Difference between revisions of "Part:BBa K1668006:Design"

 
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<partinfo>BBa_K1668006 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1668006 SequenceAndFeatures</partinfo>
  
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===Source===
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The CDS<i>plu0840</i> gene was amplified by PCR by two pieces to remove one enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>. We got the strain from Shandong University.
  
 
===Design Notes===
 
===Design Notes===
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We used seamless assembly.
 
  
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<h4>PCR</h4>
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The CDS<i>plu0840</i> gene was amplified by PCR by two pieces to remove one enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>.
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We use primer plu0840-left L and plu0840-left R to amplify the left side of gene. And we use plu0840-right L and plu0840-right R to amplify the right side. Primers are shown below.<br>
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<h4>Seamless assembly</h4>
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We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol. By this way prefix sequence, <i>plu0840</i>-left, <i>plu0840</i>-right, and suffix sequence can be ligated seamlessly.
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plu0840-left F (F, 5’-3’): AATTCGCGGCCGCTTCTAGATGTTGTATAACACCCCAGT <br>
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plu0840-left R (R, 5’-3’): CAACGAATTAAGAGGGAACAGCGGCC <br>
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plu0840-right F(F, 5’-3’): TGTTCCCTCTTAATTCGTTGATGCTGCATGGC <br>
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plu0840-right R(R, 5’-3’): ACTGCAGCGGCCGCTACTAGTATTATTATTTCGATGGGGTCAAAG <br>
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<h4>Transformation and confirmation</h4>
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After seamless assembly, standard plasmid pSB1C3 containing <i>plu0840</i> gene was transformed into <i> E.coli DH5α</i>. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.
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<h4>Plasmid map</h4>
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[[File:ZJU-CHINA_TPCDS_0840.PNG|300px|thumb|left|Fig.3 the plasmid map of BBa_K1668006]]
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===Source===
 
  
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The metK gene was amplified by PCR with genomic DNA of S. avermitilis(&#20855;&#20307;&#22411;&#21495;) as template using primers metK1 and metK2
 
  
 
===References===
 
===References===

Revision as of 19:27, 16 September 2015

CDS plu0840


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]

Source

The CDSplu0840 gene was amplified by PCR by two pieces to remove one enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain Photorhabdus luminescens TT01. We got the strain from Shandong University.

Design Notes

PCR

The CDSplu0840 gene was amplified by PCR by two pieces to remove one enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain Photorhabdus luminescens TT01.

We use primer plu0840-left L and plu0840-left R to amplify the left side of gene. And we use plu0840-right L and plu0840-right R to amplify the right side. Primers are shown below.



Seamless assembly

We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol. By this way prefix sequence, plu0840-left, plu0840-right, and suffix sequence can be ligated seamlessly.
plu0840-left F (F, 5’-3’): AATTCGCGGCCGCTTCTAGATGTTGTATAACACCCCAGT
plu0840-left R (R, 5’-3’): CAACGAATTAAGAGGGAACAGCGGCC
plu0840-right F(F, 5’-3’): TGTTCCCTCTTAATTCGTTGATGCTGCATGGC
plu0840-right R(R, 5’-3’): ACTGCAGCGGCCGCTACTAGTATTATTATTTCGATGGGGTCAAAG


Transformation and confirmation

After seamless assembly, standard plasmid pSB1C3 containing plu0840 gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.


Plasmid map

Fig.3 the plasmid map of BBa_K1668006





















References