Difference between revisions of "Part:BBa K1668011:Design"

 
 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1668011 short</partinfo>
 
<partinfo>BBa_K1668011 short</partinfo>
Line 5: Line 4:
 
<partinfo>BBa_K1668011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1668011 SequenceAndFeatures</partinfo>
  
 +
<br>
 +
<br>
 +
===Source===
  
 +
The <i>mCherry</i> gene was amplified by PCR with the template genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>. We got the strain from Shandong University.
 +
<br>
 +
<br>
 
===Design Notes===
 
===Design Notes===
−
~
 
  
 +
<h4>PCR</h4>
 +
The <i> mCherry </i> gene was amplified by PCR with the template genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>. We use primer mCherry -left F and mCherry R to amplify the left side of gene, which are shown below.
 +
<br>
 +
<h4>Seamless assembly</h4>
 +
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> mCherry </i> and suffix sequence can be ligated seamlessly.
 +
<br>
 +
<br>
 +
mCherry F(F, 5’-3’): AGAAAGAGGAGAAATACTAGATGGTGAG <br><br>
 +
mCherry R(R, 5’-3’): CCGGACTGCAGCGGCCGCTACTAGTATAAACGCAGAAAGGCC <br><br>
 +
<br>
 +
<br>
 +
<h4>Transformation and confirmation</h4>
 +
After seamless assembly, standard plasmid pSB1C3 containing <i> mCherry </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.
 +
<br>
 +
<br>
 +
<h4>Plasmid map</h4>
 +
[[File:ZJU-CHINA_TPmCherry.png|300px|thumb|left|Fig.3 the plasmid map of BBa_K1668011]]
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
  
  
−
===Source===
 
  
−
~
 
  
 
===References===
 
===References===

Latest revision as of 20:14, 16 September 2015

mCherry


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 mCherry gene was amplified by PCR with the template genomic DNA extracted from strain Photorhabdus luminescens TT01. We got the strain from Shandong University.

Design Notes

PCR

The mCherry gene was amplified by PCR with the template genomic DNA extracted from strain Photorhabdus luminescens TT01. We use primer mCherry -left F and mCherry R to amplify the left side of gene, which 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, mCherry and suffix sequence can be ligated seamlessly.

mCherry F(F, 5’-3’): AGAAAGAGGAGAAATACTAGATGGTGAG

mCherry R(R, 5’-3’): CCGGACTGCAGCGGCCGCTACTAGTATAAACGCAGAAAGGCC



Transformation and confirmation

After seamless assembly, standard plasmid pSB1C3 containing mCherry 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_K1668011





















References