Difference between revisions of "Part:BBa K1431201:Design"
 
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We have anther kind of NLS but only with one side. We chose two NLS for it may increase the probability of protein import into the cell nucleus. As for BbsI site, we have a series same design sequence. See the mechanism of BbsI site work below.
 
We have anther kind of NLS but only with one side. We chose two NLS for it may increase the probability of protein import into the cell nucleus. As for BbsI site, we have a series same design sequence. See the mechanism of BbsI site work below.
  
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https://static.igem.org/mediawiki/parts/a/ab/Our_RFC.png
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<center>https://static.igem.org/mediawiki/parts/a/ab/Our_RFC.png</center>
  
 
If you want to insert a coding sequence between NLS, the prefix of forward primer you design must contain '''GAAGACNN<span style="color: red">taaa<span style="color: black">NNNN''' and reverse must contain '''GAAGACNN<span style="color: red">agtt<span style="color: black">NNNN'''. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into two NLS seamless. The PCR product must be such sequence, including the protect bases, shows below.
 
If you want to insert a coding sequence between NLS, the prefix of forward primer you design must contain '''GAAGACNN<span style="color: red">taaa<span style="color: black">NNNN''' and reverse must contain '''GAAGACNN<span style="color: red">agtt<span style="color: black">NNNN'''. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into two NLS seamless. The PCR product must be such sequence, including the protect bases, shows below.
  
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<center>{{SUSTC-Image|wiki/images/1/1b/PCR_product_by_new_RFC.png}}</center>
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<center>https://static.igem.org/mediawiki/parts/1/1b/PCR_product_by_new_RFC.png</center>
 
<center>'''Fig.2  PCR Product Sequence'''</center>
 
<center>'''Fig.2  PCR Product Sequence'''</center>
  
 
And the reason why we designed that because we want to make a seamless gather between NLS and protein or any triple nucleotides. That depend on which will make the highest efficiency.
 
And the reason why we designed that because we want to make a seamless gather between NLS and protein or any triple nucleotides. That depend on which will make the highest efficiency.
  
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===Sequencing Results===
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We sent fresh bacteria broth for sequencing using standard Biobricks sequencing primer VF2/VR. The sequencing cooperation we used is Invitrogen Guangzhou filiale.
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Sequence of sequencing primer we used:<br>
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VF2: tgccacctgacgtctaagaa<br>
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VR: attaccgcctttgagtgagc
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The result shows the same sequence with our ideal design.
  
 
==='''Source'''===
 
==='''Source'''===
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===References===
 
===References===
  
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[http://zlab.mit.edu zhangfeng lab]
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[http://zlab.mit.edu zhangfeng lab]<br>
 
http://en.wikipedia.org/wiki/Nuclear_localization_sequence
 
http://en.wikipedia.org/wiki/Nuclear_localization_sequence

Latest revision as of 03:17, 18 October 2014

NLS, lead protein into the nucleus


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 66
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 88
    Illegal NgoMIV site found at 107
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We have anther kind of NLS but only with one side. We chose two NLS for it may increase the probability of protein import into the cell nucleus. As for BbsI site, we have a series same design sequence. See the mechanism of BbsI site work below.

Our_RFC.png

If you want to insert a coding sequence between NLS, the prefix of forward primer you design must contain GAAGACNNtaaaNNNN and reverse must contain GAAGACNNagttNNNN. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into two NLS seamless. The PCR product must be such sequence, including the protect bases, shows below.

PCR_product_by_new_RFC.png
Fig.2 PCR Product Sequence

And the reason why we designed that because we want to make a seamless gather between NLS and protein or any triple nucleotides. That depend on which will make the highest efficiency.

Sequencing Results

We sent fresh bacteria broth for sequencing using standard Biobricks sequencing primer VF2/VR. The sequencing cooperation we used is Invitrogen Guangzhou filiale.

Sequence of sequencing primer we used:
VF2: tgccacctgacgtctaagaa
VR: attaccgcctttgagtgagc

The result shows the same sequence with our ideal design.

Source

These two NLS sequence was from two sides in Cas9 sequence, plasmid px330, zhangfeng lab. For the too length and repeat A&G bases, we completely synthesis the sequence and add two BbsI site specially.

References

[http://zlab.mit.edu zhangfeng lab]
http://en.wikipedia.org/wiki/Nuclear_localization_sequence