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

(→‎Design Notes)
(→‎References)
 
(5 intermediate revisions by the same user not shown)
Line 9: Line 9:
  
 
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).
 
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===
Line 17: Line 19:
  
 
===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
 +
 +
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]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Unknown
  • 25
    INCOMPATIBLE WITH RFC[25]
    Unknown
  • 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)