Difference between revisions of "Part:BBa K2212002"
 
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This part is intended to be used in Synechococcus elongatus PCC 7942 in the pathway for the production of tagatose from sucrose. Originally, this protein is an important enzyme in plant galactose metabolism pathway.  
 
This part is intended to be used in Synechococcus elongatus PCC 7942 in the pathway for the production of tagatose from sucrose. Originally, this protein is an important enzyme in plant galactose metabolism pathway.  
  
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[[Image:SynEco pathway.png|700px|center|thumb| '''Figure 1: Proposed tagatose synthesis pathway.''']]
 
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===Usage and Biology===
 
===Usage and Biology===
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<partinfo>BBa_K2212002 parameters</partinfo>
 
<partinfo>BBa_K2212002 parameters</partinfo>
 
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==Gel Validation==
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Primers:<br />
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Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3' <br />
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Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3' <br />
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All three clones have the expected ~2.2kbp band
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[[Image:RafS gel.jpg|700px|center|thumb| '''Figure 2: Gel image of the validation of RafS.''']]
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==Sequencing==
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After gel validation, we sent one clone (clone #13, the first one from the left in the gel image above) for sequencing validation. The primers used were the same ones as used for PCR (Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3' Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3'). The alignment results are shown below.
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[[Image:RafS sequencing 5'.png|700px|center|thumb| '''Figure 3: Sequencing validation for RafS insertion. 5' end.''']]
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[[Image:RafS sequencing 3'.png|700px|center|thumb| '''Figure 4: Sequencing validation for RafS insertion. 3' end.''']]

Latest revision as of 04:49, 29 October 2017


RafS w/ pconII+RiboswitchF+DYKDDDDK+rrnBT1

This part contains coding sequence for enzyme Raffinose synthase (EC 2.4.1.82), pconII promoter, riboswitch F, DYKDDDDK-tag, rrnB T1 terminator. The unlabeled sequences are random filler sequences. The protein sequence is originated from Arabidopsis thaliana. The protein coding sequence was codon optimized for Synechocystis sp. PCC 6803.

This part is intended to be used in Synechococcus elongatus PCC 7942 in the pathway for the production of tagatose from sucrose. Originally, this protein is an important enzyme in plant galactose metabolism pathway.

Figure 1: Proposed tagatose synthesis pathway.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 410
    Illegal BamHI site found at 1471
    Illegal XhoI site found at 527
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 41
    Illegal AgeI site found at 2020
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 564
    Illegal BsaI.rc site found at 429


Gel Validation

Primers:
Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3'
Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3'

All three clones have the expected ~2.2kbp band

Figure 2: Gel image of the validation of RafS.

Sequencing

After gel validation, we sent one clone (clone #13, the first one from the left in the gel image above) for sequencing validation. The primers used were the same ones as used for PCR (Forward: 5'AGTCGGCCAATAACCCAGGGATTTCTTAGCTTTCGCTAAGGATGATTTCTG 3' Reverse: 5'GATTCTAGAAAGCTTCAAAAAGGCCACACCTTGCCCTTTTTTGCCGGA 3'). The alignment results are shown below.

Figure 3: Sequencing validation for RafS insertion. 5' end.
Figure 4: Sequencing validation for RafS insertion. 3' end.