Composite
Latex Oper

Part:BBa_K2027039

Designed by: Gordon Sun, Taylor Sihavong, Michael Becich   Group: iGEM16_Stanford-Brown   (2016-10-13)


Rubber Transferases + Small Rubber Particle Protein Cassette

This construct was conceived to transform the latex synthesis pathway into a single cell organism that could be grown in bioreactors, such as Escherichia coli. Through genetic manipulation of the endogenous methylerythritol phosphate (MEP/DOXP) pathway and transformation with rubber production genes from Hevea brasiliensis, we developed a transgenic single cell organism capable of converting glucose into cis-1,4-polyisoprene, the primary chemical constituent in latex. Not only is our modified organism capable of producing cis-polyisoprenes quickly, but also in high yield. This part accounts for the enzymes responsible for latex polymer synthesis. It contains proteins native to Hevea brasiliensis, the primary source of natural latex. The two classes of proteins this construct holds are cis-prenyltransferases and small rubber particle proteins (SRPP). When provided isopentenyl pyrophosphate(IPP), the cis-prenyltransferases will polymerize the IPP molecules into a chain by linking the 1st and 4th carbons. Although SRPP is not directly involved in the process of linking monomers, it serves as a cofactor for the cis-prenyltransferase as a rubber elongating factor. Addition of magnesium sulfate is also necessary to activate enzyme activity. Since the genes are linked to an IPTG inducible promoter, IPTG must be added to culture to initialize gene expression, and subsequently cis 1,4 polyisoprene synthesis. In short, the purpose of this construct is to enable isopentenyl pyrophosphate polymerization to polyisoprene compounds.

The parts included in this construct are HRT1(AB061234.2), HRT2(AB064661.2), and SRPP AF051317. All protein DNA sequences have been codon optimized for E. coli transfection. While the HRT1,2 proteins are involved in the process of elongating isopentenyl pyrophosphate monomers to form cis 1,4 polyisoprene, the SRPP serves as a coenzyme for HRT1,2 activity. Magnesium sulfate is also needed as a cofactor for HRT activity, and IPTG is needed to induce expression of all proteins in the construct. The conjugated tag on each protein should allow for purification and visualization with anti-FLAG antibodies, visualization with Lumio™ Green, and purification with nickel columns.

Sequences were derived from Asawatreratanakul et al. (http://www.ncbi.nlm.nih.gov/pubmed/14622254) and Oh, Kang et al. (http://www.ncbi.nlm.nih.gov/pubmed/10358068)

These parts, the cis-prenyltransferases and SRPP, have been biobricked into the following parts:

Additionally, our construct also includes the following ribosome binding sites, terminators, tags, and promoters:

For optimal yield, this construct is best used in conjunction with our DXS synthase construct (http://parts.igem.org/Part:BBa_K2027040), whose protein product converts pyruvate and G3P into isopentenyl pyrophosphate. Alternatively, an IPP supplement can be provided to the transgenic organism containing this part.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 25
    Illegal SapI.rc site found at 80


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