Difference between revisions of "Part:BBa K934001:Design"

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sequence confirmed
 
sequence confirmed
  
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To construct a part that meets Biobrick format, first, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format (BBa_K934001).
+
To get this part, first, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format (BBa_K934001).
 +
 
 +
[http://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Construction_of_pha-C1-A-B1_in_Biobrick_format#3. Link to detailed design description]
  
 
===Source===
 
===Source===
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===References===
 
===References===
 +
Jumiarti Agus, et al, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650
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 +
Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)

Latest revision as of 14:43, 13 October 2012

phaC1-A-B1 [P(3HB) synthesis]


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 916
    Illegal BglII site found at 1741
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 222
    Illegal NgoMIV site found at 293
    Illegal NgoMIV site found at 893
    Illegal NgoMIV site found at 1205
    Illegal NgoMIV site found at 1484
    Illegal NgoMIV site found at 2136
    Illegal NgoMIV site found at 2158
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4002


Design Notes

sequence confirmed

To get this part, first, we cloned the wild type gene phaC1-A-B1 from R.eutropha H16 by using PCR and inserted the gene into pSB1C3. However, wild type phaC1-A-B1 gene sequence contains one NotI and three PstI recognition sites that are not allowed in Biobrick format. To get phaC1-A-B1 sequence without these recognition sites, we ordered the chemically synthesized DNA from IDT/MBL. In this chemically synthesized DNA, coding is optimized for E.coli. We used restriction enzyme XbaI (on pSB1C3) and BsrGI (on phaC1-A-B1) to insert sequence. That is to say, we got Poly[(R)-3-hydroxybutyrate] synthesizing gene in Biobrick format (BBa_K934001).

[http://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/detail/index.htm#Construction_of_pha-C1-A-B1_in_Biobrick_format#3. Link to detailed design description]

Source

  • amplified from genome DNA from Ralstonia eutropha H16
  • gene synthesis

References

Jumiarti Agus, et al, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650

Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16, Nat Biotechnol 24:1257-62 (2006)