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Team:ETH Zurich/expresults/integrases
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== Integrases == | == Integrases == | ||
| - | The design of our XOR gates was based on integrase logic | + | The design of our [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR gates] was based on [https://2014.igem.org/Team:ETH_Zurich/modeling/xor#Biological_Principles integrase logic]. This means, depending on the input molecules, integrases can be expressed, subsequently switch a terminator sequence previously blocking gene expression, and then the output gene can be transcribed. This approach is explained [https://2014.igem.org/Team:ETH_Zurich/modeling/xor#Biological_Principles here]. |
| - | In order to characterize the integrase system described above, we first combined the pLux promoter with one of our integrase genes followed directly by mCherry to make the expression accessible. Also, this system includes an XOR buffer gate per default blocking transcription of sfGFP. Upon integrase activity and switching the gate into ON-state, the terminator should have been removed and sfGFP should have been expressed. We designed three different constructs for characterization of the recombinases and their cross-activity. However, the measurement of fluorescent proteins, with both a plate reader and a | + | In order to characterize the integrase system described above, we first combined the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)] with one of our integrase genes followed directly by mCherry to make the expression accessible. Also, this system includes an XOR buffer gate per default blocking transcription of sfGFP. Upon integrase activity and switching the gate into ON-state, the terminator should have been removed and sfGFP should have been expressed. We designed three different constructs for characterization of the recombinases and their cross-activity. However, the measurement of fluorescent proteins, with both a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 plate reader] and a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#BD_LSRFortessa.E2.84.A2_Flow_Cytometer_System flow cytometer], did not indicate GFP expression due to recombinase activity. Nevertheless, mCherry was clearly detectable upon induction in plate reader experiments indicating that the induction itself worked (see fig.4 and fig.5). |
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Revision as of 12:51, 17 October 2014
Integrases
The design of our XOR gates was based on integrase logic. This means, depending on the input molecules, integrases can be expressed, subsequently switch a terminator sequence previously blocking gene expression, and then the output gene can be transcribed. This approach is explained here.
In order to characterize the integrase system described above, we first combined the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)] with one of our integrase genes followed directly by mCherry to make the expression accessible. Also, this system includes an XOR buffer gate per default blocking transcription of sfGFP. Upon integrase activity and switching the gate into ON-state, the terminator should have been removed and sfGFP should have been expressed. We designed three different constructs for characterization of the recombinases and their cross-activity. However, the measurement of fluorescent proteins, with both a plate reader and a flow cytometer, did not indicate GFP expression due to recombinase activity. Nevertheless, mCherry was clearly detectable upon induction in plate reader experiments indicating that the induction itself worked (see fig.4 and fig.5).
As our constructs did not show the expected functionality, we decided to directly use the plasmids described by Bonnet et al. which where obtained from addgene (ref, ref). However, we were using a TOP10 strain not expressing TetR by default (as compared to DH5alphaZ1) and as a result our strain had to be co-transformed with an additional plasmid encoding TetR. Also, we used defined M9 medium with 0.4% glycerol and 1%CAA (ref) instead of proprietary defined medium (Teknova Hi-Def Azure medium). As of today, this set-up did not allow us to get the integrase XOR gate running. We are not giving up on this and are proceeding with debugging our construct further and hope to find a solution until the Giant Jamboree in Boston.
PUT GRAPH HERE
