Team:ETH Zurich/expresults/rr

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Revision as of 19:36, 15 October 2014

Riboregulators

As many former iGEM teams have encountered unwanted basal expression (leakiness) of their genes of interest (goi) (ref ETH2013, Groningen2012 amongst others), we decided to further investigate this severe obstacle towards tighly controllable systems. We started looking into the available literature and found with riboregulators a promising approach to challenge the issue of leakiness (ref isaacs). In addition, we chose this approach, to study and characterize such regulators in combination with cell-to-cell communication (ref quorum sensing).

The riboregulator systems include two parts: 1) a cis-repressed RBS in front of the goi, and 2) a co-expressed trans-activating RNA. This approach is described in the figure below.

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To characterize this system for cell-to-cell communication, we combined the riboregulator parts (ref to parts) with promoters of the quorum-sensing system used by our team (LuxI/LuxR, LasI/LasR, and RhlI/RhlR (ref to qs parts)). In our system, the riboregulators were intended for the control of integrase (ref). However, to have an easily accessible output, we used GFP as our riboregulated goi and measured the output fluorescence with a Tecan plate reader (ref to materials).

First, the experiments were conducted using pSEVA plasmids (ref pSEVA) to have a well characterized environment and an appropriate plasmid copy number. Also, the published riboregulator sequences were used. This important, because the original riboregulator sequence includes two forbidden restriction site (XbaI and SpeI). Second, the restriction sites were removed by two different approaches: a) multiple site-directed mutagenesis, b) blunting and ligation (Klenow and T4 DNA polymerase). Afterwards, the functionality was again tested to approve that no severe loss-of-function occurred due to the site-removal. In a third step, the construct was transferred into the pSB1C3 backbone to be in line with the BioBrick Standard (ref to standard). Below, you can find the corresponding graphs.

In more detail, we have characterized promoters of the three quorum-sensing systems. These are pLuxR, pLasR, and pRhlR. To have a reference, we first measured the transfer functions of the non-regulated GFP (ref constructs), i.e. the amount of fluorescence in dependence of the inducer concentration present. Next, we repeated the experiments with the corresponding riboregulated constructs (ref constructs) once with and without the forbidden restriction sites.

To conclude, we found a x-fold reduced basal GFP expression when using the riboregulator construct. Moreover, we achieved an increased signal-to-noise ratio of z-fold, as compared to the non-regulated system. Finally, the BioBrick conform part was confirmed to show no loss-of-funtion. However, the sensitivity towards the inducer was slightly reduced. This results are summarized in the graphs below.

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