Revision as of 02:23, 20 October 2016

LAB RESULTS

Our fellow iGEM colleagues will surely agree with us when we say that our precious bacteria are very stubborn and mischievous and that they don't always want to behave like we would want them to. It takes a group of very stubborn and cunning people to trick them into doing what we want. Last few months were a tug-of-war between experimentalists and Pavlov's coli. We definitely got pulled on several occasions. But we are a stubborn bunch of people, so let us present how we pulled back and got as far as we did.

Construction of nitric oxide sensor: NorV Promoter

Our initial design was focused on building a nitric oxide sensor which consists of a nitric oxide responsive promoter (pNorV) and the corresponding regulator NorR. We were happy that we were able to construct the plasmids very early on in the project. Unfortunately sequencing results for norR showed a relatively big deletion inside the norR gene. Nevertheless, we performed a preliminary experiment with our construct. To our surprise we saw a significant induction of pNorV promoter even with low doses of inducer DETA/NO. This lead us to hypothesize that a genomic version of norR is responsible for the activation our promoter. We proved the hypothesis by transforming and studying our system in a norR knockout E. coli strain.

Figure 1: PnorV dose response curve for a very low range of DETA/NO concentrations. Samples where norR was not present in the cell also got induced when the DETA/NO was added

Figure 1: Figure shows that we are able to get an induction of pNorV in the wild type Keio strain where genomic copy of norR gene was present. There was no induction of pNorV in the norR knock-out strain

We were able to build:

functional pNorV promoter

We were not able to build:

norR regulator of pNorV

However, we were able to prove that a genomic copy of norR is sufficient for an induction of pNorV

Construction of an AHL sensor: esabox/EsaR system

During our brainstorming sessions in the months before "hitting the lab" we put special focus on trying to find a system for an AHL sensor which would be based on a repressor rather than activator. We estimated this would allow us to build a better AND gate. We found the esabox/EsaR system. Esaboxes are binding sites for EsaR. We saw a potential to create many different combinations of hybrid promoters by placing esaboxes on different places and in different numbers to the promoter region. However, due to the strong secondary structure and repetitive sequences, esaboxes proved to be a challenge to create. We tried several different approaches and at the end we managed to construct five different combinations of synthetic promoters with esaboxes in different numbers and different spacings. We additionally managed to create more than 10 different AND gate promoters with esaboxes and pNorV. In total we created almost 20 different promoters just by varying the location and number of esaboxes in the promoter region. We were able to observe what is the effect of spacers and number of esaboxes on the behavior of the promoters.

When we started with testing phase of our esabox promoters we observed that while we can successfully repress transcription with our EsaR repressor, we cannot successfully release the repressor from the promoter.

We were able to build:

five different promoters with different combinations of esaboxes as roadblocks

We were not able to build:

EsaR repressor which can get released from promoter upon induction

Construction of a recombinase based switch

Permanent memory is an important aspect of our project. The most obvious way of achieving permanent memory in cells is by changing their DNA. Recombinases provide an elegant way to create such uni-directional switch.They flip a sequence flanked between two recombinase recognition sites. However, it is important that we consider the dynamics of recombinases when we try to build such switch. Intuition often fails when it comes to estimating the dynamics of a design or a system. This is where a collaboration between theory and experiments plays an important role. In our project, the interaction between theory and experiments has been important since the beginning in the construction a recombinase based switch. In their quest to minimize the potential leakiness of our system, modelers discovered a better implementation of our idea. That means that after we already spent a month in the lab designing and building our system,they provided us with important simulations of our system and offered a novel design which we immediately started to build. On the other hand, modelers needed rigorous experimental data to be able to characterize recombinases and their dynamics. This would again in turn help experimentalists create a better design. In our project we studied dynamics of two different recombinases, bxb1 and tp901 by creating reporters for the respective recombinases and perform rigorous experiment about their dynamics. We studied properties of three versions with different degradation tags for each of the recombinases.

Multiplexing

Another important aspect of our idea is that by implementing associative learning in the circuit, we are provided with the flexibility to detect several different markers. In the scope of our project we tried to demonstrate that there is potential to expand markers to additional molecules of AHL, as well as also other metabolites, which are considered important in IBD. We used directed evolution to try and evolve EsaR to change specificity to a different AHL. Unfortunately esaR turned out to be a tough nut to crack in this project. However, we were able to show that the dual selector system is a suitable selection tool if the repressor is strong enough. We were able to expand our idea beyond AHL. Preliminary results show that it is possible to build an AND gate which could be used in the associative learning circuit where we would simultaneously detect lactate (instead of AHL) and nitric oxide

Dual Selector System: CAT-UPRT Fusion Protein

Assay:

The response of the dual selector plasmid containing a CAT-UPRT fusion protein (CAT: chloramphenicol acetyltransferase, UPRT: uracil phosphoribosyltransferase) towards 5-fluorouracil was tested. The Keio-Collection strain JW2483-1 (δupp) was transformed with a plasmid constitutively expressing the fusion protein.
The cells were grown overnight in M9 medium, diluted to OD=0.1 and transferred into a flat bottom 96-well plate. They were grown for another three hours and finally different concentrations of 5-fluorouracil and chloramphenicol were added (10 µl into 190 µl of bacterial culture, final concentration are shown.)

Conclusions:

Whereas cells expressing the fusion protein are resistant against chloramphenicol, the are especially sensitive towards 5-fluorouracil.
The cells lacking the plasmid as well as do not have a chromosomal copy of upp are less inhibited but are sensitive towards chloramphenicol.

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