Difference between revisions of "Part:BBa K1668008:Design"
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===Source===
 
===Source===
  
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The CDS<i>tcdA1</i> gene was amplified by PCR by three pieces to remove three enzyme site EcoRI in its original squnce.  
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The device is composed of arabinose inducible promoter <i>pBad</i>, CDS <i> tcdA1</i> and reporter <i>mCherry</i>. As the <i>mCherry</i> is already in pSB1C3, we linearize the part with enzyme PstI as backbone. The other two genes are amplified by PCR separately from parts [https://parts.igem.org/Part:BBa_I0500] (<i>pBad</i>), [https://parts.igem.org/Part: BBa_K1668006] (CDS <i>plu0840</i>).
 
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The template is genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>. We got the strain from Shandong University.
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We got <i>pBad</i> from Part Registry and the CDS <i> tcdA1</i> is a part we have constructed ourselves.  
 
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<h4>PCR</h4>
 
<h4>PCR</h4>
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The CDS<i>tcdA1</i> gene was amplified by PCR by three pieces to remove three enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>.
+
We use primer pBad F/pBad R to amplify <i>pBad</i> and standard F/ tcdA1-right R for CDS <i> tcdA1</i>.  
 
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<h4>Seamless assembly</h4>
 
<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>tcdA1</i>-left, <i>tcdA1</i>-middle, <i>tcdA1</i>-right, and suffix sequence can be ligated seamlessly.
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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>pBad</i>, CDS <i> tcdA1</i> and front twenty bases of <i>mCherry</i> sequence can be ligated seamlessly.  
 
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tcdA1-left F (F, 5’-3’): AATTCGCGGCCGCTTCTAGATGAACTCGCCTGTAAAAGAGA <br><br>
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pBad F (F, 5’-3’): ATTCGCGGCCGCTTCTAGAGTTATGACAACTTGACG <br><br>
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tcdA1-left R (R, 5’-3’): AAATTCACAACAAGCGGATACATTACAC <br><br>
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pBad R (R, 5’-3’): GCTAGCCCAAAAAAACG <br><br>
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tcdA1-middle F(F, 5’-3’): TATCCGCTTGTTGTGAATTTAATCCGG <br><br>
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standard F (F, 5’-3’): CCGTTTTTTTGGGCTAGCAGAAAGAGGAGAAATACTAG <br><br>
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tcdA1-middle R(R, 5’-3’): TAACATTAGCGGAAAATTCCATCACATAACCTGTTGC <br><br>
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tcdA1 R (R, 5’-3’): CTAGTATTTCTCCTCTTTCTTTATTTAATGGTGTAGCGAA <br><br>
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tcdA1-right F(F, 5’-3’): GGAATTTTCCGCTAATGTTATGAATACCGAAGC <br><br>
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tcdA1-right R(R, 5’-3’): CTGCAGCGGCCGCTACTAGTATTATTATTTAATGGTGTA <br><br>
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<h4>Transformation and confirmation</h4>
 
<h4>Transformation and confirmation</h4>
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After seamless assembly, standard plasmid pSB1C3 containing <i>tcdA1</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 tcdA1_M1(a primer on the right part of tcdA1)/ VR as the universal primers for the CDS<i>tcdA1</i> is too long to amplify all the parts sequence with VF2/VR .Primers are shown below. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.
+
After seamless assembly, standard plasmid pSB1C3 containing <i> tcdA1</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 tcdA1_M1(a primer on the right part of tcdA1)/VR as the universal primers for the CDS<i>tcdA1</i> is too long to amplify all the parts sequence with VF2/VR .Primers are shown below. 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>
 
<h4>Plasmid map</h4>
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[[File:ZJU-CHINA_TPCDS_A1.PNG|300px|thumb|left|Fig.3 the plasmid map of BBa_K1668008]]
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[[File:ZJU-CHINA_TPDevice_A1.PNG|300px|thumb|left|Fig.3 the plasmid map of BBa_K1668008]]
 
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Revision as of 20:00, 16 September 2015

tcdA1-device


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 8257
    Illegal NheI site found at 8590
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1651
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 7691
    Illegal NgoMIV site found at 8351
    Illegal AgeI site found at 979
    Illegal AgeI site found at 3527
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961



Source

The device is composed of arabinose inducible promoter pBad, CDS tcdA1 and reporter mCherry. As the mCherry is already in pSB1C3, we linearize the part with enzyme PstI as backbone. The other two genes are amplified by PCR separately from parts [1] (pBad), BBa_K1668006 (CDS plu0840).

We got pBad from Part Registry and the CDS tcdA1 is a part we have constructed ourselves.

Design Notes

PCR

We use primer pBad F/pBad R to amplify pBad and standard F/ tcdA1-right R for CDS tcdA1.

We use primer tcdA1-left L and tcdA1-left R to amplify the left side of gene., tcdA1-middle L and tcdA1-middle R to amplify the middle part and tcdA1-right L and tcdA1-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, pBad, CDS tcdA1 and front twenty bases of mCherry sequence can be ligated seamlessly.

pBad F (F, 5’-3’): ATTCGCGGCCGCTTCTAGAGTTATGACAACTTGACG

pBad R (R, 5’-3’): GCTAGCCCAAAAAAACG

standard F (F, 5’-3’): CCGTTTTTTTGGGCTAGCAGAAAGAGGAGAAATACTAG

tcdA1 R (R, 5’-3’): CTAGTATTTCTCCTCTTTCTTTATTTAATGGTGTAGCGAA


Transformation and confirmation

After seamless assembly, standard plasmid pSB1C3 containing tcdA1 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 tcdA1_M1(a primer on the right part of tcdA1)/VR as the universal primers for the CDStcdA1 is too long to amplify all the parts sequence with VF2/VR .Primers are shown below. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.

tcdA1_M1(F, 5’-3’): CTGCCAATCTATGCCACACC

VR(F, 5’-3’): ATTACCGCCTTTGAGTGAGC


Plasmid map

Fig.3 the plasmid map of BBa_K1668008





















References