Difference between revisions of "Part:BBa K3580101"

Latest revision as of 01:01, 28 October 2020

Ptrc-trGPPS-LS(Limonene synthase)

It is a part composed of trGPPS and limonene synthase whose expression is regulated by Ptrc, and LacI to enable IPTG induction of expression of those enzymes. This part was created and used in a paper reported by Alonso-Gutierrez, Jorge et al. in 2013. Limonene can 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 limonene synthesis system

We confirmed limonene synthesis GC/MS analysis with SIM. In this SIM analysis, ions with four m/z values characteristic in limonene (68 and 93) and sabinene (77 and 91, 93) were analyzed. By using authentic limonene standard, we confirmed a retention time for GC and the characteristic limonene SIM signal at the specific m/z values. At the same retention time with the standard, limonene-specific m/z value (68, 93) ions were detected in the selected ions (The upper right figure of Fig. 2). We also draw a GC chart by summation of the signals from the selected ions (The lower left figure of Fig. 2). By comparison with a negative control experiment which we omitted the extract containing GPP synthase and limonene synthase, we found clear peak from our limonene synthesis.

We regard our BBa_K3580101 as an improved version from pre-existing BBa_K3052001, which has only limonene synthase. Our improved part can use the set of IPP DMAPP both of which are provided not only mevalonate pathway used here but the other famous non-mevalonate pathway. Thus, the set is produced in so many engineered pathways, including the addgene plasmid used here, as an important intermediate set for bioproduction. From this view point, we are sure that the simple combination of two enzymes in this part satisfied easy use modularity in synthetic biology than pre-existing BBa_K3052001.

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 (Fig. 3). The best yield of limonene per reaction solution with limonene synthase contained system was 0.73 µM.

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]

@@ Line 8: / Line 8: @@
 [[File:T--waseda--monoterpene_2-2-1_parts_and_metabolic_pathways_in_this_experiment_and_a_schematic_diagram_of_the_experiment.png|1500px|thumb|center|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).
+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 ([https://parts.igem.org/Part:BBa_K3580103 BBa_K3580103]).  In order to take advantage of an engineering principle of synthetic biology we provided two biobrick parts ( [https://parts.igem.org/Part:BBa_K3580101 BBa_K3580101], [https://parts.igem.org/Part:BBa_K3580102 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.
-[[File:T--Waseda--monoterpene_2-2-2_GCMS_analysis_results_of_limonene_synthesis_system.png|1500px|thumb|center|Fig. 2 GC/MS analysis results of cell-free limonene synthesis system ]]
+[[File:T--Waseda--monoterpene_2-2-2_GCMS_analysis_results_of_limonene_synthesis_system.png|1500px|thumb|center|Fig. 2 GC/MS analysis results of cell-free limonene synthesis system]]
-We confirmed limonene synthesis GC/MS analysis with SIM.  In this SIM analysis, ions with four m/z values  characteristic in limonene (68 and 93) and sabinene (77 and 91, 93) were analyzed.   By using authentic limonene standard, we confirmed a retention time for GC and the characteristic limonene SIM signal at the specific m/z values.  At the same retention time with the standard, limonene-specific m/z value (68, 93) ions were detected in the selected ions (The upper right figure of Figure. 2).  We also draw a GC chart by summation of the signals from the selected ions (The lower left figure of Figure. 2).  By comparison with a negative control experiment which we omitted the extract containing GPP synthase and limonene synthase, we found clear peak from our limonene synthesis.
+We confirmed limonene synthesis GC/MS analysis with SIM.  In this SIM analysis, ions with four m/z values  characteristic in limonene (68 and 93) and sabinene (77 and 91, 93) were analyzed.   By using authentic limonene standard, we confirmed a retention time for GC and the characteristic limonene SIM signal at the specific m/z values.  At the same retention time with the standard, limonene-specific m/z value (68, 93) ions were detected in the selected ions (The upper right figure of Fig. 2).  We also draw a GC chart by summation of the signals from the selected ions (The lower left figure of Fig. 2).  By comparison with a negative control experiment which we omitted the extract containing GPP synthase and limonene synthase, we found clear peak from our limonene synthesis.
 We regard our BBa_K3580101 as an improved version from pre-existing BBa_K3052001, which has only limonene synthase.   Our improved part can use the set of IPP DMAPP both of which are provided not only mevalonate pathway used here but the other famous non-mevalonate pathway.  Thus, the set is produced in so many engineered pathways, including the addgene plasmid used here, as an important intermediate set for bioproduction.  From this view point, we are sure that the simple combination of two enzymes in this part satisfied easy use modularity in synthetic biology than pre-existing [https://parts.igem.org/Part:BBa_K3052001 BBa_K3052001].
@@ Line 21: / Line 21: @@
 [[File:T--Waseda--monoterpene_2-2-4_Yields_of_monoterpene_per_reaction_solution_in_this_experiment.png|1500px|thumb|center|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 limonene per reaction solution with limonene synthase contained system was 0.73 µmol/L.
+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 (Fig. 3).  The best yield of limonene per reaction solution with limonene synthase contained system was 0.73 µM.
 <!-- Add more about the biology of this part here