Difference between revisions of "Team:NRP-UEA-Norwich/Collaborations/Manchester"
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<p class="space20">The system consists of two quorum sensing (QS) systems EsaR/I and CepR/I. The EsaR/I system belongs to the plant pathogen Pantoea stewartii. The second QS-System, CepR/I, belongs to the opportunistic pathogen Burkholderia cenocepacia. For details about the system and the model please look into: https://2015.igem.org/Team:Manchester-Graz/modelling.</p> | <p class="space20">The system consists of two quorum sensing (QS) systems EsaR/I and CepR/I. The EsaR/I system belongs to the plant pathogen Pantoea stewartii. The second QS-System, CepR/I, belongs to the opportunistic pathogen Burkholderia cenocepacia. For details about the system and the model please look into: https://2015.igem.org/Team:Manchester-Graz/modelling.</p> | ||
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| − | + | <p class="space10">Butyrate is converted starting from two Acetyl CoAs over several steps to Butyryl CoA. In the last step the Coenzyme A is transferred to Acetate, producing Acetyl CoA and Butyrate (1). | |
| − | + | For simplicity, the pathway was reduced to some essential parts and steps in the pathway. Acetate is converted to Acetyl CoA. The steps to Butyryl CoA are reduced to one step. Coenzyme A is recycled in the last step to Butyrate and can be reused to produce Acetyl CoA (Fig 1). </p> | |
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<p>Even though glycogen acylation has not yet been described, phosphorylation of glycogen has been studied previously in both muscle and bacterial glycogen. Tagliabracci V. S. et al showed that the phosphate in glycogen is present as C2 and C3 phosphomonoesters <sub><a data-id="ref" class="scroll-link" style = "color: #002bb8;">3</a></sub> . Phosphorylation of starch has been also characterized2. According to Blennow A. et al, the phosphate groups bind at the free C6 and C3 hydroxyl groups of the glucose units. Both groups are located at the hydrophilic surface of the double helix, which might affect the stability of the molecule <sub><a data-id="ref" class="scroll-link" style = "color: #002bb8;">4</a></sub>. | <p>Even though glycogen acylation has not yet been described, phosphorylation of glycogen has been studied previously in both muscle and bacterial glycogen. Tagliabracci V. S. et al showed that the phosphate in glycogen is present as C2 and C3 phosphomonoesters <sub><a data-id="ref" class="scroll-link" style = "color: #002bb8;">3</a></sub> . Phosphorylation of starch has been also characterized2. According to Blennow A. et al, the phosphate groups bind at the free C6 and C3 hydroxyl groups of the glucose units. Both groups are located at the hydrophilic surface of the double helix, which might affect the stability of the molecule <sub><a data-id="ref" class="scroll-link" style = "color: #002bb8;">4</a></sub>. | ||
Revision as of 13:19, 17 September 2015
Manchester-Graz Collaboration
The Manchester-Graz team have developed an expression system designed to regulate single and multi-gene pathways for an intestine expression. For controlling a wide range of pathways it is designed in a flexible and modular manner. They tested the production of butyrate in the gut. The pathway was incorporated into the expression system model to observe the expression of butyrate under the control of the developed system. The model generated helped us to understand how the system is dealing with pathways that consist of several enzymes at an intestine level.
The system consists of two quorum sensing (QS) systems EsaR/I and CepR/I. The EsaR/I system belongs to the plant pathogen Pantoea stewartii. The second QS-System, CepR/I, belongs to the opportunistic pathogen Burkholderia cenocepacia. For details about the system and the model please look into: https://2015.igem.org/Team:Manchester-Graz/modelling.
Figure 1: Overview of the reduced pathway. Coenzyme A (Co A) is recycled in the pathway by the butyryl CoA-acetyl CoA-tranferase.
Butyrate is converted starting from two Acetyl CoAs over several steps to Butyryl CoA. In the last step the Coenzyme A is transferred to Acetate, producing Acetyl CoA and Butyrate (1). For simplicity, the pathway was reduced to some essential parts and steps in the pathway. Acetate is converted to Acetyl CoA. The steps to Butyryl CoA are reduced to one step. Coenzyme A is recycled in the last step to Butyrate and can be reused to produce Acetyl CoA (Fig 1).
Even though glycogen acylation has not yet been described, phosphorylation of glycogen has been studied previously in both muscle and bacterial glycogen. Tagliabracci V. S. et al showed that the phosphate in glycogen is present as C2 and C3 phosphomonoesters 3 . Phosphorylation of starch has been also characterized2. According to Blennow A. et al, the phosphate groups bind at the free C6 and C3 hydroxyl groups of the glucose units. Both groups are located at the hydrophilic surface of the double helix, which might affect the stability of the molecule 4.
We therefore needed to model the putative changes in glycogen structure depending on the location of the modification. Our aim was to produce carbohydrate molecules with 5-10% of the residues modified since this level of butrylation (achieved by chemical modification) has positive benefits to the colon of rats 5 Our model indicates that this level of modification is only viable if the enzyme can modify any base - If it can only use the carbon-4 position it would impact on the growth of the molecule.
Glyco2D Works!
When we increase the initial percentage of random acylation, the total number of modified glucoses increased. We showed a great linear correlation between both (see figure 3)
Figure 3: A graph showing the results from Glyco2D acylation simulation.
Acylation on carbon 4
As we're using putative acyltransferases, we don't know the carbon in the glucose molecule that gets acylated. If it binds to C4, the acylation will restrict further growth of the glycogen molecule.
As we can see, by having a chance of acylation of 1% at C4, the glucose lost is around 50%. By 'glucose lost' we mean all the glucose molecules that could haven been added to the unmodified molecule (see figure 4).
Therefore, if the acyl group always binds to C4, we won't have a viable glycogen molecule (see figure 5).
Figure 4: Results from acylation simulation with acyl group added into carbon 4.
Figure 5: A modified glycogen structure where acylation will restrict growth produced by Glyco2D. Glucose molecules with acyl groups added are coloured in red.
Acylation in any available position
Another hypothesis is that the acyltransferase can add the group to any carbon on the glucose molecule. We assume a random chance of a group being added on the 4 carbons available in the molecule. If it is added to C4, it will restrict growth. If it is added to C6, it might restrict branching if it binds to the glucose molecule in the 5th or 10th position in the chain (branching points). The normal growth will not be altered if it binds C2 or C3.
We ran the simulation 300 times for each % of acylation and calculated the average for all the possible randomly generated molecules. We observed a good correlation between the final % of acylated glucose residues and the % of glucose molecules that are lost in comparison to the unmodified molecule (see figure 6).
As we can see, when a 5% of final acylation is achieved, only a 22 % of the glucose in the molecule is lost, and this give rise to a valid glycogen molecule. This model allowed us to define the optimal value so that the minimum amount of glucose units are lost from the glycogen molecule but we achieve the desired level of acylation.
Figure 6: Acylation simulation results: total glucose lost for different percentages of overall acylation percentage.
Figure 7: A modified glycogen structure where acylation may restrict growth depending on where the acyl groups are added. Glucose molecules with acyl groups added are coloured in red.
