Difference between revisions of "Part:BBa K1668005:Design"
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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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The template is genomic DNA extracted from strain <i>Photorhabdus luminescens</i> TT01. We got the strain from Shandong University.
 
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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>.   
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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</i> TT01.   
 
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<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.
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After seamless assembly, standard plasmid pSB1C3 containing <i>tcdA1</i> gene was transformed into <i> E.coli</i> 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 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>
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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_TPCDS_A1.PNG|300px|thumb|left|Figure 3 the plasmid map of BBa_K1668008]]
 
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Revision as of 09:44, 18 September 2015

CDS tcdA1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7035
    Illegal NheI site found at 7368
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 429
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 6469
    Illegal NgoMIV site found at 7129
    Illegal AgeI site found at 2305
  • 1000
    COMPATIBLE WITH RFC[1000]



Source

The CDStcdA1 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 Photorhabdus luminescens TT01. We got the strain from Shandong University.

Design Notes

PCR

The CDStcdA1 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 Photorhabdus luminescens TT01.

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, tcdA1-left, tcdA1-middle, tcdA1-right, and suffix sequence can be ligated seamlessly.

tcdA1-left F (F, 5’-3’): AATTCGCGGCCGCTTCTAGATGAACTCGCCTGTAAAAGAGA

tcdA1-left R (R, 5’-3’): AAATTCACAACAAGCGGATACATTACAC

tcdA1-middle F(F, 5’-3’): TATCCGCTTGTTGTGAATTTAATCCGG

tcdA1-middle R(R, 5’-3’): TAACATTAGCGGAAAATTCCATCACATAACCTGTTGC

tcdA1-right F(F, 5’-3’): GGAATTTTCCGCTAATGTTATGAATACCGAAGC

tcdA1-right R(R, 5’-3’): CTGCAGCGGCCGCTACTAGTATTATTATTTAATGGTGTA


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

Figure 3 the plasmid map of BBa_K1668008





















References