Generator

Part:BBa_K3580102

Designed by: Yuya Otsuki   Group: iGEM20_Waseda   (2020-10-23)
Revision as of 13:27, 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).

Results

Similar to a very recent study from Jwett lab (Dudly et al 2019) who mixed 7 extracts, we synthesized limonene using only two extracts. Although fine-tuning can be possible when a larger number of extracts is prepared, we are sure that entry projects in iGEM should be simple but has engineering principle in order to expand iGEM sucess to an educational tool. This is why we selected the division into two extracts.

Fig. 2 GC/MS analysis results of cell-free sabinene synthesis system

We confirmed the world's first report of sabinene synthesis using cell extracts. Although elaborative reconstituted system by mixing of purified enzymes has been reported (Srividya Narayanan et al 2015), (Korman Tyler P et al 2017). Extract-base system with not only simple but engineering principle must provide much contribution to iGEM and DIY biology which as large number of players. Furthermore, extract systems do not require expensive coenzymes (Dudly et al 2019). As well as limonene detection of BBa_K3580101, we firstly confirmed sabinene retention time and SIM signal at the m/z values (77, 91, and 93). From our cell-free production, we then detected sabinene SIM signal and chromatogram peak which cannot find from the negative control. Also, from the system containing sabinene synthase and GPP synthase, a peak with the same retention time as the standard limonene product and ions with m/z values ​​characteristic in limonene at that retention time were detected. This was consistent with the fact that the paper that referred to the sabinene synthase used in this experiment also reported the synthesis of limonene as a by-product (Srividya Narayanan. et al 2015).

The part BBa_K3580102 used in our sabinene production is a new part for Parts Registry. One amino acid mutation was introduced into limonene synthase of Mentha spicata to create sabinene synthase, by using information reported in a 2015 paper by Srividya and Narayanan et al.

Fig. 3 Yields of monoterpene per reaction solution in this experiment

Finally, each of monoterpenes were quantified based on peaks of substance having 93 m/z and each monoterpene standard curves. Taking advantage of the modularity of the combination of extracts, we confirmed whether the yield of monoterpenes could be changed by changing the mixing ratio of the extract containing the enzyme of the mevalonate pathway and the extract containing GPP synthase and monoterpene synthase. As a result, changes in the yield of monoterpenes due to the mixing ratio of the extracts were observed (Figure. 3). The best yield of sabinene per reaction solution with sabinene synthase contained system was 4.60 [µmol/L].

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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