Difference between revisions of "Part:BBa K338003:Design"
 
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===Design Notes===
 
===Design Notes===
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When ligated upstream of [[Team:Caltech/BBa_K338004|BBa_K338004]], the completed construct was designed to express all three PHA synthase genes required to make PHB oligomers from soybean oil. The three genes would be transcribed polycistronically on a single mRNA transcript under the IPTG-inducible control of the <partinfo>BBa_K215000</partinfo> promoter. Naturally, each gene is preceded by a standard RBS (<partinfo>B0034</partinfo>) and the transcript finishes with a strong terminator (<partinfo>B0015</partinfo>), for a total size of about 3500bp.
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When ligated upstream of <partinfo>BBa_K338004</partinfo>, the completed construct was designed to express all three PHA synthase genes required to make PHB oligomers from soybean oil. The three genes would be transcribed polycistronically on a single mRNA transcript under the IPTG-inducible control of the <partinfo>BBa_K215000</partinfo> promoter. Naturally, each gene is preceded by a standard RBS (<partinfo>B0034</partinfo>) and the transcript finishes with a strong terminator (<partinfo>B0015</partinfo>), for a total size of about 3500bp.
  
 
Note that these three genes should only cause the production of PHB ''oligomers'' in cells, not hardened plastic. A crosslinking agent is required to link the oligomers and form the final plastic product. Over-expression of the ''phaC1'' gene could cause some crosslinking, but this has not been experimentally verified.
 
Note that these three genes should only cause the production of PHB ''oligomers'' in cells, not hardened plastic. A crosslinking agent is required to link the oligomers and form the final plastic product. Over-expression of the ''phaC1'' gene could cause some crosslinking, but this has not been experimentally verified.

Latest revision as of 11:15, 26 October 2010

PHA Synthase Composite, Part 1/2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 987
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 953
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 326
    Illegal NgoMIV site found at 338
    Illegal AgeI site found at 701
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

When ligated upstream of BBa_K338004, the completed construct was designed to express all three PHA synthase genes required to make PHB oligomers from soybean oil. The three genes would be transcribed polycistronically on a single mRNA transcript under the IPTG-inducible control of the BBa_K215000 promoter. Naturally, each gene is preceded by a standard RBS (BBa_B0034) and the transcript finishes with a strong terminator (BBa_B0015), for a total size of about 3500bp.

Note that these three genes should only cause the production of PHB oligomers in cells, not hardened plastic. A crosslinking agent is required to link the oligomers and form the final plastic product. Over-expression of the phaC1 gene could cause some crosslinking, but this has not been experimentally verified.

SY Lee describes how a similar gene construct (pSYL105) was used to produce very large amounts of PHB, up to 80-90% of the dry cell weight, under certain conditions. Synthesis of PHB is related to the amount of acetyl-CoA available - synthesis was bolstered in the presence of complex nitrogen sources, amino acids, or oleic acid. He also mentions that PHB production was highly dependent on the particular bacterial strain used. [1]

Source

Registry of Standard Biological Parts

References

  1. Lee, SY. Bacterial polyhydroxyalkanoates. Biotechnology and Bioengineering 49, 1-14 (1996).