Generator

Part:BBa_K3580102

Designed by: Yuya Otsuki   Group: iGEM20_Waseda   (2020-10-23)
Revision as of 13:20, 27 October 2020 by Yuya Otsuki (Talk | contribs) (→‎Experiment)


Ptrc-trGPPS-SS(Sabinene synthase)

It is a part composed of trGPPS and sabinene synthase whose expression is regulated by Ptrc, and LacI to enable IPTG inductionof expression of those enzymes.This part was designed based on the Ptrc-trGPPS-LS(BBa_K3580101)used in the 2013 paper by Alonso-Gutierrez, Jorge et al. The introduction of the one-amino acid mutation reported in a 2015 paper by Srividya, Narayanan et al. changed the limonene synthase of Ptrc-trGPPS-LS to sabinene synthase.Sabinenecan be synthesized by combining this part with an upstream pathway that can supply the products of the mevalonate pathway (IPP and DMAPP).

Experiment

Fig. 1 Parts and metabolic pathways in the experiment of monoterpene synthesis and a schematic diagram of the experiment

In this cell-free monoterpene synthesis, we mixed two E. coli extracts each of which has either first 7 or last 2 enzymes of a pathway from Ac-CoA, which is a major intermediate of cell central metabolism. Through mevalonate pathway, the former extract one (derived from E. coli into which pBbA5c-MevT-MBI has been introduced) can provide IPP and DMAPP, which can also be used as intermediates for other important biosynthesis. Here we indeed supplemented only glucose and acetate as carbon sources. We obtained expression system for those seven genes from addgene and have converted this into Biobrick RFC 1000 format by synonymous replacement (BBa_K3580103). In order to take advantage of an engineering principle of synthetic biology we provided two biobrick parts ( BBa_K3580101, BBa_K3580102) for the source for the latter extract. BBa_K3580101 has GPP synthase (GPPS) and limonene synthase. Although GPP synthase is shared with BBa_K3580101, BBa_K3580102 has sabinene synthase, which has one point mutation in limonene synthase (Srividya Narayanan et al 2015) and a new coding sequence for Parts registry of iGEM (See here for more details on this experiments).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1980
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1980
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2366
    Illegal XhoI site found at 4022
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1980
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1980
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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