anything said in [https://static.igem.org/mediawiki/parts/e/e0/Cre_PCR3.pdf '''this summary'''] trumps the registary site.
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'''Reporter  Gene'''
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The part contains a RFP reporter which is transcribed in the 3’-5’ direction. This means that un-activated parts will fluoresce red and activated parts will not fluoresce. This allows you to see that the part is working in your system. It also allows you to observe the efficiency of activation of the part in your system. The part runs the other way to the rest of the system to prevent any fluorescence due to external Pops passing into the device
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'''Lox Sites'''
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The Cre lox system is very complex and it will not be described fully. There are multiple Lox sites which exist - we have chosen Lox sites 66 and 71. They are both placed in the forward direction on the sense strand of the DNA. This causes the DNA between the lox sites to be excised rather than reversed. Lox 66 and 71 have been mutated so that the reversed strand of DNA cannot be re-inserted by cre. Lox 66 should be upstream of Lox 71. The DNA between the Lox sites is seen as a plasmid in the cells once it has been excised however it has no origin of replication so will be quickly degraded inside the cell.
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'''Terminator Sequences'''
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There are two main terminator sequences in the registry: 1) B0015 which consists of two separate sequences B0010 and B0012. 2) B0012 which is a single terminator sequence. B0015 is much more efficient than B0021, 98.4% and 60% respectively. The two terminator sequences in this part cannot be the same as when you try to PCR them they will bind together and form a 131 base pair hairpin loop which the polymerase cannot pass through. Therefore the two terminator sequences must be different. We just have to accept that the stopping will not be as efficient. In theory we could have multiple copies of B0021 but this would require several ligations.
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http://openwetware.org/images/9/9a/Cre2.PNG
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===Cre recombinase===
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*A Cre recombinase device has been designed in the registry - [https://parts.igem.org/wiki/index.php/Part:BBa_J37030 J37030]
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*This device produces the enzyme Cre recombinase under the control of the lacI promoter. Therefore by introducing IPTG, one would be able to induce the production of Cre.
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*It is also possible to obtain commercially available Cre plasmids as well
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 +
Cre sequence and primers that will be used to remove the sequence [http://openwetware.org/images/f/ff/Cre_Primers.doc here]
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http://openwetware.org/images/a/a1/Cre.PNG
  
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John
 
  
 
===Source===
 
===Source===
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[https://parts.igem.org/wiki/index.php/Part:BBa_I13521 BBa_I13521]
 
[https://parts.igem.org/wiki/index.php/Part:BBa_I13521 BBa_I13521]
  
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'''Published Works'''
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===References===
  
 
Lox Sites:
 
Lox Sites:
 
Zuwen Zhang and Beat Lutz (2002) Cre Recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein NUCLEAIC ACIDS RESEARCH
 
Zuwen Zhang and Beat Lutz (2002) Cre Recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein NUCLEAIC ACIDS RESEARCH
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===References===
 

Latest revision as of 20:47, 30 October 2006

Pops Blocker (Cre/Lox system)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 340
    Illegal AgeI site found at 452
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part contains coding regions on both strands so cannot be correctly represented in this version of the registry. The supplied sequence is correct however. Further information can be found here


Reporter Gene

The part contains a RFP reporter which is transcribed in the 3’-5’ direction. This means that un-activated parts will fluoresce red and activated parts will not fluoresce. This allows you to see that the part is working in your system. It also allows you to observe the efficiency of activation of the part in your system. The part runs the other way to the rest of the system to prevent any fluorescence due to external Pops passing into the device

Lox Sites

The Cre lox system is very complex and it will not be described fully. There are multiple Lox sites which exist - we have chosen Lox sites 66 and 71. They are both placed in the forward direction on the sense strand of the DNA. This causes the DNA between the lox sites to be excised rather than reversed. Lox 66 and 71 have been mutated so that the reversed strand of DNA cannot be re-inserted by cre. Lox 66 should be upstream of Lox 71. The DNA between the Lox sites is seen as a plasmid in the cells once it has been excised however it has no origin of replication so will be quickly degraded inside the cell.

Terminator Sequences

There are two main terminator sequences in the registry: 1) B0015 which consists of two separate sequences B0010 and B0012. 2) B0012 which is a single terminator sequence. B0015 is much more efficient than B0021, 98.4% and 60% respectively. The two terminator sequences in this part cannot be the same as when you try to PCR them they will bind together and form a 131 base pair hairpin loop which the polymerase cannot pass through. Therefore the two terminator sequences must be different. We just have to accept that the stopping will not be as efficient. In theory we could have multiple copies of B0021 but this would require several ligations.


http://openwetware.org/images/9/9a/Cre2.PNG

Cre recombinase

  • A Cre recombinase device has been designed in the registry - J37030
  • This device produces the enzyme Cre recombinase under the control of the lacI promoter. Therefore by introducing IPTG, one would be able to induce the production of Cre.
  • It is also possible to obtain commercially available Cre plasmids as well

Cre sequence and primers that will be used to remove the sequence [http://openwetware.org/images/f/ff/Cre_Primers.doc here]

http://openwetware.org/images/a/a1/Cre.PNG


Source

From Registry:

BBa_B0015

BBa_I13521


References

Lox Sites: Zuwen Zhang and Beat Lutz (2002) Cre Recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein NUCLEAIC ACIDS RESEARCH