Difference between revisions of "Part:BBa K1138000"

Latest revision as of 00:25, 25 October 2016

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.

2016 new application: modeling continued

After observing in 2015 (see below) that excess carbon could result in an increase of geraniol production and specific growth rate, we continued to use the M. maripaludis S2 metabolic model (iMM518) to optimize geraniol production. In 2016 we explored the possibility of both formate and acetate as secondary carbon sources to carbon dioxide. We further modified the model by adjusting the lower bounds of both acetate and formate exchange as to permit uptake. We began this endeavor, because it was noted in primary literature that both formate and acetate could replace carbon dioxide as a carbon source. We changed the values for both of these and observed their effect on the productions of biomass and geraniol.
Figure 1.Flowchart of Testing and Results

Results

Figure 2.Effect of Acetate on Specific Growth Rate 
 We discovered that the uptake of acetate had an inverse relationship, negatively affecting the formation of biomass.
 Our maximum biomass corresponded to no uptake of acetate, and as we forced the system to uptake acetate, biomass decreased.

Figure 3.Effect of Acetate on Geraniol Production 
 With regards to acetate, geraniol responded the same as biomass production. 
 There was an inverse relationship, as the uptake of acetate led to the decrease of geraniol production.

Figure 4.Effect of Formate on Specific Growth Rate
With regards to formate, we discovered that biomass production increased until it reached a maximum value (0.748 formate uptake) and then decreased linearly.

Figure 5.Effect of Formate on Geraniol Production
Geraniol production responded similarly to Biomass production. They both increased until Geraniol reached a maximum value and then decreased in an inverse relationship.

Discussion

Although possible, when the system is given the choice in optimizing both biomass and geraniol, it fluctuates the uptake of carbon dioxide and does not uptake formate or acetate. We discovered that even if we constrained the uptake rate of carbon dioxide to zero and forced the system to accept formate or acetate, we still would get no biomass and no production of geraniol. If biologically relevant, this could signify that carbon dioxide is a necessary carbon source. Nevertheless, the model was developed in such a way that carbon dioxide is the only carbon source that can be utilized. However, we then discovered that if we set carbon dioxide to a constant amount, we could then manipulate the amounts of formate and acetate and examine the effect they would have on our target products.

In manipulating the amount of formate, we found that both biomass formation and geraniol production increased with the increase of formate to a certain value and then began decreasing. It makes sense that geraniol and biomass would be affected in the same way since geraniol production is always proportional to the biomass formation. That increase in formate could result in an increase of geraniol and biomass to a point can be explained by the fact that formate is not only a carbon source, but is also an energy source for the cell. This relationship between formate and biomass formation/geraniol production has not been tested experimentally.

In manipulating the amount of acetate, we found that both biomass formation and geraniol production decreased. As mentioned, it makes sense that geraniol and biomass would be affected in the same way since geraniol production is always proportional to the biomass formation. Acetate's effect seems to have this negative effect because of how the incorporation of acetate as a carbon source requires ATP and is an energy expensive investment for the cell.

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.

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*)

2. A. Increasing CO2 + maintaining NH4 and observing change in geraniol production (with corresponding change in specific growth rate*)

B. Increasing CO2 + maintaining NH4 and observing change in geraniol production (with same specific growth rate*)

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.

• 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