Part:BBa_K2841500
Legionella Sensing LacI Repressing Alpha Toxicity Experimental Device
In order to ensure that our dCAS9 (BBa_K2841510) gRNA (BBa_K2841514) sequence had minimal off target effects, we decided to test the ability of the cell to synthesise the riboregulated gRNA without an impact upon fluorescence. We used IPTG to induce synthesis of an mRNA strand upstream of a riboregulated gRNA, allowing us to quantify the fluorescence of the cell, and ensure that the gRNA was not causing undesired toxicity reducing GFP expression. Our data shows that the OD600 of the cells were all similar, where the induced cells actually grew at an improved rate towards the end. The fluorescence of the cells per OD600 was also significant, and we were able to demonstrate that the cells responded to the LacI repressor normally. The data also helped ensure all of our cells had the necessary protein expression for further use: the fluorescence showed the cell was overall healthy, and the effective response to the LacI demonstrated that our MG1655 Z1 cells still maintained their Z1 cassette (which also includes the TetR required for our experiments). The data retrieved from our own preliminary test kit ensured that our cells were suitable for use with the riboregulated gRNA detection system we intended to implement.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1077
Illegal NgoMIV site found at 1106 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 856
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