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Team:ETH Zurich/expresults/rr
From 2014.igem.org
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==Riboregulators== | ==Riboregulators== | ||
| - | As many former iGEM teams have encountered basal expression (leakiness) of their genes of interest (ref ETH2013, Groningen2012 amongst others), we decided to investigate this severe obstacle. We started looking into the available literature and found with | + | As many former iGEM teams have encountered basal expression (leakiness) of their genes of interest (goi) (ref ETH2013, Groningen2012 amongst others), we decided to further investigate this severe obstacle. 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 quroum sensing). |
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| + | 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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| + | OUT FIGURE HERE | ||
| + | |||
| + | 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). | ||
Revision as of 19:15, 15 October 2014
Riboregulators
As many former iGEM teams have encountered basal expression (leakiness) of their genes of interest (goi) (ref ETH2013, Groningen2012 amongst others), we decided to further investigate this severe obstacle. 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 quroum 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.
OUT FIGURE HERE
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).
