Main results

Simple amplification loop • Why an AND gate ? • Biological results • Issues encountered • Coming up next

Simple amplification loop

In a first place, biologists and modellers worked on a simple amplification loop.

The modellers proved that a simple amplification loop could not work. There is no state where GFP is not expressed.

Why an AND gate ?

The team thought that an AND gate could be our solution. It could add a biological noise filter and resolve the problem of the continuous expression of GFP.

Modelling results confirmed that built system will work.

Biological results

Biological AND gate test.

As you can see the paraBAD works as expected. We began the construction of the amplification loop (paraBAD_gfp_cyaA).

Issues encountered

We have not yet constructed that part. We had some problem with the ligation procedure. Another explanation could be that our bacteria are transformed with the right construction leading to an over-expression of Adenylate cyclase which is lethal: this can be attributed to the fact that overproduction of cAMP is lethal to E. coli possibly due to an accumulation of methylglyoxal [1] [2].

Coming up next

In the future weeks (according to ours results) we will try to make the culture of our transformed bacteria in a medium complemented with glucose and acetate.
A possible track would be to add another regulation system on the adenylate cyclase production with the riboswitch RsmA/rsmY.

As we have had issues constructing paraBAD_gfp_cyaA using the standard restriction/ligation procedure, we are going to try another approach. We want to insert it on pSB3C5 using the Gibson Assembly procedure (we have the brick but we are failing the insertion).
Once this construction is achieved, we will transform BW25113 ΔcyaA E. Coli strain to test the designed amplification feed-forward loop using the same protocol as for the AND gate.

We also plan to test our engineered membrane receptor (TapZ) in BW25113 ΔEnvZ E. Coli strain. To test it, we are going to use an mCherry reporter under the promotion of pOmpC.

In order to estimate the functionality of our new riboswitch, we are planning to transform one strain of BW25113 with plac_fha1_mcherry (on a low-copy plasmid) and in a second strain the previous construct plus plac_RBS_rsmA (medium-copy plasmid). In a last strain we will add the third partner (plac_rsmY on a high-copy plasmid) to estimate its inhibition release action. To characterize this we will use flow cytometry experiments.

Team:Grenoble/Project/Main Results

From 2012.igem.org