Part:BBa_K1138000

pAW42-gs (ugaiGEM2)

The plasmid pAW42-gs(ugaiGEM2)consists of the gs gene inserted into the backbone of pAW42-vector. The gs gene encodes for geraniol synthase enzyme. This vector has puramycin marker for selection in Methanogens and ampicilin marker for selection in E. coli.

2015 new application: modeling"

Our 2014 UGA-Georgia iGEM team mapped out the isoprenoid biosynthesis pathway for Methanococcus maripaludis, as we can take of this pathway’s production of high-carbon compounds. Additionally we adopted and adapted the M. maripaludis S2 metabolic model (iMM518) to contain exchange and formation reactions for geraniol synthase (Table 1). By incorporating the geraniol synthase gene, were able to calculate the rate of production of geraniol in a M. maripaludis cell using flux balance analysis.

Table 1

. The geraniol synthase metabolites and reactions added to the original M. maripaludis metabolic model (iMM518) from BioModels Database.

<” https://static.igem.org/mediawiki/2015/4/45/UGA-Georgia_Modeling_Table_1-6.png”>

This year, our 2015 UGA-Georgia iGEM team used our modified model to observe the rate of geraniol production after altering specific growth substrates, carbon dioxide (CO2) and ammonium (NH4). Shown below is the progression of flux balance analyses.

Target Growth Substrates:

Carbon source: CO2 Nitrogen Source: NH4 These two common growth substrates for M. maripaludis were chosen as our constraints, as they can be easily manipulated for in vivo experiments.

Target Products:

Biomass production Geraniol production The production of biomass for cells is essential and is significantly from isoprenoids. Geraniol is an isoprenoid derivative, so when targeting geraniol, we must also consider a reasonable biomass production rate.

Preliminary Observation:

Changes in CO2/NH4 ratio result in influenced biomass and geraniol production.

Exploring the Evidence:

Upon observing the relationship between the CO2/NH4 ratio and the production of our target products, biomass and geraniol, we began to specifically explore the ratio of these substrates. We explored two different options, (1) limiting nitrogen and (2) excess carbon. 1. Reducing NH4 + maintaining CO2 and observing change in geraniol production: (with same specific growth rate*) <” https://static.igem.org/mediawiki/2015/7/7c/UGA-Georgia_Modeling_Table_1-2.jpeg”> 2. A. Increasing CO2 + maintaining NH4 and observing change in geraniol production (with corresponding change in specific growth rate*) <” https://static.igem.org/mediawiki/2015/1/1c/UGA-Georgia_Modeling_Table_3.jpeg”> B. Increasing CO2 + maintaining NH4 and observing change in geraniol production (with same specific growth rate*) <” https://static.igem.org/mediawiki/2015/1/1e/UGA-Georgia_Modeling_Table_4.jpeg”>

Results:

1. Although CO2/NH4 ratio is increased by decreasing NH4, there was no change in geraniol production (no change in specific growth rate* as well) 2. The increase in CO2/NH4 by increasing CO2 A. with corresponding increase in specific growth rate* resulted in decreased geraniol production B. with same specific growth rate* resulted in increased geraniol production. <” https://static.igem.org/mediawiki/2015/5/50/UGA-Georgia_Modeling_Figure_1.png”>

• The constraints for specific growth rate were obtained from simulating the original iMM518 model to represent the specific growth rate of wild-type M. maripaludis.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1553
  Illegal XbaI site found at 1883
  Illegal XbaI site found at 1892
  Illegal SpeI site found at 6597
  Illegal PstI site found at 2421
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1553
  Illegal SpeI site found at 6597
  Illegal PstI site found at 2421
  Illegal NotI site found at 2608
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1553
  Illegal BglII site found at 1870
  Illegal BamHI site found at 2402
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1553
  Illegal XbaI site found at 1883
  Illegal XbaI site found at 1892
  Illegal SpeI site found at 6597
  Illegal PstI site found at 2421
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1553
  Illegal XbaI site found at 1883
  Illegal XbaI site found at 1892
  Illegal SpeI site found at 6597
  Illegal PstI site found at 2421
  Illegal AgeI site found at 538
  Illegal AgeI site found at 2444
  Illegal AgeI site found at 2620
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 4778
  Illegal BsaI.rc site found at 3161
  Illegal BsaI.rc site found at 3490
  Illegal SapI site found at 6442