Difference between revisions of "Part:BBa K2212006"
 
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<partinfo>BBa_K2212006 parameters</partinfo>
 
<partinfo>BBa_K2212006 parameters</partinfo>
 
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==Gel Validation==
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The insertion is validated by enzyme digestion analysis with enzymes EcoRI and SacI, and CutSmart buffer.
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The clones highlighted have the expected ~2kbp and 7kbp bands
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[[Image:composite gel.png|700px|center|thumb| '''Figure 2: Gel image of the validation''']]
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==Sequencing==
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After gel validation, we sent one clone (clone #8, the eighth from the left in the gel image above) for sequencing validation. The primer used were: <br />
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Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3' <br />
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Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3' <br />
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The alignment results are shown below.
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[[Image:composite sequencing 5'.png|700px|center|thumb| '''Figure 3: Sequencing validation. 5' end.''']]
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[[Image:composite sequencing 3'.png|700px|center|thumb| '''Figure 4: Sequencing validation. 3' end.''']]

Latest revision as of 07:09, 29 October 2017


GolS+RafS+GLA+LAI

This part was made by compositing parts BBa_2212002, BBa_2212003, BBa_2212004, and BBa_2212005 through 3A assembly. It contains the coding sequence for enzymes inositol 3-alpha-galactosyltransferase (EC 2.4.1.123), Raffinose synthase (EC 2.4.1.82), alpha-galactosidase (EC 3.2.1.22), and L-arabinose isomerase (EC 5.3.1.4). Each sub-part is a complete transnational unit, which contains promoter pconII, riboswitch F including ribosome binding site, enzyme gene sequence, DYKDDDDK-tag, and rrnB T1 terminator. This part is intended to be used in Synechococcus elongatus PCC 7942 for the production of tagatose. The part contains the enzymes sufficient for S. elongatus PCC 7942 to convert tagatse from sucrose.

Figure 1: Proposed tagatose synthesis pathway.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4234
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4123
    Illegal BamHI site found at 1649
    Illegal BamHI site found at 2710
    Illegal XhoI site found at 1766
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 6493
    Illegal NgoMIV site found at 6659
    Illegal AgeI site found at 41
    Illegal AgeI site found at 367
    Illegal AgeI site found at 1280
    Illegal AgeI site found at 3259
    Illegal AgeI site found at 3749
    Illegal AgeI site found at 5267
    Illegal AgeI site found at 6592
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1803
    Illegal BsaI site found at 5600
    Illegal BsaI.rc site found at 1668
    Illegal BsaI.rc site found at 4088


Gel Validation

The insertion is validated by enzyme digestion analysis with enzymes EcoRI and SacI, and CutSmart buffer.

The clones highlighted have the expected ~2kbp and 7kbp bands

Figure 2: Gel image of the validation

Sequencing

After gel validation, we sent one clone (clone #8, the eighth from the left in the gel image above) for sequencing validation. The primer used were:
Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3'
Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3'
The alignment results are shown below.

Figure 3: Sequencing validation. 5' end.
Figure 4: Sequencing validation. 3' end.