Team:Edinburgh/modelling(generegulatorynetwork)

Personal note

When I first heard about iGEM, I bored sitting in a computing lab doing uni work and thinking “Why am I doing this?” I had never heard of synthetic biology but always knew that I wanted to go down the biological/medical side of engineering so when I received the email I knew that is what I wanted to do this summer. When I first started I had no idea about biology, what a promoter was, what a repressor was, however over the course of the summer I have learnt this and a whole lot more. iGEM has shown me some of the advancements that synthetic biology can make to the world and it will be very interesting to see how all these weird and wonderful things are put into practise in the future.
Rachel

The systems were modelled in Cellucidate using a set of rules, and initial conditions shown in the appendix below. The rules are based on Ty Thomson’s framework for creating modular and reusable models of individual BioBrick parts. From the beginning of the project, it was thought that we would use pniR, a nitrite-sensitive promoter. Thus modelling of different setups was done with pniR first, but we did not get this to work in the wet lab whereas, another promoter we had, pYeaR did, so this was then modelled. The promoters are similar both being repressed by nsrR, but pniR is repressed by an nsrR that is not naturally expressed in E. coli, whereas pYeaR is. Therefore, nsrR had to be expressed in the pniR systems.

Here are the different setups modelled when using the pniR promoter:

1.

2.

3.

4.

5.

Here are the different setups modelled using the pYeaR promoter:

1.

2.

3.

The genes that are under a ‘green’ promoter are constitutively weakly expressed.

The omp-C promoter is controlled by the presence of TNT. This promoter will activate the genes for PETNR enzyme and EYFP. For the TNT system, the TNT R1 receptor, and the TrZ fusion protein need to be expressed as described in the biology section.

The pYeaR promoter is activated in the presence of nitrates and nitrites. This promoter will activate the genes for luciferase (luxA and luxB).

It is the number of molecules being expressed that decides the optimal setup. When this was decided, the rest of the lux operon for the constant aldehydes was added into the model.

All models start at 0 seconds, and at 2000 seconds is when the bacteria lands in the soil where nitrates/nitrites and TNT are present.

Results

pniR setups:

1. The expression levels are high, with the receptor, fusion protein, and repressor being weakly expressed. Fine setup.

2. The repressor, nsrR is being expressed higher than setup 1, and repressing the synthesis of the luciferase proteins to a lower level.

3. Expression of the receptor, and fusion protein is higher, thus reducing the levels of other more significant proteins.

4. This is very similar to setup 1.

5. Expression of the receptor, fusion protein, and repressor is higher, thus reducing the levels of other more significant proteins.

pYeaR results (similar results to pniR setups):

1. Expression of the receptor, fusion protein, and repressor is higher, thus reducing the levels of other more significant proteins.

2. Expression of the receptor, fusion protein, and repressor is higher, thus reducing the levels of other more significant proteins.

3. The expression levels are high, with the receptor, fusion protein, and repressor being weakly expressed. Fine setup.

High expression levels of receptors, fusion protein, and repressors are not required, and reduce the levels of the others due to increase competition for ribosome’s, and a greater metabolic burden on the bacteria.

Setup 4 of pniR, and setup 3 of pYeaR would be the optimal setups as gene expression levels are high for the important genes. If time allowed, then setup 3 of pYeaR would be built into a single plasmid in the wet lab.

Setup 3 including the lux operon:



When everything is put together this is how it should work:

No nitrites/nitrates or TNT present: Just the aldehydes are expressed.

When nitrites/nitrates are present: luxA and luxB are also expressed giving off a blue light.

When TNT is present: EYFP and PETNR enzymes are expressed, but no light is seen as EYFP is not activated yet by the GFP wavelength

When nitrates/nitrites and TNT are present: Everything is expressed, and a yellow light is seen as the GFP fusion protein of the luciferase activates the EYFP.

Set of rules:

Edinburgh University iGEM Team 2009